ESMO recommendations on the standard methods to detect RET fusions and mutations in daily practice and clinical research.

Aberrant activation of RET is a critical driver of growth and proliferation in diverse solid tumours. Multikinase inhibitors (MKIs) showing anti-RET activities have been tested in RET-altered tumours with variable results. The low target specificity with consequent increase in side-effects and off-target toxicities resulting in dose reduction and drug discontinuation are some of the major issues with MKIs. To overcome these issues, new selective RET inhibitors such as pralsetinib (BLU-667) and selpercatinib (LOXO-292) have been developed in clinical trials, with selpercatinib recently approved by the Food and Drug Administration (FDA). The results of these trials showed marked and durable antitumour activity and manageable toxicity profiles in patients with RET-altered tumours. The European Society for Medical Oncology (ESMO) Translational Research and Precision Medicine Working Group (TR and PM WG) launched a collaborative project to review the available methods for the detection of RET gene alterations, their potential applications and strategies for the implementation of a rational approach for the detection of RET fusion genes and mutations in human malignancies. We present here recommendations for the routine clinical detection of targetable RET rearrangements and mutations.

Placental programmed cell death: insights into the role of aquaporins.

STUDY HYPOTHESIS: Are the placental aquaporins (AQPs) involved in the apoptosis of human trophoblast? STUDY FINDING: The general blocking of placental AQPs with HgCl2 and, in particular, the blocking of AQP3 activity with CuSO4 abrogated the apoptotic events of human trophoblast cells. WHAT IS KNOWN ALREADY: Although apoptosis of trophoblast cells is a natural event involved in the normal development of the placenta, it is exacerbated in pathological processes, such as pre-eclampsia, where an abnormal expression and functionality of placental AQPs occur without alterations in the feto-maternal water flux. Furthermore, fluctuations in O2 tension are proposed to be a potent inducer of placental apoptotic changes and, in explants exposed to hypoxia/reoxygenation (H/R), transcellular water transport mediated by AQPs was undetectable. This suggests that AQPs might be involved in processes other than water transport, such as apoptosis. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: Explants from normal term placentas were maintained in culture under conditions of normoxia, hypoxia and H/R. Cell viability was determined by assessing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide incorporation. For the general or specific inhibition of AQPs, 0.3 mM HgCl2, 5 mM CuSO4, 0.3 mM tetraethylammonium chloride (TEA) or 0.5 mM phloretin were added to the culture medium before explants were exposed to each treatment. Oxidative stress parameters and apoptotic indexes were evaluated in the presence or absence of AQPs blockers. AQP3 expression was confirmed by western blot and immunohistochemistry. MAIN RESULTS AND THE ROLE OF CHANCE: First, we observed that in H/R treatments cell viability decreased by 20.16 ± 5.73% compared with those explants cultured in normoxia (P = 0.009; n = 7). Hypoxia did not modify cell viability significantly. Both hypoxia and H/R conditions induced oxidative stress. Spontaneous chemiluminescence and thiobarbituric acid reactive substance levels were significantly increased in explants exposed to hypoxia (n = 6 per group, P = 0.0316 and P = 0.0009, respectively) and H/R conditions (n = 6 per group, P = 0.0281 and P = 0.0001, respectively) compared with those cultured in normoxia. Regarding apoptosis, H/R was a more potent inducer of trophoblast apoptosis than hypoxia alone. Bax expression and the number of apoptotic nuclei were significantly higher in explants cultured in H/R compared with normoxia and hypoxia conditions (n = 12, P = 0.0135 and P = 0.001, respectively). DNA fragmentation was only observed in H/R and, compared with normoxia and hypoxia, the activity of caspase-3 was highest in explants cultured in H/R (n = 12, P = 0.0001). In explants exposed to H/R, steric blocking of AQP activity with HgCl2 showed that DNA degradation was undetectable (n = 12, P = 0.001). Bax expression and caspase-3 activity were drastically reduced (n = 12, P = 0.0146 and P = 0.0001, respectively) compared with explants cultured in H/R but not treated with HgCl2. Similar results were observed in explants exposed to H/R when we blocked AQP3 activity with CuSO4. DNA degradation was undetectable and the number of apoptotic nuclei and caspase-3 activity were significantly decreased compared with explants cultured in H/R but not treated with CuSO4 (n = 12, P = 0.001 and P = 0.0001, respectively). However, TEA and phloretin treatments, to block AQP1/4 or AQP9, respectively, failed in abrogate apoptosis. In addition, we confirmed the expression and localization of AQP3 in explants exposed to H/R. LIMITATIONS, REASONS FOR CAUTION: Our studies are limited by the number of experimental conditions tested, which do not fully capture the variability in oxygen levels, duration of exposure and alternating patterns of oxygen seen in vivo. WIDER IMPLICATIONS OF THE FINDINGS: Our results suggest that any alteration in placental AQP expression might disturb the equilibrium of the normal apoptotic events and may be an underlying cause in the pathophysiology of placental gestational disorders such as pre-eclampsia. Furthermore, the dysregulation of placental AQPs may be one of the crucial factors in triggering the clinical manifestations of pre-eclampsia. LARGE SCALE DATA: n/a. STUDY FUNDING AND COMPETING INTERESTS: This study was supported by UBACyT 20020090200025 and 20020110200207 grants and PIP-CONICET 11220110100561 grant, and the authors have no conflict of interest to declare.

