Hesperidin protects renal and hepatic tissues against free radical-mediated oxidative stress during DMBA-induced experimental breast cancer

Hesperidin has been reported to have an excellent and wide variety of biological activities. This property has brought the compound to a new stage in the treatment of various oxidative stress-mediated diseases. The present investigation was aimed to evaluate the therapeutic potential of hesperidin by assaying the activities of antioxidant enzymes, lipid peroxidation, membrane bound marker enzymes, adenosine triphosphates, and TCA cycle enzymes, especially in kidney tissues during 7,12-dimethybenz(a)anthracene-induced breast cancer. Daily oral administration of hesperidin (30 mg/kg body wt) to breast cancer-bearing rats for 45 days demonstrated a significant (P < .05) decline in renal lipid peroxidation and membrane bound marker enzymes, as well as a remarkable increase in adenosine triphosphatases, mitochondrial functional enzymes, and renal antioxidants. Furthermore, histological studies of liver and kidney provided evidence of biochemical alterations. Thus, the protective effects of hesperidin on attenuating the peroxidation reaction and membrane bound marker enzyme activities as well as upregulation of adenosine triphosphatases, TCA cycle enzymes, and antioxidants suggest promising uses of flavonoglycoside hesperidin in the future treatment of oxidative stress-mediated diseases.     

Modulating effect of Withania somnifera on TCA cycle enzymes and electron transport chain in azoxymethane-induced colon cancer in mice

The aim of the present investigation was to evaluate the efficacy of Withania somnifera on tricarboxylic acid (TCA) cycle enzymes and electron transport chain in azoxymethane-induced experimental colon cancer in mice. Azoxymethane at the dose of 15?mg/kg body weight was induced intraperitoneally once in a week for 28?days. The progression in colon tumor development was correlated with the appearance of the histological biomarker and aberrant crypt foci (ACF). Azoxymethane-induced colon cancer animals were treated with 400?mg/kg body weight of W. somnifera once in a week orally for 28?days. After the experimental period, the animals were killed and analyzed for TCA cycle key enzymes, such as isocitrate dehydrogenase (ICDH), succinate dehydrogenase (SDH), malate dehydrogenase (MDH), and α-keto glutarate dehydrogenase (α-KGDH). The modulating effect of W. somnifera on TCA cycle key enzymes and electron transport chain complexes were investigated against colon cancer induced by azoxymethane in Swiss albino mice. Decreased activities of TCA cycle key enzymes such as ICDH, SDH, MDH, and α-KGDH in colon cancer bearing animals were observed. W. somnifera administration normalized these enzyme levels in azoxymethane-induced experimental mice. These results suggested that W. somnifera is the promising chemotherapeutic agent for the treatment of colon cancer.     

Age-related changes in glucose metabolizing enzymes in spleen, thymus, and pulmonary lavage cells from F344 rats

Healthy male Fischer 344 rats were sampled at 6, 12, 18, and 24 months of age. There was no gross pathological evidence or deviations in body weight, hematology, or clinical chemistry that were indicative of disease. Mixed populations of thymus, spleen, and pulmonary cells were obtained for enzymatic analyses. Key enzymes from the hexose monophosphate shunt, glycolysis and the tricarboxylic acid cycle were evaluated to determine if there were tissue-specific or pathway-specific changes that occurred during aging. The enzyme responses among the tissues were not consistent during the aging process. Generally the activities of the glucose metabolizing enzymes in thymus and pulmonary lavage cells decreased with age whereas they increased in the spleen cells. Between 18 and 24 months enzymes representative of all three glucose metabolic pathways decreased in pulmonary lavage cells, whereas the decreases in thymic cells were mainly restricted to glycolytic enzymes. By contrast there were two- to ten-fold increases during aging in all of the splenic enzymes measured except malate dehydrogenase. The alterations in tissue enzyme activities probably reflected the changing cellular populations during aging, and in the thymic and pulmonary lavage cellular environment resulted in a loss of energy production by glucose oxidation, compared to the vigorous activity maintained in spleen.     

