Early exposure to Aroclor 1254 in vivo disrupts the functional synaptic development of newborn hippocampal granule cells.

Neurogenesis in the dentate gyrus is sensitive to endogenous and exogenous factors that influence hippocampal function. Ongoing neurogenesis and the integration of these new neurons throughout life thus may provide a sensitive indicator of environmental stress. We examined the effects of Aroclor 1254 (A1254), a mixture of polychlorinated biphenyls (PCBs), on the development and function of newly generated dentate granule cells. Early exposure to A1254 has been associated with learning impairment in children, suggesting potential impact on the development of hippocampus and/or cortical circuits. Oral A1254 (from the 6th day of gestation to postnatal day 21) produced the expected increase in PCB levels in brain at postnatal day 21, which persisted at lower levels into adulthood. A1254 did not affect the proliferation or survival of newborn neurons in immature animals nor did it cause overt changes in neuronal morphology. However, A1254 occluded the normal developmental increase in sEPSC frequency in the third post-mitotic week without altering the average sEPSC amplitude. Our results suggest that early exposure to PCBs can disrupt excitatory synaptic function during a period of active synaptogenesis, and thus could contribute to the cognitive effects noted in children exposed to PCBs.

[Endocrine disruption: a challenge in research, public health and clinical practice]. / La perturbation endocrinienne: entre enjeux de recherche, enjeux de santé publique et enjeux de pratique quotidienne.

Epidemiological and experimental data highlight the fetal and early postnatal life as critical periods for the effects of endocrine disrupting chemicals (EDCs), since exposure to EDCs during these periods can predispose to disease later in life. EDCs' effects include disorders of the reproductive system throughout life (abnormalities of sexual differentiation, infertility or subfertility and some neoplasia) and disorders of energy balance (obesity and metabolic syndrome). They could also influence the development of the cerebral cortex. However, the demonstration of the involvement of a single EDC remains difficult in human since we are virtually exposed to a mixture of several ubiquitous EDCs which are variably persistent in the environment and the body and have lifelong consequences. Moreover, since their dose-response relationship can be non-monotonic, setting a threshold dose for EDCs effects has become meaningless. Pregnant women, newborns and young children appear to be mostly at risk. However, the role of the physician remains difficult and raises several questions: how can we formulate justified, applicable and updated recommendations that are not counterproductive or alarmist...in a society that has to take the necessary steps to regulate production and protect the population?

Nitroxide stable radicals protect beating cardiomyocytes against oxidative damage.

The protective effect of stable nitroxide radicals against oxidative damage was studied using cardiomyocyte cultures obtained from newborn rats. Monolayered cardiomyocytes were exposed to H2O2 and the effect on spontaneous beating and leakage of LDH was determined. Hydrogen peroxide irreversibly blocked rhythmic beating and resulted in a significant membrane injury as shown by release of LDH. The injury was prevented by catalase which removes H2O2 and by cell-permeable, metal-chelating agents such as desferrioxamine or bipyridine. In contrast, reagents which are excluded from the cell such as superoxide dismutase or DTPA did not protect the cells against H2O2. Five- and six-membered ring, stable nitroxide radicals which have previously been shown to chemically act as low-molecular weight, membrane-permeable, SOD-mimetic compounds provided full protection. The nitroxides prevented leakage of LDH and preserved normal cardiomyocyte contractility, presumably by intercepting intracellular O2-radicals. Alternatively, protection may result through nitroxides reacting with reduced transition metal ions or by detoxifying secondary organic radicals.

Exposure to endocrine disrupting chemicals and neurodevelopmental alterations.

The developing brain is remarkably malleable as neural circuits are formed and these circuits are strongly dependent on hormones for their development. For those reasons, the brain is very vulnerable to the effects of endocrine-disrupting chemicals (EDCs) during critical periods of development. This review focuses on three ubiquitous endocrine disruptors that are known to disrupt the thyroid function and are associated with neurobehavioral deficits: polychlorinated biphenyls, polybrominated diphenyl ethers, and bisphenol A. The human and rodent data suggesting effects of those EDCs on memory, cognition, and social behavior are discussed. Their mechanisms of action go beyond relative hypothyroidism with effects on neurotransmitter release and calcium signaling.