[Effect of VKORC1 and CYP2C9 variants on dosage of oral anticoagulants in Chilean individuals]. / Efecto de las variantes de VKORC1 y CYP2C9 sobre la dosis de anticoagulantes orales en individuos chilenos.

BACKGROUND: The dose of oral anticoagulants (OAC) shows great variability among patients. Pharmacogenetic studies have shown that common variants in genes CYP2C9 (*2 and *3) and VKORC1 (-1639G>A) are associated with lower requirements of OAC. AIM: To study the association between average maintenance doses of oral anticoagulant therapy required to maintain a stable INR and CYP2C9 and VKORC1 gene variants in Chilean adults. MATERIAL AND METHODS: Prospective study of patients on anticoagulant treatment and with a stable international normalized ratio (INR) for prothrombin time for at least three months. Patients were classified as having high or low acenocoumarol or warfarin requirements. Peripheral blood DNA genotyping was performed by polymerase chain reaction and restriction fragment polymorphism or sequencing and electrophoresis. RESULTS: The study included 185 patients, 125 on acenocoumarol and 60 on warfarin. Patients with VKORC1-1639A allele were more likely to require lower doses of both drugs than patients with the G allele (Odds ratio [OR] for acenocoumarol 9.06, and OR for warfarin = 18.7). There was no association between CYP2C9*2 and*3 and acenocoumarol or warfarin requirements. CONCLUSIONS: There is an association between VKORC1-1639A variant and anticoagulant doses.

Alzheimer disease and cognitive impairment associated with diabetes mellitus type 2: associations and a hypothesis.

INTRODUCTION: Epidemiological studies have demonstrated that patients with diabetes mellitus have an increased risk of developing Alzheimer disease, but the relationship between the 2 entities is not clear. DEVELOPMENT: Both diseases exhibit similar metabolic abnormalities: disordered glucose metabolism, abnormal insulin receptor signalling and insulin resistance, oxidative stress, and structural abnormalities in proteins and ß-amyloid deposits. Different hypotheses have emerged from experimental work in the last two decades. One of the most comprehensive relates the microvascular damage in diabetic polyneuritis with the central nervous system changes occurring in Alzheimer disease. Another hypothesis considers that cognitive impairment in both diabetes and Alzheimer disease is linked to a state of systemic oxidative stress. Recently, attenuation of cognitive impairment and normalisation of values in biochemical markers for oxidative stress were found in patients with Alzheimer disease and concomitant diabetes. Antidiabetic drugs may have a beneficial effect on glycolysis and its end products, and on other metabolic alterations. CONCLUSIONS: Diabetic patients are at increased risk for developing Alzheimer disease, but paradoxically, their biochemical alterations and cognitive impairment are less pronounced than in groups of dementia patients without diabetes. A deeper understanding of interactions between the pathogenic processes of both entities may lead to new therapeutic strategies that would slow or halt the progression of impairment.