Anticarcinogenic activity of kalpaamruthaa,a modified Siddha preparation,against aflatoxin–B1-induced hepatocellular carcinoma in rats

To investigate the potential anticancer effect of kalpaamrutha (KA), a modified Siddha preparation, against aflatoxin–B₁(AFB₁)-induced hepatocellular carcinoma (HCC) in rats, KA (200 mg/kg body weight/day) was administered to AFB₁-induced (2 mg/kg body weight i.p.) HCC rats, orally for 28 days. At the end of the experimental period, changes in body weight were recorded; the levels of alpha-fetoprotein and total protein was estimated in the serum; activities of marker enzymes were assayed in serum and total protein; DNA and RNA contents were estimated in liver tissue; activities of glycolytic enzymes, mitochondrial Krebs cycle enzymes, and respiratory chain enzymes were assayed in liver tissue of control and experimental rats. Further histopathological examination of the liver sections was carried out to support the anticancer effect of KA against AFB₁-induced HCC. Administration of KA overall increase in glycolytic enzymes with a subsequent reduction in gluconeogenic enzymes, mitochondrial Krebs cycle enzymes, and respiratory chain enzymes was observed in HCC-induced rats. These altered enzyme activities were effectively counteracted by supplementation with KA and also prevented the body weight loss by enhancing the host energy metabolism. Histological studies supported the biochemical findings. The results of the present study reveal that the drug KA has potential anticancer effect against AFB₁-induced HCC rats.     

Anaplerosis of the citric acid cycle: role in energy metabolism of heart and skeletal muscle

Efficient energy transfer in heart and skeletal muscle requires a series of moiety-conserved cycles. The intermediaries of the metabolic cycles are finely regulated to maintain a dynamic state of equilibrium. In heart muscle, depletion of the citric acid cycle (TCA cycle) through a block of 2-oxoglutarate dehydrogenase results in a rapid decline of contractile function, which is reversed by the addition of substrates promoting flux through the carboxylating enzymes, malic enzyme, pyruvate carboxylase and propionyl-CoA carboxylase. Anaplerosis describes a pathway, which replenishes a metabolic cycle. We show that enzymes for anaplerosis of the TCA cycle are expressed in heart and skeletal muscles. The role of anaplerosis of the TCA cycle in skeletal muscle is not entirely clear, but there is substantial evidence for its operational control during exercise. While the anaplerotic flux of carbon into the TCA cycle exceeds the removal of cycle intermediates, this process is only transient and reverses with prolonged exercise. It remains to be determined, however, whether the initial increase in TCA cycle intermediates is obligatory in order to attain high rates of TCA cycle flux, or primarily reflects a mass action phenomenon owing to increased substrate availability for anaplerotic pathways.     

Therapeutic potential of Dendrophthoe falcata (L.f) Ettingsh on 7,12-dimethylbenz(a)anthracene-induced mammary tumorigenesis in female rats: effect on antioxidant system, lipid peroxidation, and hepatic marker enzymes

In this study, the therapeutic potential of the hydroalcoholic extract of Dendrophthoe falcata (L.f) Ettingsh (Loranthaceae; HEDF) on 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary carcinoma was investigated in Wistar female rats at 55 days of age. Thirty female rats were divided into five groups with six animals in each group: control, DMBA (25 mg in 0.5 ml olive oil by air pouch technique), and DMBA + HEDF (250, 475, and 950 mg/kg). After 90 days of induction, HEDF were administered for 28 days, by gastric intubations. The levels of lipid peroxides and activities of enzymic and nonenzymic antioxidants were measured in serum, liver, kidney, and breast of both control and experimental groups. In addition to this, liver marker enzymes were also assessed. Rats treated with DMBA showed an increase in lipid peroxidation accompanied by high malondialdehyde levels along with lowered activities of enzymic antioxidants (superoxide dismutase, catalase, glutathione peroxidase) and nonenzymic antioxidants (glutathione, ascorbic acid, and α-tocopherol). A significant decrease in alanine aminotransferase, aspartate aminotransferase with a sharp increase in alkaline phosphatase, acid phosphatase, and 5′-nucleotidase was observed in the liver of mammary cancer-bearing animals. HEDF treatment caused the activity of these liver marker enzymes’ return to almost normal control levels. Furthermore, the breast tumor weight decreased significantly in the DMBA + HEDF-treated groups. This result suggests that HEDF shows antioxidant activity and play a protective role against DMBA-induced breast carcinogenesis.     

