Mechanism of arsenic-induced neurotoxicity may be explained through cleavage of p35 to p25 by calpain.

In recent studies we have demonstrated that arsenic (As) metabolites change the composition of neuronal cytoskeletal proteins in vivo and in vitro. To further examine the mechanism of arsenic-induced neurotoxicity with various arsenate metabolites (iAsV, MMAV and DMAV) and arsenite metabolites (iAsIII, MMAIII and DMAIII), we investigated the role of the proteolytic enzyme calpain and its involvement in the cleavage of p35 protein to p25, and also mRNA expression levels of calpain, cyclin-dependent kinase 5 (cdk5) and glycogen synthase kinase 3 beta (gsk3ss). A HeLa cell line transfected with a p35 construct (HeLa-p35) was used as a model, since all other proteins such as calpain, CDK5 and GSK3beta are already present in HeLa cells as they are in neuronal cells. HeLa-p35 cells were incubated with various As metabolites and concentrations of 0, 10 and 30 microM for duration of 4 h. Subsequently the cells were either lysed to study their relative quantification levels of these genes or to be examined on their p35-protein expression. P35-RNA expression levels were significantly (p<0.01) increased by arsenite metabolites, while p35 protein was cleaved to p25 (and p10) after incubation with these metabolites. The cleavage of p35 is caused by calcium (Ca2+) induced activation of calpain. Inhibition of calpain activity by calpeptin prevents cleavage of p35 to p25. These results suggest that cleavage of p35 to p25 by calpain, probably As-induced Ca2+-influx, may explain the mechanism by which arsenic induces its neurotoxic effects.

Comparative toxicity of arsenite metabolites in wild type CHO cells and in cells deficient in excision repair cross-complementing 1 and 2.

Arsenic (As) has been shown to alter one or more DNA repair processes. Excision repair cross-complementing 1 and 2 (ERCC1 and ERCC2) have shown to be associated with arsenic-induced toxicity and carcinogenicity. In this study, we investigated cytotoxic effects of various As metabolites in relation to two nucleotide excision repair genes: ERCC1 and ERCC2. Various arsenate (pentavalent) and arsenite (trivalent) metabolites were tested in ERCC1, ERCC2 deficient and wild type cells. Our results showed that in the selected concentration range pentavalent As metabolites; iAs(V), MMA(V) and DMA(V) were not cytotoxic, unlike the trivalent As metabolites; iAs(III), MMA(III) and DMA(III). The measured LC(50) demonstrated a significant difference (p<0.01) for iAs(III) between the three cell lines, while MMA(III) and DMA(III) are more cytotoxic to all three cell lines. UV5 (ERCC2 deficient) cells also showed a lower resistance to iAs(III) in comparison to AA8 (wild type) and UV20 (ERCC2 deficient) cells. This might be explained through the generation of hydrogen peroxide (H(2)O(2)), which is generated by increase of intracellular Ca(2+) level. Generation of H(2)O(2) in UV5 cells after incubation with iAs(III) is significantly higher than AA8 and UV20 cells (p<0.01). In conclusion, absence of ERCC2 leads to a increased generation of H(2)O(2) by iAs(III) in UV5 cells, which is in contrast to AA8 and UV20 cells.

Arsenic metabolites affect expression of the neurofilament and tau genes: an in-vitro study into the mechanism of arsenic neurotoxicity.

Neurological studies indicate that the central (CNS) and peripheral nervous system (PNS) may be affected by arsenic (As). As-exposed patients show significantly lower nerve conduction velocities (NCVs) in their peripheral nerves in comparison to healthy subjects. As may play a role in the disruption of neuroskeletal integrity, but the mechanisms by which it exerts a toxic effect on the peripheral and central nervous system are still unclear. In the present study, we examined the neurotoxic effects of various arsenic metabolites (iAs(III), iAs(V), MMA(V) and DMA(V)) on two different cell lines derived from the peripheral (ST-8814) and central (SK-N-SH) nervous system. The effects of the arsenic metabolites were examined on the relative quantification levels of the cytoskeletal genes, neurofilament-light (NEFL), neurofilament-medium (NEF3), neurofilament-heavy (NEFH) and microtubule-associated protein-tau (MAPT), using real-time PCR. Our results show that iAs(III) and iAs(V) have no significant effects on either cell lines. On the other hand, MMA(V) and DMA(V) cause significant changes in expression levels of NEF3 and NEFL genes, while the expression level of the NEFH gene is significantly increased in both cell lines.

Arsenic neurotoxicity--a review.