The oxidative stress induced in vivo by Shiga toxin-2 contributes to the pathogenicity of haemolytic uraemic syndrome.

Typical haemolytic uraemic syndrome (HUS) is caused by Shiga toxin (Stx)-producing Escherichia coli infections and is characterized by thrombotic microangiopathy that leads to haemolytic anaemia, thrombocytopenia and acute renal failure. Renal or neurological sequelae are consequences of irreversible tissue damage during the acute phase. Stx toxicity and the acute inflammatory response raised by the host determine the development of HUS. At present there is no specific therapy to control Stx damage. The pathogenic role of reactive oxygen species (ROS) on endothelial injury has been largely documented. In this study, we investigated the in-vivo effects of Stx on the oxidative balance and its contribution to the development of HUS in mice. In addition, we analysed the effect of anti-oxidant agents as therapeutic tools to counteract Stx toxicity. We demonstrated that Stx induced an oxidative imbalance, evidenced by renal glutathione depletion and increased lipid membrane peroxidation. The increased ROS production by neutrophils may be one of the major sources of oxidative stress during Stx intoxication. All these parameters were ameliorated by anti-oxidants reducing platelet activation, renal damage and increasing survival. To conclude, Stx generates a pro-oxidative state that contributes to kidney failure, and exogenous anti-oxidants could be beneficial to counteract this pathogenic pathway.

Thymic carcinoma with Lynch syndrome or microsatellite instability, a rare entity responsive to immunotherapy.

IMPORTANCE: Thymic carcinoma (TC) is a rare aggressive tumour occurring in adults characterised by one of the lowest tumor mutational burdens (TMB). Microsatellite instability (MSI) is a mutational signature, caused by defects in the DNA MisMatch Repair (MMR) system, that predicts benefit from immunotherapy and causes high TMB. Fragmentary and unstructured evidence of these conditions co-occurring are reported in literature. OBJECTIVE: Review available data on the co-occurrence of these two conditions and determine its frequency in our institute case series. DESIGN: We performed a systematic analysis of literature and a retrospective evaluation of all the cases of TET treated at our institution from 2000 to 2020, selecting patients with a medical history of multiple tumours to enhance a priori probability of identifying cases with underlying predisposition. RESULTS: Literature yielded 3 cases of patients with MSI TC, for which MMR gene alteration was reported. None of them received immunotherapy. Of 366 patients with TETs treated in our institute, 32 had a medical history of multiple tumours and 25 of 32 (19 thymomas and 6 TCs) had available tissue for MMR analysis. One patient with TC showed a high TMB, and MSI due to MLH1 mutation and was treated in a phase II study with avelumab and axitinib combination obtaining a long-lasting partial response. MLH1 alterations are shared across MSI TC cases. CONCLUSIONS AND RELEVANCE: This analysis highlights the usefulness of MSI testing in patients with TC. The observation of cases of TC occurring in patients with Lynch syndrome and the unexpected homogeneity of gene alterations support further investigation.

Simulation of urban boundary and canopy layer flows in port areas induced by different marine boundary layer inflow conditions.

Computational fluid dynamics (CFD) simulations and wind-tunnel (WT) tests can be considered as boundary-value problems, where the inlet boundary condition, which is usually obtained inferring inlet mean wind profiles from on-site measurements or other type of experimental data, represents the large-scale atmospheric forcing exerted at the outer limit of the urban model. It is not clear, however, to which extent the choice of different inflow wind speed profiles may affect WT and CFD results in the urban environment. In the present study, this aspect is investigated through the comparison of the wind flow fields simulated numerically and tested experimentally in an atmospheric boundary layer wind tunnel (ABLWT) within a district of Livorno city, Italy, called "Quartiere La Venezia". Three different shapes of inflow profiles were tested using the CFD technique and the results were compared with each other: one is based on the approach-flow profiles measured upstream of the urban model in the WT test section (WT profile) and two are based on anemometric data corresponding to the approach-flow profile measured by means of a LiDAR wind profiler (LiDAR profile 1 and 2). The analysis showed that using different wind speed profiles does not affect significantly the results in the urban canopy layer (UCL), where correlations of 95% and 98% were found between the LiDAR profile 1 and 2 data and the WT profile data (at z = 0.02 m above the bottom), respectively. Conversely, the different inflow profiles strongly affected the results above the UCL. This means that the local-scale effects induced on the wind field in the UCL by the urban texture are dominated mainly by the larger-scale forcing, as within the canopy the flow remains topologically invariant despite the different inflow conditions.