Modulation of tricarboxylic acid cycle dehydrogenases during hepatocarcinogenesis induced by hexachlorocyclohexane in mice

The sequential distribution of key tricarboxylic acid (TCA) cycle enzymes have been investigated during hexachlorocyclohexane (HCH)-induced hepatocarcinogenesis in Swiss mice. Animals were continuously exposed to HCH (500 ppm) for 2, 4, and 6 months until liver tumor developed. The activity of TCA cycle enzymes such as isocitrate dehydrogenase (ICDH), succinate dehydrogenase (SDH), and malate dehydrogenase (MDH) have been studied. The activity of all the enzymes declined after 2 months of exposure of HCH in the liver. The neoplastic nodules and tumors developed after an exposure of HCH for 4 and 6 months, respectively. Neoplastic nodule and tumor showed wide variations in the activity and distribution of TCA cycle enzymes. The decreasing pattern in the activity of enzymes persisted in the non-neoplastic and non-tumor regions of the liver except SDH. However, the cells in nodular area and tumor showed intense enzymatic activities at cellular level. In the nodular region SDH activity declined prominently, whereas the non-nodular area showed positive reaction. Conspicuously, the tumor showed islands of positive and negative zones for TCA cycle dehydrogenases. The significance and relevance of such a distribution pattern still remains a mystery. The results are discussed in the light of HCH-induced toxicity on energy metabolism in exposed animals and possible role of such enzymes in the tumor formation.     

Time course of changes in human skeletal muscle succinate dehydrogenase and cytochrome oxidase activities and maximal oxygen uptake with physical activity and inactivity.

Succinate dehydrogenase (SDH) and cytochrome oxidase activities in the lateral vastus of the human quadriceps femoris muscle together with total body VO2 max were followed during an 8-10 week period of endurance training (n = 13) and a successive 6 week period without training (n = 8). During the training period there was a gradual increase in both VO2 max and muscle oxidative enzyme activities, all being significantly different from the pre-training levels after 3 weeks of training. After 8 weeks of training VO2 max was 19%, vastus lateralis SDH 32%, and cytochrome oxidase activity 35% above the pre-training levels respectively. 6 weeks post training VO2 max was still 16% above the pre-training level, and not significantly different from the level at the end of training (p greater than 0.2). In contrast vastus lateralis SDH activity had returned to the pre-training level. Cytochrome oxidase activity had returned to the pre-training level within two weeks post-training. The significantly faster post-training decline in skeletal muscle oxidative enzyme activities in contrast to that of the VO2 max indicates that an enhancement of the oxidative potential in skeletal muscle is not a necessity for a high VO2 max. Moreover, the fast return to the pre-training level of both SDH and cytochrome oxidase activities indicate a high turnover rate of enzymes in the TCA cycle as well as the respiratory chain.     

Effect of chronic caloric restriction on hepatic enzymes of intermediary metabolism in the male Fischer 344 rat

