Repeated dose 90-day oral toxicity test of G-7% NANA in rats: An application of new criterion for toxicity determination to test article-induced changes.

G-7% NANA is N-acetylneuraminic acid(NANA) containing 7% sialic acid isolated from glycomacropeptide (GMP), a compound of milk. Since NANA is likely to have immunotoxicity, the need to ensure safety for long-term administration has been raised. In this study, a 90-day repeated oral dose toxicity test was performed in rats using G-7% NANA in the dosages of 0, 1250, 2500 and 5000 mg/kg/day.A toxicity determination criterion based on the significant change caused by the administration of the substancewas developed for estimating NOEL, NOAEL and LOAELapplied to this study. When analyzing the immunological markers, no significant changes were observed, even if other significant changes were observed in the high dose group. In accordance with the toxicity determination criterion developed, the NOEL in male and female has been determined as 2500 mg/kg/day, and the NOAEL in females has been determined as 5000 mg/kg/day. The toxicity determination criterion, applied for the first time in the repeated dose toxicity tests, could provide a basis for distinguishing NOEL and NOAEL more clearly; nevertheless, the toxicity determination criterion needs to be supplemented by adding differentiating adverse effects and non-adverse effects based on more experiences of the repeated dose toxicity tests.

Short-term intra-nasal erythropoietin administration with low sialic acid content is without toxicity or erythropoietic effects.

The objective of this investigation was to assess the toxicological potential of nasal formulation of erythropoietin with low sialic acid content (Neuro EPO) after 28 days of intra-nasal dosing in rats besides to evaluate the immunogenicity and erythropoietic effect of the test substance. Healthy Wistar rats of both sexes were used for 28 days subacute toxicity and immunogenicity assays. Doses evaluated were 3450, 4830 and 6900 UI/kg/day. The toxicological endpoints examined included animal body weight, food consumption, hematological and biochemical patterns, antibodies determination, selected tissue weights and histopathological examination. Reversibility of toxic effects was evaluated at high dose 14 days after treatment period. Female B6D2F1 mice were used for evaluated erythropoietic effect of the nasal formulation. Hematological endpoints were examined every week during 28 days of intra-nasal dosing of 6900 UI/kg/day. Variations of hematological patterns were not observed after 28 days of intranasal dosing. A slight increase in glucose level of treated animals within the normal range was observed. This effect was not dose related and was reversible. Antibody formation was not observed in any of the test doses. Histopathological examination of organs and tissues did not reveal treatment induced changes. The administration of Neuro EPO in normocythaemic mice did not produce erythropoietic effect. These results suggest that Neuro EPO could be used as a neuroprotective agent, without significant systemic haematological side effects.

Alteraciones bioquímicas en individuos expuestos al arsénico en el agua de bebida en Tucumán, Argentina / Biochemical alterations in individuals consuming high levels of arsenic in drinking water in Tucuman, Argentina

El objetivo de esta investigación fue evaluar los niveles del ácido siálico (S) y enzimas hepáticas en individuos que consumieron agua con arsénico (As) relacionándolos con la presencia ("I con H") o no de hepatomegalia ("I sin H"). Se incluyeron 200 individuos, 85 correspondieron al Grupo Control (GC), 32 "I con H" y 83 "I sin H" quienes habían consumido agua con niveles mayores a 0,01 mg/L. Se les tomó una muestra de sangre venosa y se les realizó el dosaje del S y de las enzimas alaninaninotransferasa (ALT), aspartatoaminotransferasa (AST), fosfatasa alcalina (FAL), gamma glutamil transferasa (GGT), bilirrubina directa (BD) y total (BT), lactato deshidrogenasa (LDH) y 5' nucleotidasa (5'Nu). En los individuos que consumieron agua contaminada se encontró un aumento de los niveles del S. En el grupo "I con H", la FAL, la GGT y la LDH se encontraron aumentadas. En el grupo "I sin H", la GGT y la LDH tuvieron niveles elevados. En los individuos expuestos al As, no se encontraron alteraciones en los otros parámetros bioquímicos estudiados y la prevalencia de hepatomegalia no fue significativa. Los cambios bioquímicos encontrados fueron compatibles con la presencia de un patrón colestásico. Estos datos muestran que la concentración del S sérico podría servir como un indicador de exposición al arsénico que podría ser utilizado en forma conjunta con otros marcadores.