Redox dyshomeostasis in the experimental chronic hepatic overloads with iron or copper.

Male rats of 80-90 g were overloaded with either Fe(II) or Cu(II) for 42 days by high concentrations of FeCl2 or CuSO4 in the drinking water. The animals were fed with a commercial rodent diet of 2780 kcal/100 g. Both metal treatments led to a liver redox imbalance and dyshomeostasis with oxidative stress and damage and the concomitant enhancement of oxidative processes as indicated by in vivo surface liver chemiluminescence, the sensitive and organ non-invasive assay for oxidative free radical reactions, and by ex vivo determined processes of phospholipid peroxidation and protein oxidation. In parallel, marked decreases in the antioxidant defense were observed. Liver reduced glutathione (GSH) content and the reduced/oxidized glutathione ratio (GSH/GSSG) were early indicators of oxidative metabolic disturbance upon the metal overloads. Thus, GSH plays a central role in the defense reactions involved in the chronic toxicity of Fe and Cu. Chronic overloads of Fe or Cu in rats afford an experimental animal model of hemochromatosis and of Wilson's disease, respectively. These two animal models could be useful in the study and development of the beneficial effects of pharmacological interventions in the two human diseases.

Brain oxidative stress in rat with chronic iron or copper overload.

Male rats of 80-90 g that were fed 42 days with a commercial rodent diet of 2780 kcal/100 g and received chronic overloads of either Fe(II) or Cu(II) in the drinking water. The two metals produced brain oxidative stress and damage with marked increases in the indicators of oxidative processes: in vivo brain surface chemiluminescence (the sensitive organ non-invasive assay for oxidative free radical reactions), and the ex vivo processes of phospholipid peroxidation and protein oxidation. Brain redox imbalance was also indicated by marked decreases in the cellular indicators of oxidative metabolic stress: reduced glutathione (GSH) content and reduced/oxidized glutathione ratio (GSH/GSSG). Brain decreased GSH content has a central role in the biochemical oxidative processes associated with Fe and Cu chronic damage. The understanding of biochemical oxidative imbalances in the rat brain with chronic Fe(II) or Cu(II) overloads may be useful for the establishment of pharmacological therapies for human pathologies associated to Fe and Cu cellular imbalances.

HP-Xe to go: Storage and transportation of hyperpolarized (129)Xenon.

Recently the spin-lattice relaxation time T1 of hyperpolarized (HP)-(129)Xe was significantly improved by using uncoated and Rb-free storage vessels of GE180 glass. For these cells, a simple procedure was established to obtain reproducible wall relaxation times of about 18 h. Then the limiting relaxation mechanism in pure Xe is due to the coupling between the nuclear spins and the angular momentum of the Xe-Xe van-der-Waals-molecules. This mechanism can be significantly reduced by using different buffer gases of which CO2 was discovered to be the most efficient so far. From these values, it was estimated that for a 1:1 mixture of HP-Xe with CO2 a longitudinal relaxation time of about 7 h can be expected, sufficient to transport HP-Xe from a production to a remote application site. This prediction was verified for such a mixture at a total pressure of about 1 bar in a 10 cm glass cell showing a storage time of T1≈9 h (for T1(wall)=(34±9) h) which was transported inside a magnetic box over a distance of about 200 km by car.

High concentrations of pralidoxime are needed for the adequate reactivation of human erythrocyte acetylcholinesterase inhibited by dimethoate in vitro.