Intracellular lipase activities in heart and skeletal muscle homogenates. The absence of trierucin cleavage by the heart: a possible biochemical basis for erucic acid lipidosis.

Rat heart and skeletal muscle homogenates were compared for their intracellular lipolytic activity towards a series of saturated and unsaturated triglycerides from trilaurin (C12:0) to trierucin (C22:1). It is shown that for all triglycerides esterified with fatty acids from C12 to C18, lipolytic activity in heart homogenates was higher than in skeletal muscle homogenates. For these triglycerides there was no relationship between the fatty acid chain length and the lipolytic activity. In both homogenates cleavage of unsaturated triglycerides was higher than cleavage of the homologous saturated triglyceride. Lipolysis of tri-delta-11-eicosenoin (C20:1) was similar in both homogenates but much lower than lypolysis of other triglycerides. Although cleavage of trierucin (C22:1) was very low in skeletal muscle homogenates, it was undetectable in heart homogenates, even when enzyme concentration was increased. A mixture of triglycerides did not show preferential hydrolysis of any simple triglyceride. Trierucin was the only triglyceride that did not complete for lipolytic activity and only with heart homogenates, which shows that that lipase(s) do not cleave trierucin. The absence of lipolytic activity towards trierucin in heart homogenates could explain the selective accumulation of erucic acid-rich triglycerides in hearts of animals fed a diet with a high erucic acid content.

Interactions of cardiac glycosides with cultured cardiac cells. II. Biochemical and electron microscopic studies on the effects of ouabain on muscle and non-muscle cells.

Electron microscopic and biochemical studies revealed a salient difference in the response to toxic doses of ouabain by cultured cardiac muscle and non-muscle cells from neonatal rats. Progressive cellular injury in myocytes incubated with 1 . 10(-4)--1 . 10(-3) M ouabain ultimately leads to swelling and necrosis. The morphological damage in myocytes was accompanied by a drastic decrease in 14CO2 formation from 14C-labeled stearate or acetate but not glucose. Neither morphological nor biochemical impairments were observed in non-muscle cells. The interaction between ouabain and the cultured cells, using therapeutic doses of ouabain (i.e., less than 1 . 10(-7) M), was characterized. Two binding sites were described in both classes of cells, one site is a saturable K+-sensitive site whereas the other is non-saturable and K+-insensitive. The complexes formed between the sarcolemma receptor(s) and ouabain, at low concentrations of the drug (e.g., 7.52 . 10(-9) M), had Kd values of 8.9 . 10(-8) and 2.3 . 10(-8) M for muscle and non-muscle cells, respectively. The formation and dissociation of the complexes were affected by temperature and potassium ions.

High oligomycin concentrations augment 6-keto-PGF1 alpha production in ventricular cardiomyocytes.

Incubation of cultured ventricular cardiomyocytes with high oligomycin concentrations (100 micrograms/ml), either alone or combined with 2-deoxyglucose (20 mM), led to the rapid depletion of cellular ATP. Inositol (poly)phosphate production decreased, and 6-keto PGF1 alpha production was increased. In cells depleted of ATP, either by low oligomycin concentrations or by sodium azide, 6-keto PGF1 alpha was not appreciably increased. There was a 25% rise in the release of fatty acids from the sn-2 position in glycerophospholipids. We suggest that oligomycin at high concentrations causes the release of free arachidonic acid from phospholipids either by non-PIP2-specific PLC and DG lipase or by phospholipase D, phosphatidic acid phosphatase and DG lipase. The effect is unrelated to decreased cellular ATP content.

Oxygen and extracellular fluid restriction in cultured heart cells: electron microscopy studies.