The aim of this study was to investígate the relationship between the levels of serum sialic acid (S) and hepatic enzymes in individuáis who drink As contaminated water. Two hundred individuáis were selected: 85 were the control group, 32 presented hepatomegaly (I with H) and 83 did not present hepatomegaly (I without H) who had consumed drinking water containing As levels higher than O.Ol mg/L. Blood samples were collected for the determination of S and hepatic enzymes in serum: alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (FAL), gamma glutamil transpeptidase (GGT), direct (BD) and total bilirrubin (BT), lactate deshydrogenase (LDH) and 5'nucleotldase (5'Nu). The populatlon exposed to As in drinking water presented high levels of S, FAL, GGT, LDH and GGT, LDH were increased in I with H and I without H respectively. No significant changes were observed in the other parameters studied. Prevalence of hepatomegaly was not significantly higher and the hepatic biochemical changes were related with the presence of cholestasis in As-exposed people. These data show that people with high As level intake would have an increased sialoprotein replacement which could be an marker with other one of the alterations caused by arsenic.

A theoretical interpretation of the transient sialic acid toxicity of a nanR mutant of Escherichia coli.

This article reports on experimental evidence that an Escherichia coli nanR mutant shows inhibited growth in N-acetylneuraminic acid. This effect is prevented when inocula are grown in an excess of glucose, but not in an excess of glycerol. The nanATEK operon is controlled by catabolite repression, suggesting that diminished expression of the nanATEK operon in the presence of glucose explains the inocula effects. Neither double nanR-nagC nor nanR dam mutants show growth inhibition in the presence of N-acetylneuraminic acid. A theoretical model of N-acetylneuraminic acid metabolism (i.e., in particular of the nanATEK and nagBACD operons) is presented; the model suggests an interpretation of this effect as being due to transient high accumulations of GlcNAc-6P in the cell. This accumulation would lead to suppression of central metabolic functions of the cell, thus causing inhibited growth. Based on the theoretical model and experimental data, it is hypothesised that the nanATEK operon is induced in a two-step mechanism. The first step is likely to be repressor displacement by N-acetylneuraminic acid. The second stage is hypothesised to involve Dam methylation to achieve full induction.

N-acetylneuraminic acid coupled human recombinant TNFalpha exhibits enhanced anti-tumor activity against Meth-A fibrosarcoma and reduced toxicity.

In order to study the effect of glycosylation on its biological activities and to develop tumor necrosis factor alpha (TNFalpha) with less deleterious effects, N-acetylneuraminic acid (NeuAc) with a C9 spacer was chemically coupled to human recombinant TNFalpha. NeuAc-coupled TNFalpha (NeuAc-TNFalpha) exhibited reduced activities in vitro by about threefold compared to native TNFalpha. In this study, we examined a variety of TNFalpha activities in vivo. NeuAc-TNFalpha reduced activities in the up-regulation of serum levels of IL-6 and NOx, but comparable activity as native TNFalpha in the down-regulation of the serum level of glucose. However, NeuAc-TNFalpha was more potent than TNFalpha in the up-regulation of the serum level of serum amyloid A (SAA). NeuAc-TNFalpha was less toxic to mice. In addition, NeuAc-TNFalpha exhibited an augmented anti-tumor activity against Meth-A fibrosarcoma without hemorrhagic necrosis. These results indicate that coupling with NeuAc enabled us to develop neoglycoTNFalpha with selective activities in vivo, including enhanced anti-tumor activity but reduced toxicity.

Polysulfated sialic acid derivatives as anti-human immunodeficiency virus.

We report the synthesis of a novel alkyl polysulfated sialic acid derivative denoted as NMSO3. NMSO3 exhibited potent inhibition against both laboratory and clinical human immunodeficiency virus type 1 (HIV-1). The anti-viral activity of this compound (1 uM) was compared to dextran sulfate (3 uM), and was found to be more potent against HIV-1IIIb than AZT (10 uM). The anti-coagulation time was more than 15-fold shorter than that of dextran sulfate. An in vivo anti-viral study of NMSO3 in NOD-SCID-PBL mice HIV model showed complete protection of the animals from virus challenge at the concentration of 10 mg/kg. This suggests that NMSO3 can be effective in the treatment of HIV-infected individuals.