Due to the current controversy about the real effectiveness of the oximes in the treatment of organophosphate poisoning, the reactivation capacity of pralidoxime has been evaluated in vitro on human erythrocyte acetylcholinesterase inhibited by dimethoate. In the in vitro model, a partial recovery of acetylcholinesterase activity was observed with concentrations from 0.066 mM pralidoxime, probably useful enough to prevent death in most cases in vivo. However, much more effectiveness was observed with concentrations up to 0.70 mM pralidoxime. Although pralidoxime should be applied as soon as possible after organophosphate exposure, the application of the antagonist can be useful even 24h after, particularly for organophosphates with biological half-life longer than one day. The protective capacity of pralidoxime after the application was reduced up to 50% in 6h and disappeared almost completely in 24h. Furthermore, the pesticide and its metabolites remained active and were able to inhibit the enzyme as soon as pralidoxime reduced its antagonist capacity. Our results in conjunction with the short half-life of pralidoxime suggest that the maintenance of higher plasmatic concentrations than the currently used should be considered in the management of severe poisoned patients, although adverse effects could be expected.

Effect of cyclosporin A (CyA) on the in vitro growth of hemopoietic progenitors from normal marrow.

Unfractionated normal bone marrow cells (NBM), adherent-cell-depleted NBM, and E-rosette-depleted NBM were plated in vitro for CFU-c or BFU-E formation at plateau concentrations of colony-stimulating activity (CSA) or erythropoietin, untreated or after preincubation with cyclosporin A (CyA). At concentrations of 1000 or 2000 ng/ml, CyA enhanced CFU-c growth up to 137 +/- 32% and 147 +/- 31% of expected baseline growth respectively (p = 0.005 and 0.001). When CyA was added to NBM depleted of T cells by rosetting once with sheep red blood cells (SRBC) there was no CFU-c enhancement at CyA concentrations of 1000 ng/ml, but enhancement could be seen at 2000-5000 ng/ml. The enhancing effect of CyA was completely abolished, however, when the SRBC rosetting procedure was repeated twice (95 +/- 37% of expected growth). On the other hand, removal of adherent cells was without effect on CFU-c enhancement mediated by CyA. The addition of CyA to NBM also enhanced the growth of BFU-E (256 +/- 182% of expected growth) (p = 0.001). The results of this study suggest that CyA can increase the plating efficiency of NBM cells, possibly by inhibiting an endogenous, T-cell-mediated, suppressor mechanism.

Human serum-dependent survival of GM-CFCs in vitro from patients with chronic granulocytic leukemia.

Bone marrow (BM) cells from 15 patients with chronic granulocytic leukemia (CGL) and 12 normal donors were placed in liquid culture for 4-7 days in the presence of fetal calf serum (FCS) or human AB serum. BM cells were plated in agar for GM-CFC growth at day 0, day 4, day 7 of culture, in the presence of human placenta conditioned medium (HPCM). The recovery of GM-CFCs on day 4 in FCS was 86 +/- 18 and 15 +/- 18% from normal or CGL BM cells respectively (p = 0.005). The recovery of GM-CFCs on day 4 in human AB serum was comparable for normal (66 +/- 48%) and CGL (69 +/- 32%) BM cells. Similar results were obtained on day 7 of culture. Cytochemical staining of agar plates showed a sharp drop of macrophage colonies in CGL BM cells kept in FCS cultures on day 4-7, when compared to both normal BM cells and baseline colonies. These data suggest that the survival in liquid culture of GM-CFCs from patients with CGL is dependent on a factor present in human and not in fetal calf serum. This is not the case for normal GM-CFCs.

Androgen receptor gene (CAG)n repeat analysis in the differential diagnosis between Kennedy disease and other motoneuron disorders.