STUDY OBJECTIVE: To evaluate the effects of "simulated ischaemia" on the structure of cultured heart cells. DESIGN: Cultured heart cells were subjected for 2 h either to anoxia or to anoxia with simultaneous extracellular volume restriction ("simulated ischaemia"). Cells maintained under normoxic conditions served as controls. The cells were then fixed in situ in Petri dishes with formaldehyde-glutaraldehyde. EXPERIMENTAL MATERIALS: Heart cells from one day old rats on day 5 in culture were used. MEASUREMENTS AND RESULTS: Electron microscope studies were carried out on control and injured cells. "Mildly ischaemic" cells featured raffled and invaginated cell surfaces, reduced matrix density, disorientated mitochondrial cristae due to swelling, and giant mitochondria. Dilatation of rough endoplasmic reticulum and electron dense membrane bound vesicles were observed in the cytoplasm. CONCLUSIONS: The model of simulated ischaemia is in keeping with the classical picture of irreversible cell damage caused by ischaemic injury.

Studies on oxygen and extracellular fluid restrictions in cultured heart cells: high energy phosphate metabolism.

Although cultured heart cells are increasingly used for the study of cardiac metabolism, relatively little is known about their energy turnover. We studied the effects of anoxia with simultaneous restrictions of the volume of the extracellular medium ("ischaemia") on high energy phosphate catabolism in cells from neonatal rat ventricles, cultured for 5 days. The cells were incubated for up to 4 h in Ham-F10 medium either in the presence or in the absence of glucose. High energy phosphates in cell extracts and AMP catabolites in the incubation medium were measured by high pressure liquid chromatography. ATP and creatine phosphate content in normoxic cells did not change significantly, either in the presence or absence of glucose, and the values were similar to those found in the heart in vivo. Energy rich phosphates decreased during anoxia, and were more rapidly depleted during simultaneous oxygen deprivation and volume restriction. Glucose delayed the decline in high energy phosphates. In the presence of glucose, hypoxanthine uptake was higher during normoxia than in anoxia, whereas in "ischaemic" conditions some hypoxanthine was produced. In the absence of glucose, only minor changes were observed in hypoxanthine levels during anoxia, but hypoxanthine production was marked when anoxia was coupled with extracellular volume restriction. Adenosine levels were below the limit of detection. Inosine release was relatively low under all conditions, Xanthine release did not show variation, and anoxia suppressed urate production. Oxygen and glucose deprivation thus led to various degrees of ATP and creatine phosphate breakdown in cultured neonatal heart cells both during anoxia and in simulated "ischaemia".

Upregulation of vascular endothelial growth factor expression induced by myocardial ischaemia: implications for coronary angiogenesis.

OBJECTIVE: The process of coronary collateral development is poorly understood. It is assumed that particular angiogenic factors are upregulated during episodes of myocardial ischaemia and act as a trigger for neovascularisation. However, the identity of these factors is unknown. The angiogenic factor vascular endothelial growth factor (VEGF) has been shown to be hypoxia inducible, so this factor may mediate ischaemia induced angiogenesis in the heart. The aim of this study was to examine hypoxia inducibility of VEGF in cultured myocardial cells as well as in normally perfused and ischaemic porcine myocardium. METHODS: (1) In vitro experiment: cultured rat myocardial cells were subjected to hypoxia, and steady state levels of VEGF mRNA were measured after 2 and 4 h of hypoxia. (2) In vivo experiment: myocardial ischaemia in pigs hearts was induced by repeated 2-10 min left anterior descending coronary artery occlusions, separated by 20 min of reperfusion. Hearts were retrieved after 6 h of intermittent ischaemia. Total RNA was extracted from normal and ischaemic zones of the heart and processed for RNA blot hybridisation analysis. RESULTS: In vitro experiment: as soon as 2-4 h after exposure of cultures to hypoxia, VEGF mRNA levels were significantly raised (6-10-fold). In vivo experiment: VEGF expression was significantly augmented in the ischaemic territory of the myocardium (three- to fivefold induction). Furthermore, polymerase chain reaction amplification of the reverse transcribed mRNA showed increased production of multiple forms of differentially spliced VEGF mRNA in the ischaemic myocardium. CONCLUSIONS: VEGF production in the myocardium is significantly upregulated by hypoxia in vitro and by ischaemia in vivo. These results suggest that VEGF is a likely mediator in the natural process of ischaemia induced myocardial neovascularisation.