It is well established that caloric restriction extends life span and significantly retards the rate of occurrence of most age-associated degenerative disease processes. A paucity of data exists relative to the mechanisms by which caloric restriction accomplishes these events. We have examined the effect of caloric restriction in rats on several hepatic enzymes of intermediary metabolism. The activities of glycolytic and supporting enzymes including lactate dehydrogenase, pyruvate kinase, sorbitol dehydrogenase, and alcohol dehydrogenase were all decreased in response to caloric restriction. Fructose 1-phosphate aldolase and creatine phosphokinase were not altered. Likewise, enzymes associated with lipid metabolism (malic enzyme and glycerokinase) were reduced (fatty acid synthetase was reduced, but not to a statistically significant degree). Activities of enzymes supporting gluconeogenesis (glutamate oxaloacetate transaminase, tyrosine aminotransferase, glutamate pyruvate transaminase, glutamate dehydrogenase, amino acid oxidase, malate dehydrogenase, and glucose 6-phosphatase) were either unchanged or increased significantly by caloric restriction. Glucagon levels were decreased. Comparisons between young ad libitum fed and older calorically restricted rats revealed similar but not identical metabolic activity. These results suggest that caloric restriction produces an effect on intermediary metabolism, favoring the role of glucagon and glucose synthesis; but limiting the role of insulin and glucose catabolism in the liver. The former observation provides for the efficient support of peripheral tissues and the latter a level of energy production necessary only for self maintenance. Limited lipid metabolism suggests decreased potential for fatty acid epoxide formation and free radical damage to cellular macromolecules. Additionally, caloric restriction may delay the progressive age associated changes in the activities of some of the enzymes investigated.     

A renewed focus on the interplay between viruses and mitochondrial metabolism

Mitochondria fulfil several key functions within cellular metabolic and antiviral signalling pathways, including their central role in ATP generation. Viruses, as intracellular parasites, require from their cellular host the building blocks for generation of their viral progeny and the energy that drives viral replication and assembly. While some viruses have adopted ways to manipulate the infected cell such that cellular metabolism supports optimal virus production, other viruses simply exhaust cellular resources. The association of viruses with mitochondria is influenced by several important factors such as speed of the viral replication cycle and viral dependence on cellular enzymes and metabolites. This review will highlight the complex interconnectivity of viral life cycles with the three main mitochondrial metabolic pathways, namely β-oxidation, the tricarboxylic (TCA) cycle, and oxidative phosphorylation. This interconnectivity has the potential to reveal interesting points for antiviral therapy with either prometabolites or antimetabolites and highlights the importance of the viral association with mitochondrial metabolism.     

Cardiac system bioenergetics: metabolic basis of the Frank-Starling law

The fundamental principle of cardiac behaviour is described by the Frank-Starling law relating force of contraction during systole with end-diastolic volume. While both work and respiration rates increase linearly with imposed load, the basis of mechano-energetic coupling in heart muscle has remained a long-standing enigma. Here, we highlight advances made in understanding of complex cellular and molecular mechanisms that orchestrate coupling of mitochondrial oxidative phosphorylation with ATP utilization for muscle contraction. Cardiac system bioenergetics critically depends on an interrelated metabolic infrastructure regulating mitochondrial respiration and energy fluxes throughout cellular compartments. The data reviewed indicate the significance of two interrelated systems regulating mitochondrial respiration and energy fluxes in cells: (1) the creatine kinase, adenylate kinase and glycolytic pathways that communicate flux changes generated by cellular ATPases within structurally organized enzymatic modules and networks; and (2) a secondary system based on mitochondrial participation in cellular calcium cycle, which adjusts substrate oxidation and energy-transducing processes to meet increasing cellular energy demands. By conveying energetic signals to metabolic sensors, coupled phosphotransfer reactions provide a high-fidelity regulation of the excitation–contraction cycle. Such integration of energetics with calcium signalling systems provides the basis for 'metabolic pacing', synchronizing the cellular electrical and mechanical activities with energy supply processes.     