An increase in the number of (CAG)n repeats in the first coding exon of the androgen receptor (AR) gene has been strongly associated with Kennedy disease (KD) (spinal and bulbar muscular atrophy). This is an X-linked hereditary disorder characterized by motoneuron degeneration occurring in adults together with gynecomastia and hyperestrogenemia. We have performed AR gene molecular analysis in several members of a large family with KD as well as in 25 sporadic patients suffering from heterogeneous motoneuron disease (MND). An increase in the length of the (CAG)n repeats was detected, as expected, in all the affected males and in obligatory carrier females, some of which had minor signs of lower motoneuron involvement. There was only one possible exception, one young male with initial signs of the disease, who had an apparent normal length allele. An increased pathological allele was also found in 3 patients with MND. This indicates that the analysis of (CAG)n repeats of the AR gene plays a role in the differential diagnosis of this heterogeneous group of neurological diseases.

Acute myelomonocytic leukemia. Demonstration of pulmonary involvement by bronchoalveolar lavage.

Massive pulmonary infiltration by leukemic cells resulting in respiratory symptoms is a rare complication of acute leukemia. We report the findings in a patient with acute myelomonocytic leukemia presenting with acute onset of fever, dyspnea, and nonproductive cough, in whom the diagnosis of pulmonary invasion by leukemic cells was made by cytochemical analysis of bronchoalveolar cells recovered by lavage.

The antioxidant enzymatic blood profile in Alzheimer's and vascular diseases. Their association and a possible assay to differentiate demented subjects and controls.

A study of several elements of the antioxidative system: Cu-Zn superoxide dismutase (SOD), catalase (CAT), glutathione system (GLU), chemiluminescence (CHE), and antioxidant capacity (AOX), was conducted in 20 demented probable Alzheimer's (DAT), and 15 vascular demented (VD) patients, 19 control (C) subjects, and 11 relatives (F) of one DAT patient. A significant association was found between the variables of the antioxidant system, measured in blood samples, and the neurological pathologies VD and DAT: Kruskal-Wallis test; p = 0.0006 (p = 0.014 when the analysis did not include SOD). This demonstrated that VD and DAT diseases are accompanied by oxidative disorders. The VD and DAT diseases are differentially distinguishable by changes in blood profiles. A graphical method for classification, the Principal Components Analysis (PCA), distinguished between demented and non-demented subjects on the basis of their laboratory variables. A numerical method, Discriminant Functions (DF), constructed to separate the clinical groups on the basis of the same variables, obtained relatively high percentages of success: 92% of demented were detected against healthy subjects; of the latter 82% have been correctly identified as non-demented. Discrimination between VD and DAT patients was achieved for 100% of VD and 86% of DAT patients. DF were similarly successful in detecting the healthy condition of DAT relatives. Possible different mechanisms involved in H2O2 elimination in DAT and VD patients are proposed, where CAT is the responsible enzyme of this reaction in DAT patients, while in VD this function would be achieved mainly through the action of GLU. It seems that SOD levels are stable, at least, within one year. Variations appear to be linked with clinical changes.

Oxidative stress in blood of HIV infected patients.

The oxidative stress in human erythrocytes was studied in asymptomatic and symptomatic patients infected by the human immunodeficiency virus (HIV), and patients with the acquired immunodeficiency syndrome (AIDS). tert-Butyl hydroperoxide initiated chemiluminescence, superoxide dismutase and catalase activities, and total glutathione were evaluated in the erythrocytes and the total antioxidant capacity in the plasma of control, patients infected with HIV that have not yet developed acquired immunodeficiency syndrome, and patients in the later stage of AIDS. tert-Butyl hydroperoxide initiated chemiluminescence was increased by 33% in asymptomatic (stage A1) and symptomatic patients (stage B2) infected with HIV and 82% for patients with AIDS (stage B3) (P < 0.05). While catalase activity did not show any difference between patients and controls, other indices showed differences that, in some cases, reached statistical significance. Superoxide dismutase activity was increased by 24% in stages A1 and B2 of HIV infection and 65% in patients in stage B3 (P < 0.05). Glutathione was decreased by 20% in stages A1 and B2, and by 32% in stage B3 patients (P < 0.05). Total plasma antioxidant capacity was increased in 30 and 57% for the asymptomatic and AIDS patients groups, respectively (P < 0.05). The data indicate that erythrocyte's oxidative stress is associated with the progressive development of HIV disease. Parameters indicating oxidative stress could be an interesting form to screen the evolution of these patients and their response to anti-oxidant therapies.