Arsenic (As) is one of the oldest poisons known to men. Its applications throughout history are wide and varied: murder, make-up, paint and even as a pesticide. Chronic As toxicity is a global environmental health problem, affecting millions of people in the USA and Germany to Bangladesh and Taiwan. Worldwide, As is released into the environment by smelting of various metals, combustion of fossil fuels, as herbicides and fungicides in agricultural products. The drinking water in many countries, which is tapped from natural geological resources, is also contaminated as a result of the high level of As in groundwater. The environmental fate of As is contamination of surface and groundwater with a contaminant level higher than 10 particle per billion (ppb) as set by World Health Organization (WHO). Arsenic exists in both organic and inorganic species and either form can also exist in a trivalent or pentavalent oxidation state. Long-term health effects of exposure to these As metabolites are severe and highly variable: skin and lung cancer, neurological effects, hypertension and cardiovascular diseases. Neurological effects of As may develop within a few hours after ingestion, but usually are seen in 2-8 weeks after exposure. It is usually a symmetrical sensorimotor neuropathy, often resembling the Guillain-Barré syndrome. The predominant clinical features of neuropathy are paresthesias, numbness and pain, particularly in the soles of the feet. Electrophysiological studies performed on patients with As neuropathy have revealed a reduced nerve conduction velocity, typical of those seen in axonal degeneration. Most of the adverse effects of As, are caused by inactivated enzymes in the cellular energy pathway, whereby As reacts with the thiol groups of proteins and enzymes and inhibits their catalytic activity. Furthermore, As-induced neurotoxicity, like many other neurodegenerative diseases, causes changes in cytoskeletal protein composition and hyperphosphorylation. These changes may lead to disorganization of the cytoskeletal framework, which is a potential mechanism of As-induced neurotoxicity.

Arsenic-induced toxicity: effect on protein composition in sciatic nerve.

Exposure to arsenic compounds may lead to skin and lung cancer and various disorders such as vascular disease and peripheral neuropathy in humans. Peripheral arsenic neurotoxicity has been demonstrated clinically and in electrophysiological studies. Patients intoxicated with arsenic show neurological symptoms in their feet and hands. These patients show significantly lower nerve conduction velocities (NCVs) in their peripheral nerves in comparison with controls. The mechanism of arsenic peripheral nervous system (PNS) toxicity, however, has never been described before. This is the first study to investigate the toxicity of arsenic on the PNS. Male Wistar rats were exposed to arsenite given as a single dose i.v. After sacrifice, sciatic nerves were excised and the protein composition was analysed. Protein analysis of sciatic nerves showed disappearance of neurofilament and fibroblast proteins in rats treated with arsenite doses of 15 and 20 mg/kg in comparison with the control groups. Some fibroblast protein bands had disappeared in the 20-mg/kg dose group. The analysed neurofilament-M and -L proteins decreased dose dependency over time. arsenic affects the composition of proteins in the rat sciatic nerve, especially the neurofilaments. The reduction of signals in Western blot analysis reveals changes in cytoskeletal composition, which may well lead to neurotoxic effects in vivo.

Renal damage in mice after sequential cisplatin and irradiation: the influence of prior irradiation on platinum elimination.

Doses of 4-6 mg kg-1 c-DDP given 6 months before renal irradiation caused only a modest increase in functional radiation damage (DEF 1.1). These effects could be explained by additive toxicities and the damage was much less than when c-DDP was given 3-6 months after irradiation. Pharmacokinetic studies did not demonstrate any decrease in the rate of platinum elimination after previous low-dose renal irradiation.

Aluminum affects interconnections between aggregates of cultured hippocampal neurons. Neurotoxicity of aluminum in vitro.

The effect of aluminum (Al) was studied in an in vitro system of cultured hippocampal neurons from fetal rats in a chemically defined medium. Neuronal aggregates interconnected by neurites were detected light and electron microscopically in control and Al-treated cultures. In the Al-treated cultures disruptions of the fibers were observed proximal to the aggregates; the number of disruptions increased with The Al concentration applied. It is concluded that interference of intracellular Al with the neuronal skeleton occurs, leading to an enhanced fragility of the interconnecting neurites. The concentration-dependent Al-induced disruption of interconnecting neurites may serve as an in vitro model for further research in Al neurotoxicity.

Biological monitoring of aluminium in renal patients.