In vitro effect of quinolinic acid on energy metabolism in brain of young rats

Quinolinic acid (QA) is found at increased concentrations in brain of patients affected by various common neurodegenerative disorders, including Huntington's and Alzheimer's diseases. Considering that the neuropathology of these disorders has been recently attributed at least in part to energy deficit, in the present study we investigated the in vitro effect of QA (0.1-100 microM) on various parameters of energy metabolism, such as glucose uptake, (14)CO(2) production and lactate production, as well as on the activities of the respiratory chain complexes I-V, the citric acid cycle (CAC) enzymes, creatine kinase (CK), lactate dehydrogenase (LDH) and Na(+),K(+)-ATPase and finally the rate of oxygen consumption in brain of 30-day-old rats. We initially observed that QA significantly increased glucose uptake (55%), whereas (14)CO(2) generation from glucose, acetate and citrate was inhibited (up to 60%). Furthermore, QA-induced increase of brain glucose uptake was prevented by the NMDA receptor antagonist MK-801. Complex II activity was also inhibited (up to 35%) by QA, whereas the other activities of the respiratory chain complexes, CAC enzymes, CK and Na(+),K(+)-ATPase were not affected by the acid. Furthermore, inhibition of complex II activity was fully prevented by pre-incubating cortical homogenates with catalase plus superoxide dismutase, indicating that this effect was probably mediated by reactive oxygen species. In addition, lactate production was also not altered by QA, in contrast to the conversion of pyruvate to lactate catalyzed by LDH, which was significantly decreased (17%) by this neurotoxin. We also observed that QA did not change state III, state IV and the respiratory control ratio in the presence of glutamate/malate or succinate, suggesting that its effect on cellular respiration was rather weak. The data provide evidence that QA provokes a mild impairment of brain energy metabolism in vitro and does not support the view that the brain energy deficiency associated to certain neurodegenerative disorders could be solely endorsed to QA accumulation.     

In vivo metabolic flux profiling with stable isotopes discriminates sites and quantifies effects of mitochondrial dysfunction in C. elegans

Mitochondrial respiratory chain (RC) disease diagnosis is complicated both by an absence of biomarkers that sufficiently divulge all cases and limited capacity to quantify adverse effects across intermediary metabolism. We applied high performance liquid chromatography (HPLC) and mass spectrometry (MS) studies of stable-isotope based precursor–product relationships in the nematode, C. elegans, to interrogate in vivo differences in metabolic flux among distinct genetic models of primary RC defects and closely related metabolic disorders.MethodsC. elegans strains studied harbor single nuclear gene defects in complex I, II, or III RC subunits (gas-1, mev-1, isp-1); enzymes involved in coenzyme Q biosynthesis (clk-1), the tricarboxylic acid cycle (TCA, idh-1), or pyruvate metabolism (pdha-1); and central nodes of the nutrient-sensing signaling network that involve insulin response (daf-2) or the sirtuin homologue (sir-2.1). Synchronous populations of 2000 early larval stage worms were fed standard Escherichia coli on nematode growth media plates containing 1,6-¹³C₂-glucose throughout their developmental period, with samples extracted on the first day of adult life in 4% perchloric acid with an internal standard. Quantitation of whole animal free amino acid concentrations and isotopic incorporation into amino and organic acids throughout development was performed in all strains by HPLC and isotope ratio MS, respectively. GC/MS analysis was also performed to quantify absolute isotopic incorporation in all molecular species of key TCA cycle intermediates in gas-1 and N2 adult worms.ResultsGenetic mutations within different metabolic pathways displayed distinct metabolic profiles. RC complex I (gas-1) and III (isp-1) subunit mutants, together with the coenzyme Q biosynthetic mutant (clk-1), shared a similar amino acid profile of elevated alanine and decreased glutamate. The metabolic signature of the complex II mutant (mev-1) was distinct from that of the other RC mutants but resembled that of the TCA cycle mutant (idh-1) and both signaling mutants (daf-2 and sir-2.1). All branched chain amino acid levels were significantly increased in the complex I and III mutants but decreased in the PDH mutant (pdha-1). The RC complex I, coenzyme Q, TCA cycle, and PDH mutants shared significantly increased relative enrichment of lactate+1 and absolute concentration of alanine+1, while glutamate+1 enrichment was significantly decreased uniquely in the RC mutants. Relative intermediary flux analyses were suggestive of proximal TCA cycle disruption in idh-1, completely reduced TCA cycle flux in sir-2.1, and apparent distal TCA cycle alteration in daf-2. GC/MS analysis with universally-labeled ¹³C-glucose in adult worms further showed significantly increased isotopic enrichment in lactate, citrate, and malate species in the complex I (gas-1) mutant.ConclusionsStable isotopic/mass spectrometric analysis can sensitively discriminate primary RC dysfunction from genetic deficiencies affecting either the TCA cycle or pyruvate metabolism. These data are further suggestive that metabolic flux analysis using stable isotopes may offer a robust means to discriminate and quantify the secondary effects of primary RC dysfunction across intermediary metabolism.     