Regional arterial chemotherapy of liver tumours. I. Performance comparison between a totally implantable pump and a conventional access system.

Thirty-nine patients with liver tumours have been submitted to regional arterial chemotherapy by means of either totally implantable Infusaid-400 pumps (22 c.) or implantable ports (17 c.). The latter were subsequently perfused with external pumps. There was one single major operative complication and no operative deaths. Most patients underwent continuous Fudr infusion. Access related complications occurred in both groups. Treatment was stopped for access related complications in 18.4% and 29.4% of cases out of the pump and port groups respectively. In most of those cases, however, several cycles of chemotherapy had already been performed. The Infusaid-400 pumps showed a 12-month functional duration of 57% with a 13-month median, the 10-month duration of ports being 67%. The difference was not significant. The new implantable systems give better results in comparison with traditional regional access methods, the functional performances of the port systems appearing very similar to the totally implantable pumps, with an obvious advantage for the pumps as far as quality of life is concerned.

Comparative in vitro effects of sodium arsenite and sodium arsenate on neuroblastoma cells.

The toxic effects of arsenic at different cellular levels were assessed using two inorganic chemical species: sodium arsenite and sodium arsenate, representing the trivalent and pentavalent states of arsenic, respectively. Mouse neuroblastoma cell cultures (Neuro-2a) were exposed for 24 h, and cytotoxic effects evaluated were: cell proliferation by quantification of total protein content; cytoplasmic membrane integrity to cytosolic lactate dehydrogenase leakage; lysosomal hexosaminidase release; lactate dehydrogenase activity; mitochondrial succinate dehydrogenase activity; relative neutral red uptake by lysosomes; lysosomal hexosaminidase sphingolipid degradation activity; and acetylcholinesterase activity. As(III) was found to be five times more toxic than As(V) to neuroblastoma cell proliferation, but the relative extent of other alterations differed. Special sensitivity was detected for lactate dehydrogenase inhibition. Hexosaminidase activity was also very susceptible, being inhibited at low concentrations and stimulated at high concentrations. Less sensitive were the inhibition of cell proliferation, relative neutral red uptake, and acetylcholinesterase activity. As(III) was lysosomotropic, with secretion of hexosaminidase, but the release was decreased by As(V). Mitochondrial succinate dehydrogenase was inhibited by As(III) and stimulated by As(V). Minor sensitivity to cytoplasmic lactate dehydrogenase leakage for both compounds also shows that functional metabolic alterations produced by arsenic are more important than structural damage.

Comparative effects of the metabolic inhibitors 2,4-dinitrophenol and iodoacetate on mouse neuroblastoma cells in vitro.

The toxic effects of two metabolic inhibitors, dinitrophenol and iodoacetic acid, were compared. Mouse neuroblastoma cell cultures (Neuro-2a) were exposed to different concentrations of the toxic compounds for 24, 48 and 72 h to study basal toxicity effects (cell proliferation by quantification of total protein content (PR) and relative neutral red uptake (RNRU) by lysosomes). The following biochemical indicators assessed in the in vitro test system were: cytosolic phosphofructokinase (PFK) and enolase (ENL) activities in glycolysis; mitochondrial succinate dehydrogenase (SDH) activity in the citric acid cycle; lysosomal beta-galactosidase (GAL) activity; and neuronal acetylcholinesterase (AChE) activity. The effects of the two metabolic inhibitors on the various indicators differed. Iodoacetic acid was found to be far more toxic than dinitrophenol to neuroblastoma cell proliferation at 24 h exposure. Though 2,4-dinitrophenol and iodoacetic acid both inhibited cell proliferation of the neuroblastoma cells, their effects on the other endpoints were opposite. Dinitrophenol was a general activator of the metabolism, particularly affecting lysosomal function. Iodoacetic acid did not significantly alter general metabolism, but considerably modified lysosomal function and AChE activity. The modification of lysosomal function of Neuro-2a cells by the two compounds was quite different: dinitrophenol increased RNRU and GAL activity, and iodoacetic acid decreased both parameters.