Iron chelation.

Adequate iron chelation in thalassaemia has resulted in a striking improvement in survival, with a reduction of cardiac mortality at age 15 years from 14-3%, and a predicted survival at age 36 years of 85%. Long term desferrioxamine (DF) therapy in thalassaemic children should be started between 2-4 years of age. In addition to daily 8-12 h subcutaneous infusions, intermittent high dose (9-16 g) i.v. supplementation over 24-48 h may be given on the occasion of blood transfusions. In established myocardiopathy continuous i.v. DF infusion at 100-125 mg/kg/d may result in improved myocardial function. In addition, there is considerable current interest in the use of DF in conditions unrelated to iron overload by preventing the formation of free-radicals in inflammatory reactions, or by S-phase inhibition of cell proliferation. Although at present highly experimental, this novel approach may have important implications for the management of patients with inflammatory conditions and perhaps in the control of protozoal infections. Over the last decade several hundred candidate compounds have been studied in cell cultures and in animal models and a number of orally effective iron chelators have been identified, all of which are superior to DF in their in vivo iron chelating effect. Although we do not yet have a new drug which is immediately available for replacing DF in clinical practice, significant progress has already been made, and some of the most promising candidate drugs are currently undergoing extensive toxicity tests in anticipation of their development for large-scale clinical use.

Anoxic injury accelerates phosphatidylcholine degradation in cultured cardiac myocytes by phospholipase C.

In neonatal cultured cardiac myocytes under normoxic conditions, 32Pi incorporation pattern into various phospholipids, and double-labeling experiments with 32Pi and [3H]methyl choline, suggest that phosphatidylcholine and phosphatidylinositol are turned over rapidly, whereas the turnover of phosphatidylethanolamine is probably much slower. While increased levels of the corresponding lysophospholipids were not found under anoxia, release of diacylglycerol and phosphorylcholine was observed. These data strongly suggest that phospholipase C, and not phospholipase A2, is involved in phospholipid degradation in cultured cardiomyocytes under anoxic conditions.

Both hydroxylamine and nitroxide protect cardiomyocytes from oxidative stress.

The unique anti-oxidative activity of nitroxide radicals protecting against reactive oxygen-derived species (ROS) has been recently demonstrated in several model systems. The present study focuses on the activity of nitroxide and of its reduced form in cultured rat ventricular cardiomyocytes exposed to O2.- and H2O2 generated by hypoxanthine (HX) and xanthine oxidase (XO). To evaluate cell injury, spontaneous beating, leakage of lactate dehydrogenase (LDH), and depletion of cellular ATP were determined. The protective effect of 4-OH-2,2,6,6-tetramethyl-piperidine-N-oxyl (TPL) was compared with that of 4-OH-2,2,6,6-tetramethyl-1-hydroxypiperidine (TPL-H) and of several common anti-oxidants. A rapid exchange between TPL and TPL-H, is mediated by cellular metabolism and through reactions with ROS. In particular, TPL under O2.- flux is oxidized to oxo-ammonium cation (TPL+) which comproportionates with TPL-H yielding two nitroxide radicals. Because this exchange limits the distinction between the biological activities of TPL and TPL-H, NADH which can reduce TPL+ was included in order to maintain the nitroxide in its reduced form. The results demonstrate that both TPL and TPL-H protect cardiomyocytes against beating loss and LDH leakage. Conversely, cellular ATP depletion induced by HX/XO is inhibited by TPL-H, though not by TPL, suggesting that different mechanisms underlie their protective activities. Through a flip-flop between the two forms, which coexist in the system, the levels of TPL-H and TPL are continuously replenished. The conversion, upon reaction, of each antioxidant into the other one enables them, contrary to common antioxidants which operate in a stoichiometric mode, to act catalytically.