Enzyme histochemistry of aging rat kidney.

Aging-dependent changes of some enzymatic activities related to the glycolytic anaerobic pathway (lactate-dehydrogenase, LDH), to the Krebs's cycle (succinate dehydrogenase, SDH) and to the activity of the respiratory oxidative chain (NADH2 - tetrazolium reductase, NADH-D) were studied in the nephron of 4-(young) and 24- (aged) month-old female Wistar rats. In the renal glomeruli LDH and NADH-D activities were reduced with aging, while SDH did not undergo aging-dependent changes. In both the proximal and the distal convoluted tubules, LDH reactivity slightly increased while NADH-D remarkably decreased in old rats; SDH did not show aging-dependent changes. In the loop of Henle LDH was slightly increased, SDH was unchanged and NADH-D was decreased in aged in comparison to young rats. The three enzyme activities investigated were significantly reduced in the collecting tubules of aged animals. The above results are suggestive of the existence of aging changes in mechanisms related with energy transduction in the rat kidney, affecting at a different extent the cell populations of the nephron.     

Proliferating cell nuclear antigen (PCNA) immunolocalization in paraffin sections: an index of cell proliferation with evidence of deregulated expression in some neoplasms

Proliferating cell nuclear antigen (PCNA) is a 36 kD nuclear protein associated with the cell cycle. A monoclonal antibody, PC10, that recognizes a fixation and processing resistant epitope has been used to investigate its tissue distribution. Nuclear PCNA immunoreactivity is found in the proliferative compartment of normal tissues. PCNA immunoreactivity is induced in lectin stimulated peripheral blood mononuclear cells in parallel with bromodeoxyuridine incorporation and the number of cells with PCNA immunoreactivity is reduced by induction of differentiation in HL60 cells. In non-Hodgkin's lymphomas a linear relation between Ki67 and PCNA staining was demonstrated. These data suggest that in normal tissues and lymphoid neoplasms, PCNA immunolocalization can be used as an index of cell proliferation. However, in some forms of neoplasia, including breast and gastric cancer and in vitro cell lines, the simple relation between PCNA expression and cell proliferation is lost. In some breast and pancreatic tumours there is apparent deregulation of PCNA with increased expression in tissues adjacent to the tumours. The over-expression in some tumours and in adjacent morphologically normal tissue may represent autocrine or paracrine growth factor influence on PCNA gene expression.     

ACLY: A biomarker of recurrence in breast cancer

ObjectiveACLY is a cytoplasmic metabolic enzyme involved in lipid synthesis. It also affects proliferation and metastasis of breast cancer. However, the correlation of ACLY expression with breast cancer recurrence is unclear.MethodsThe Oncomine and TCGA databases were used to investigate the mRNA expression of ACLY in breast cancer. Immunohistochemistry (IHC) was used to evaluate ACLY expression level in tumor tissues and normal tissues from 127 breast cancer patients. Next, the prognostic role of ACLY was explored by analyzing the clinicopathological features and prognosis during follow-up. The role of ACLY in breast cancer cells drug resistance was further detected by CCK-8 assays and quantitative real-time polymerase chain reaction (qRT-PCR).ResultsACLY mRNA and protein expression was significantly increased in the breast cancer tissues compared to normal tissues. Clinically, high ACLY levels were associated with ER status, PR status, tumor size, TNM stage, and lymph node invasion. Upregulated ACLY predicted worse tumor relapse-free survival (RFS) of breast cancer patients in univariate analyses and in multivariate models. In subgroup analysis, patients with high ACLY expression showed worse RFS in the TNM III or ER positive subgroups. Moreover, ACLY over-expression induced the resistance of breast cancer cells to docetaxel and promoted the expression of multi-drug resistant protein ABCB1/ABCG2.ConclusionsOur study highlights the possibility of ACLY as a potential and independent biomarker for the recurrence prediction in breast cancer patients. It may be related to ACLY promoting drug resistance in breast cancer cells.     