During the past decade, aluminium (Al) has been shown to possess a potential for systemic toxicity. Renal patients form a high-risk group for Al poisoning, and biological monitoring is indicated to diagnose and prevent toxicity. Electrothermal atomic absorption spectrometry is the analytical method of choice for measuring Al levels. Special precautions have to be taken to prevent contamination with environmental Al or adsorption of Al to glassware during analysis. Since this metal is almost evenly distributed between plasma and erythrocytes, either plasma or whole blood may be used to estimate exposure to Al. Measurement in dialysis fluid is suitable to assess parenteral uptake. Hair analysis is of no value. The normal value in plasma is 7.3 +/- 2.0 micrograms/l (mean +/- SD, n = 10); toxic symptoms are mainly associated with levels above 100 micrograms/l. A dialysate level not exceeding 5 micrograms/l may be considered safe; intestinal absorption is then the only remaining, highly variable source of Al uptake.

Analysis of bismuth in serum and blood by electrothermal atomic absorption spectrometry using platinum as matrix modifier.

Bismuth-containing medicines have been used for years, but there is a lack of clinically applicable methods for measuring bismuth in body fluids. We describe a sensitive, accurate and precise method for analysis of bismuth in blood and serum, which is suitable both for monitoring purposes and for further investigations into the biokinetics and safety of bismuth. Bismuth was quantitated with electrothermal atomic absorption spectrometry with Zeeman background correction. In the furnace programme a cool-down step was introduced and platinum was used as a matrix modifier. Recovery for 40 micrograms/l is 93.7 +/- 4.6% (mean +/- SD) from serum and 92.8 +/- 5.4% from blood; within-day precision (n = 10) at 40 micrograms/l is 3.2% for serum and 4.2% for blood. Day-to-day precision at 40 micrograms/l (n = 10) was 4.5% for serum and 4.0% for blood. The detection limit is 0.7 microgram/l for serum and 1.0 microgram/l for blood. Blood samples have to be collected in glass tubes and stored at -20 degrees C.

Effect of aluminum chloride, -citrate, and -maltol on the calcium-mediated degradation of neurofilament proteins.

Aluminum (Al) has been observed to cause neurofilament protein accumulation in both experimental animals and cultured cells. Impairment of axonal transport is thought to be a mechanism of toxicity. Inhibition of the degradation of neurofilament proteins, however, resulting in accumulation of these proteins may be an alternative mechanism for Al toxicity. In the present study, the effect of calcium (Ca) on the proteolysis of the neurofilament triplet proteins by calcium-activated neutral proteases (CANP) was studied in the isolated sciatic nerve explants. The extent of the degradation was found to be dependent on the Ca concentration. The effect of Al chloride, -citrate and -maltol on the calcium-induced degradation was studied. No effect of any of the Al compounds was observed, suggesting that the metal may exert its neurotoxic effect via a mechanism other than impairment of neurofilament proteolysis. Maltol itself was found to enhance the effect of Ca on the degradation of neurofilament proteins, probably by facilitating the movement of Ca across the neuronal membrane.

Absorption of bismuth from several bismuth compounds during in vivo perfusion of rat small intestine.

Evaluation of the amount of bismuth (Bi) absorbed from the gastrointestinal tract is important for assessment of the possible risks associated with the use of Bi compounds in the treatment of gastrointestinal disorders. We compared the absorption of Bi from media containing the equivalent of 1 g of elemental Bi from either Bi subnitrate (BSN), Bi subsalicylate (BSS), colloidal Bi subcitrate (CBS), Bi chloride (BiCl3), or Bi citrate (BCit) by an in vivo perfusion system of rat small intestine. The intestinal absorption was < 1% for all compounds, but higher from BCit and CBS than from BSN, BSS, and BiCl3. The dose dependency of Bi absorption from both CBS and BiCl3 in citrate buffer showed a nonlinear relationship between the concentration of Bi in perfusate and the concentration of Bi in blood after 60 min.

Idiopathic haemochromatosis: magnetic resonance signal intensity ratios permit non-invasive diagnosis of low levels of iron overload.

OBJECTIVE: The detection of low levels of iron overload by magnetic resonance imaging. METHODS: Eight consecutive patients suspected of having idiopathic haemochromatosis. Comparison of signal intensity ratios and absolute iron content of liver. RESULTS: There was a good correlation between the signal intensity ratios and iron content in the range 2-30 micrograms Fe/mg dry weight. CONCLUSIONS: The ability of a non-invasive technique to detect low levels of iron overload could be useful in the assessment of therapy and in the screening of relatives of probands with idiopathic haemochromatosis.

Monitoring of aluminum in whole blood, plasma, serum, and water by a single procedure using flameless atomic absorption spectrophotometry.