Technetium-99m-RBC venography in the diagnosis of deep venous thrombosis of the lower extremity: a systematic review of the literature.

We systematically reviewed the six articles from the English-language medical literature, since 1979, which compared 99mTc-RBC venography with contrast venography for the diagnosis of deep venous thrombosis (DVT) of the lower extremity. The studies were generally small in size and poorly compliant with methodologic standards for diagnostic test research. There was considerable variation in both how the 99mTc-RBC venograms were performed and how they were interpreted. Sufficient clinical information on the patients was not provided. Although the overall sensitivities and specificities were high with a mean sensitivity of 0.89 and a mean specificity of 0.84, the small numbers of patients resulted in wide 95% confidence intervals. For distal disease, with only a total of 14 patients studied, the 95% confidence intervals were particularly broad. Although 99mTc-RBC venography is a promising technique, future studies with larger numbers of patients and closer adherence to methodologic standards are required.

Arachidonic acid channelling in the phospholipid fractions and subcellular compartments of cultured myocardial cells.

Arachidonic acid (AA) channeling in cultured heart cells was studied following pulse labelling for 1 h. AA was shown to be esterified immediately and equally distributed between the neutral lipids and phospholipids. A rapid constant flow to various phospholipid classes occurred thereafter, while the AA oxidation was only between 12%. The subcellular distribution of AA was studied by nitrogen cavitation followed by fractionation on 6.7% percoll in sucrose-EDTA. After 1 h pulse labeling and 2 h post-pulse incubation, most of the radioactivity was found in the sarcolemmal fraction with a much smaller amount in the mitochondrial fraction.

Mechanical injury increases eicosanoid production in cultured cardiomyocytes.

The release of three stable metabolites of the arachidonic acid cascade was determined in cultures of cardiac myocytes and of non-muscle cells. In both cell types, the main product was 6-keto-PGF1 alpha much less PGE2 was released, while TXB2 was only detected in muscle cells. Preincubation with arachidonic acid increased the release of all the PGs in both types of culture. Mechanical injury had a synergistic effect on the increased PG release in AA-preincubated cells. However, TXB2 was not detected in F-cells in any experimental conditions. These results suggest that PG production serves a functional role in heart preservation during injury.

Oxygen deprivation and reoxygenation augment prostacyclin synthesis in cultured ventricular myocytes.

Prostacyclin production in cultured cardiomyocytes is not induced by cellular ATP depletion per se, suggesting that the mechanism of ischemic injury is more complex. In the present study we subjected cultured ventricular myocytes to 'simulated ischemia' followed by reoxygenation. A slight increase in 6-keto-PGF(1 alpha) (the stable metabolite of PGI(2)) was found during 'ischemia', which continued to increase markedly during reoxygenation. PGE(2) levels were pronouncedly enhanced during ischemia but decreased during reoxygenation, and TXB(2) levels remained undetectable throughout. These findings reflect a cardiomyocyte response to anoxic injury, suggesting that they act to protect against cardiac injury by producing the potent vasodilators PGI(2) and PGE(2) during ischemia and reoxygenation.

The role of iron and iron chelators in anthracycline cardiotoxicity.

The redox cycling of anthracyclines promotes the formation of free radicals which are believed to play a central role in their cardiotoxicity. A number of observations indicate that the mechanism of the antineoplastic effect of anthracyclines is independent of their cardiotoxic effect and that it may be possible to prevent toxicity without interfering with therapeutic effect. Iron plays an important role in anthracycline toxicity by promoting the conversion of superoxide into highly toxic hydroxyl radicals through the Haber-Weiss reaction. Conversely, iron deprivation by its high-affinity binding to iron chelating compounds may inhibit anthracycline toxicity by interfering with free radical formation. ICRF-187, a bispiperazonedione which is hydrolyzed intracellularly into a bidentate chelator resembling EDTA, is able to decrease adriamycin-induced free hydroxyl radical formation and to prevent the development of clinical cardiac toxicity in patients receiving long-term anthracycline therapy. Our studies in rat heart cell cultures have shown that iron overload aggravates anthracycline toxicity and that this interaction can be prevented by prior iron chelating treatment. Since iron overload caused by multiple blood transfusions and bone marrow failure is a common condition in patients requiring anthracycline therapy, these observations may have significant clinical implications to the prevention of anthracycline cardiotoxicity.