Lysosomal enzymes and initiation of breast cancer

Lysosomes and lysosomal enzymes are known to be involved in cancer processes. However, integrated biochemical and cell biology studies are necessary to understand how lysosomal enzymes could initiate cancer. Most breast cancer is initiated in the milk ducts. The hypothesis presented here is that lysosomal enzymes are exocytosed into the milk ducts where these hydrolytic enzymes damage cells leading to the initiation of cancer. Lysosomal enzymes include: many cathepsins, acid phosphatases, DNAases, ribonucleases, sulfatases, glucuronidase, lipases, neuramidase, lysozyme, fucosidase, phosphodiesterases, glucosidases, galactosidases, mannosidase, and glucosaminidase. Risk factors for breast cancer that could initiate activity of lysosomal enzymes include: ionizing radiation, oxidative stress, estrogen, environmental toxicants and dietary components. Measurements of multiple lysosomal enzyme activities and their biochemical pathways are vital to the understanding of protectors to inhibit lysosomal enzyme activities that might be leading to breast cancer. Non-invasive screening assays could be developed to measure in vivo milk duct lysosomal enzyme activities. Lysosomal enzyme activities may be precursors to the onset of other kinds of cancer with other similar non-invasive screening techniques possible.     

DMBA-induced mammary pathologies are angiogenic in vivo and in vitro

We have previously shown that human pre-invasive diseases of the breast are angiogenic. In addition, normal epithelium from women with coincident or subsequent invasive breast cancer is more vascular than normal epithelium from women with no breast cancer. To develop a model in which to study the regulation of angiogenesis in pre-invasive mammary pathologies, we examined 7,12-dimethylbenz[a]anthracene (DMBA)-induced rat mammary tissues for the presence of neovascularization in pre-invasive histopathologies. These studies included morphometric analysis of tissue vascularity in pre-invasive lesions. In addition, we isolated fresh tumors and histologically normal epithelium (organoids) from DMBA or vehicle-treated control rats to test their ability to induce endothelial cell tubule formation in vitro. Finally, we examined tumors for their ability to produce vascular endothelial cell growth factor. The morphometric studies documented that with epithelial progression, the ability of individual cells to elicit angiogenesis increases. The in vitro studies showed that isolated tumors from these animals stimulate angiogenesis. Furthermore, normal epithelium from DMBA-treated rats is more angiogenic than epithelium from control animals. Finally, DMBA-induced tumors produce vascular endothelial growth factor (VEGF) mRNA, therefore, DMBA-induced mammary tumorigenesis is one model in which to test the dependency of progression on angiogenesis.     

Ligula intestinalis: Intermediary carbohydrate metabolism in plerocercoids and adults

Comparison of organic acid production in vitro, endogenous metabolite concentrations and enzyme activities suggest that glycolysis is the predominant energy synthesising pathway in non-activated (taken from the body cavity of fish) plerocercoids and adults ofLigula intestinalis. In contrast, glycolysis and carbon dioxide fixation with a partial reversal of the tricarboxylic acid (TCA) cycle are important in activated (incubated at 40°C) plerocercoids, the transition stage between larvae and adults. Carbon balances suggest that the TCA cycle, operating in the forward direction, probably plays only a minor role in the metabolism of activated plerocercoids and is likely to be of even less significance in adults.Hexokinase, phosphofructokinase and pyruvate kinase are probably regulatory enzymes in plerocercoids and adults. The roles of these and other key enzymes, notably phosphoenolpyruvate carboxykinase, are discussed in relation to the mechanism by which the carbohydrate metabolism ofL. intestinalis may be controlled in vivo.