Mechanistic effect of NMSO3 on replication of human immunodeficiency virus.

NMSO3, a sulphated sialyl lipid, was evaluated for its efficacy against human immunodeficiency virus type 1 (HIV-1). The compound exhibited concentration-dependent inhibition of HIV-1 replication in primary infection cell culture systems. Substantial inhibition was observed at concentrations of NMSO3 that showed little cytotoxicity. NMSO3 also exhibited anti HIV-1 activity in chronically HIV-1 infected cultures. The production of progeny viruses was completely abolished without cytotoxicity by continuous addition of NMSO3 to chronically infected U937 cells. Furthermore, in attempting to define the inhibitory mechanism of NMSO3, we investigated its effect on several steps of the HIV-1 replication cycle. NMSO3 competes with gp120 for binding to CD4 receptors on cells and inhibits the entry of HIV-1. By epitope analysis of the human CD4 molecule, NMSO3 inhibits the binding of antibodies, which recognize the D1 domain of CD4. Moreover, semi-quantitative reverse transcribed polymerase chain reaction (RT-PCR) showed that the integrated provirus is transcriptionally inactive in NMSO3-treated cells, supporting the lack of progeny in the culture supernatant of chronically HIV-1-infected cells treated with NMSO3. These findings indicate that NMSO3 has a unique mechanism of action against HIV-1 in both primary and chronic infection, and may be a valuable compound for the treatment of HIV-1 infection.

Wheatgerm agglutinin-mediated toxicity in pancreatic cancer cells.

Lectin binding specificities for carbohydrate allow phenotypic and functional characterization of membrane-associated glycoproteins expressed on cancer cells. This analysis examined wheatgerm agglutinin binding to pancreatic cancer cells in vitro and the resulting toxicity. Membrane preparations of nine human pancreatic carcinoma cell lines were studied for lectin binding using wheatgerm agglutinin (WGA), concanavalin A (ConA) and phytohaemagglutinin-L (PHA-L) in a lectin blot analysis. Cell proliferation in vitro was measured by thymidine incorporation in the absence or presence of lectins at various concentrations. Sialic acid binding lectins or succinyl-WGA (succWGA) served as controls. WGA toxicity was tested after swainsonine or neuraminidase pretreatment. Binding and uptake of fluorescein-labelled lectins was studied under fluorescence microscopy. All pancreatic cell lines displayed high WGA membrane binding, primarily to sialic acid residues. Other lectins were bound with weak to moderate intensity only. Lectin toxicity corresponded to membrane binding intensity, and was profound in case of WGA (ID50 at 2.5-5 microg ml(-1)). WGA exposure induced chromatin condensation, nuclear fragmentation and DNA release consistent with apoptosis. Important steps for WGA toxicity included binding to sialic acid on swainsonine-sensitive carbohydrate and lectin internalization. There was rapid cellular uptake and subsequent nuclear relocalization of WGA. In contradistinction to the other lectins studied, WGA proved highly toxic to human pancreatic carcinoma cells in vitro. WGA binding to sialic acid residues of N-linked carbohydrate, cellular uptake and subsequent affinity to N-acetyl glucosamine appear to be necessary steps. Further analysis of this mechanism of profound toxicity may provide insight relevant to the treatment of pancreatic cancer.

Comparative genotoxicity of six salicylic acid derivatives in bone marrow cells of mice.

In vivo sister chromatid exchange (SCE) and chromosome aberrations (CA) were carried out for six salicylic acid derivatives in bone marrow cells of mice. Six salicylic acid derivatives, namely acetyl salicylic acid (aspirin), salicylic acid, salicylamide, sodium salicylate, diflunisal and niclosamide, were used for these experiments. Drugs were administered both intraperitoneally (i.p.) and orally by gavage. Out of these six salicylic acid derivatives tested, only diflunisal and niclosamide showed genotoxicity as measured by both SCE and CA assays. Acetyl salicylic acid and sodium salicylate showed weak genotoxicity as measured by SCE and CA, respectively, only at the highest dose tested.