The effect of oral contraceptives on serum lipids.

PIP: In the latter part of pregnancy total lipids, cholesterol, triglycerides, phospholipids, and free fatty acids increase progressively. A study involving 375 women aged 19-30 taking various oral contraceptives, both combined and sequential, showed a mean value of 712 mg/100 ml blood of total lipids as contrasted with 596 mg/100 ml for controls. Serum triglycerides also showed marked elevation -- 122 mg/100 ml as compared with 70 mg/100 ml for controls. However, there was no significant change in the level of cholesterol. There was no significant difference between the combined and sequential users or between users of pills of different steroid composition. Total serum lipids and triglyceride levels were similar to those seen in the last 2 trimesters of pregnancy; however, in pregnancy the cholesterol level also increases. Estrogens are known to elevate serum triglycerides, and in hypercholesteremic subjects, estrogen administration lowers serum cholesterol concentrations.^ieng

Synergism among oxidants, proteinases, phospholipases, microbial hemolysins, cationic proteins, and cytokines.

A striking similarity exists between the pathogenetic properties of group A streptococci and those of activated mammalian professional phagocytes (neutrophils, macrophages). Both types of cells are endowed by the ability to adhere to target cells; to elaborate oxidants, hydrolases, and membrane-active agents (hemolysins, phospholipases); and to freely invade tissues and destroy cells. From the evolutionary point of view, streptococci might justifiably be considered the forefathers of "modern" leukocytes. Our earlier findings that synergy between a streptococcal hemolysin (streptolysin S, SLS) and a streptococcal thiol-dependent proteinase and between cytotoxic antibodies+complement and streptokinase-activated plasmin readily killed tumor cells, led us to hypothesize that by analogy to the pathogenetic mechanisms of streptococci, the mechanisms of tissue destruction initiated by activated leukocytes in inflammatory sites, as well as in tissues undergoing episodes of ischemia and reperfusion, might also be the result of the synergistic effects among leukocyte-derived oxidants, phospholipases, proteinases, cytokines, and cationic proteins. The current report extends our previous synergy studies with endothelial cells to two additional cell types--monkey kidney epithelial cells and rat beating heart cells. Monolayers of 51Cr-labeled cells that had been treated by combinations of sublytic amounts of hydrogen peroxide (generated either by glucose oxidase, xanthine-xanthine oxidase, or by paraquat) and with sublytic amounts of a variety of membrane-active agents (streptolysin S, phospholipases A2 and C, lysophosphatides, histone, chlorhexidine) were killed in a synergistic manner (double synergy). Crystalline trypsin markedly enhanced cell killing by combinations of oxidant and the membrane-active agents (triple synergy). Injury to the cells was characterized by the appearance of large membrane blebs that detached from the cells and floated freely in the media, looking like lipid droplets. Cytotoxicity induced by the various combinations of agonists was depressed, to a large extent, by scavengers of hydrogen peroxide (catalase, dimethyl thiourea, and by Mn2+) but not by SOD or by deferoxamine. When cationic agents were employed together with hydrogen peroxide, polyanions (heparin, polyanethole sulfonate) were also found to inhibit cell killing. It is proposed that in order to effectively combat the deleterious toxic effects of leukocyte-derived agonists on cells and tissues, antagonistic "cocktails" comprised of cationized catalase, cationized SOD, dimethylthiourea, Mn(2+)+glycine, proteinase inhibitors, putative inhibitors of phospholipases, and polyanions might be concocted. The current literature on synergistic phenomena pertaining to mechanisms of cell and tissue injury in inflammation is selectively reviewed.