A simple, time-saving procedure to measure aluminum (AI) in whole blood, plasma, and water samples of low ionic strength by flameless atomic absorption spectrometry is presented. With this procedure, an analagous pretreatment is given to all samples. Moreover, the pretreated samples are analyzed using an identical program of the atomic absorption spectrophotometer. The detection limit is 1.3 micrograms/L for aqueous solutions and 1.9 micrograms/L, 1.8 micrograms/L, and 2.3 microL for serum, plasma, and blood samples respectively. The precision of the method varies between 2.8% and 4.2% of all analyses. Using this method, a comparison was made between Al levels in whole blood (AlB) and plasma (AlP) in rats and AlB and AlP in renal patients. The AlB and AlP were strongly correlated. It is concluded that the monitoring of AlB may have a similar prognostic value for toxicity as the monitoring of AlP, but more experimental advantages.

Fluoride enhances the effect of aluminium chloride on interconnections between aggregates of hippocampal neurons.

The role of fluoride in aluminium neurotoxicity was studied using an in vitro system of cultured hippocampal neurons from foetal rats. Sodium fluoride (50 microM) and aluminium chloride (12.5 microM) were administered alone or in a specific combination (50 + 12.5 microM) in a 14-day culture in a chemically defined medium before staining of neurofilaments. Neuronal aggregates interconnected by neuritic fibers were detected light microscopically in control cultures. The aggregates and the fibers stained positive for neurofilament proteins. In cultures treated with aluminium chloride the development of the interconnecting fibers was affected, resulting in a fusion pattern of the aggregates. This phenomenon was enhanced when sodium fluoride was given together with aluminum chloride. It was concluded that aluminium interferes with the metabolism of the neuronal cytoskeleton and that this interference is potentiated by fluoride.

Aluminium intoxication in a dog.

A two-year-old male Barsoi dog was presented after a two-week period of muscle twitching and convulsions during exercise, which worsened to a state of tetraparesis and coma. Removal of a gastric foreign body, containing aluminium, resolved the presenting signs. Parallel with this clinical recovery the elevated serum levels of aluminium decreased to values of two normal control dogs, suggesting that the neurological signs were due to A1 intoxication.

Arsenic-induced neurotoxicity in relation to toxicokinetics: effects on sciatic nerve proteins.

In our previous study in rats acutely exposed to As, we observed an effect of As on neurofilaments in the sciatic nerve. This study deals with the effects of inorganic As in Wistar rats on the cytoskeletal protein composition of the sciatic nerve after subchronic intoxication. Sodium meta-arsenite (NaAsO2) dissolved in phosphate-buffered saline (PBS) was administered daily in doses of 0, 3 and 10 mg/kg body weight/day (n=9 rats/group) by intragastric route for 4, 8 and 12 week periods. Toxicokinetic measurements revealed a saturation of blood As in the 3- and 10-mg/kg dose groups at approximately 14 microg/ml, with an increase in renal clearance of As at increasing doses. After exsanguination, sciatic nerves were excised and the protein composition was analyzed. Analysis of the sciatic nerves showed compositional changes in their proteins. Protein expression of neurofilament Medium (NF-M) and High (NF-H) was unchanged. Neurofilament protein Low (NF-L) expression was reduced, while mu- and m-calpain protein expression was increased, both in a dose/time pattern. Furthermore, NF-H protein was hypophosphorylated, while NF-L and microtubule-associated protein tau (MAP-tau) proteins were (hyper)-phosphorylated. In conclusion, we show that expression of mu- and m-calpain protein is increased by exposure to As, possibly leading to increased NF-L degradation. In addition, hyperphosphorylation of NF-L and MAP-tau by As also contribute to destabilization and disruption of the cytoskeletal framework, which eventually may lead to axonal degeneration.

Complex cardiac defect with hypoplastic right ventricle in a fetus with valproate exposure.

We describe a fetus with a hypoplastic right ventricle detected by prenatal ultrasound examination. A possible causal relationship with prenatal valproate exposure is discussed.

Intestinal absorption of aluminium.

The intestinal absorption of aluminium can contribute significantly to systemic exposure to this element. Aluminium can be absorbed not only from oral pharmaceuticals but also from solid food and drinking water. The absorption process is not restricted to patients with kidney disorders; other groups of patients and healthy subjects are not excluded. Details of the absorptive mechanism are mainly obtained from in vitro (everted gut sac) and animal studies (intestinal perfusion) rather than from controlled human studies and case reports. The process of absorption depends on the intraluminal speciation, the intraluminal quantity, the presence of competing (iron, calcium) or complexing (citrate) substances and the intraluminal pH. The condition of the exposed organism with respect to the gut also determines intestinal absorption (iron status, calcium [vitamin D, parathyroid hormone] status, age and kidney function). Various absorption sites and passage routes, both transcellular and paracellular, have been reported, each apparently related to a different aluminium species (hydrated ionic species, aluminium citrate complex etc.). No uniform mechanistic model allowing extrapolation to the clinical situation has yet emerged.