Neurological symptoms among Sri Lankan farmers occupationally exposed to acetylcholinesterase-inhibiting insecticides.

BACKGROUND: In many agricultural districts in Sri Lanka, pesticide poisoning is a leading cause of death. This study aims to evaluate the impact of pesticide use on Sri Lankan farmers' health. METHODS: A total of 260 subjects were surveyed in both a low and a high exposure period. Acetylcholinesterase activity was measured and data on symptoms were collected with questionnaires. RESULTS: Twenty-four percent of surveyed farmers had suffered at least once from acute pesticide poisoning. Farmers showed significantly more inhibition of cholinesterase activity than controls. Acute symptoms indicative for exposure to cholinesterase-inhibiting pesticides were associated with farming and a higher degree of cholinesterase suppression (more than 13% inhibition). Integrated Pest Management (IPM) training seemed to result in less insecticide use, and less cholinesterase inhibition. CONCLUSIONS: Our results suggest that occupational acetylcholinesterase-inhibiting insecticide exposures have a negative impact on Sri Lankan farmers' health. Overall reduction in pesticide use seems the best option to protect farmers from the adverse effects of pesticides.

Analysis of interleukin-1alpha (IL-1alpha) and interleukin-8 (IL-8) expression and release in in vitro reconstructed human epidermis for the prediction of in vivo skin irritation and/or sensitization.

In the present study, reconstructed human epidermis (RHE) was used as an in vitro model to discriminate 1-chloro-2,4-dinitrobenzene (DNCB), nickel sulfate (NiSO(4)), oxazolone (OXA), 2,4-dinitrofluorobenzene (DNFB) and 2,4,6-trinitrobenzenesulfonic acid (TNBS) as skin sensitizers from benzalkonium chloride (BC), benzoic acid (BA) and sodium lauryl sulfate (SLS) as skin irritants. Our criteria were (a) the differential IL-1alpha and IL-8 synthesis and release (b) cytotoxicity assessment by MTT assay. When the RHE are topically treated with the sensitizers, very low levels of extra- and intracellular IL-1alpha are observed although they induce significant cytotoxicity. In contrast, they exhibit a sharp maximum of IL-8 release. In the presence of the tested irritants, we observe the inverse cytokine release profile, although they induce dose-dependent cytotoxicity profiles similar to those observed with the sensitizers. Finally, IL-1alpha mRNA upregulation is observed after topical application of both sensitizers and irritants, but only the latter significantly increase extracellular IL-1alpha. In conclusion, our results suggest that the associated determination of IL-8, with IL-1alpha, and MTT conversion are at least necessary to discriminate and classify, in a single assay, irritant and sensitizing agents and represent a potential in vitro alternative to two classical in vivo assays.

Hypoxia-induced increase in intracellular calcium concentration in endothelial cells: role of the Na(+)-glucose cotransporter.

Hypoxia is a common denominator of many vascular disorders, especially those associated with ischemia. To study the effect of oxygen depletion on endothelium, we developed an in vitro model of hypoxia on human umbilical vein endothelial cells (HUVEC). Hypoxia strongly activates HUVEC, which then synthesize large amounts of prostaglandins and platelet-activating factor. The first step of this activation is a decrease in ATP content of the cells, followed by an increase in the cytosolic calcium concentration ([Ca(2+)](i)) which then activates the phospholipase A(2) (PLA(2)). The link between the decrease in ATP and the increase in [Ca(2+)](i) was not known and is investigated in this work. We first showed that the presence of extracellular Na(+) was necessary to observe the hypoxia-induced increase in [Ca(2+)](i) and the activation of PLA(2). This increase was not due to the release of Ca(2+) from intracellular stores, since thapsigargin did not inhibit this process. The Na(+)/Ca(2+) exchanger was involved since dichlorobenzamil inhibited the [Ca(2+)](i) and the PLA(2) activation. The glycolysis was activated, but the intracellular pH (pH(i)) in hypoxic cells did not differ from control cells. Finally, the hypoxia-induced increase in [Ca(2+)](i) and PLA(2) activation were inhibited by phlorizin, an inhibitor of the Na(+)-glucose cotransport. The proposed biochemical mechanism occurring under hypoxia is the following: glycolysis is first activated due to a requirement for ATP, leading to an influx of Na(+) through the activated Na(+)-glucose cotransport followed by the activation of the Na(+)/Ca(2+) exchanger, resulting in a net influx of Ca(2+).

The tetraspanin CD9 associates with the integrin alpha6beta4 in cultured human epidermal keratinocytes and is involved in cell motility.

Integrins are involved in several ways in keratinocyte physiology, including cell motility. CD9 is a member of the tetraspanin protein family which is found in association with other transmembrane proteins like the integrins. CD9 is expressed in the epidermal tissue, but this expression is not regulated by differentiation. The present work focuses on association of CD9 with the integrin alpha6beta4 in keratinocytes. In vivo, CD9 does not co-localize with alpha6beta4, and is not internalized with the integrin upon basal detachment with dispase. In vitro, CD9 is found partly in co-localization with alpha6beta4 and is internalized with the integrin after keratinocyte detachment with dispase. Using blocking antibodies in a phagokinetic tracks assay, we show that CD9, and to a lesser extent alpha6beta4, but not the tetraspanin CD82, promote motility of subconfluent keratinocytes on collagen I. Our observations also suggest that CD9 is involved in the formation of lamellipodia. We also report that the phorbol ester TPA has no effect on CD9 expression and association with alpha6beta4, but increases keratinocyte motility, possibly through modulation of integrin subunits expression, or through upregulation of collagenase-1 expression. Together, these results confirm that CD9 associates with alpha6beta4 in cultured keratinocytes, possibly in order to regulate the function of the integrin, and that CD9 is involved in keratinocyte motility on collagen. The data suggest that regulation of adhesion characteristics by CD9 in keratinocytes may play a role in epidermal repair.

Effect of Ruscus extract and hesperidin methylchalcone on hypoxia-induced activation of endothelial cells.

BACKGROUND: Ruscus aculeatus extract and the flavonoid hesperidin methylchalcone (HMC) are drugs used in the treatment of chronic venous insufficiency. METHODS: In the present study, we investigated their effects on the activation of endothelial cells by hypoxia, a condition which mimics venous blood stasis. RESULTS: We observed that Ruscus extract was able to inhibit the activation of endothelial cells by hypoxia: the decrease in ATP content, the activation of phospholipase A2 as well as the subsequent increase in neutrophil adherence with a maximal protection obtained at 50 microg/ml. HMC was also able to inhibit the hypoxia-induced decrease in ATP content. Furthermore, the effects of Ruscus extract and of HMC on this decrease seem to be additive. CONCLUSIONS: The biochemical mechanism evidenced in this work might explain some of the beneficial therapeutic effects of these products in the treatment of chronic venous insufficiency patients.

Differential expression and release of cytokines by an in vitro reconstructed human epidermis following exposure to skin irritant and sensitizing chemicals.

In the present study, a model of reconstructed human epidermis (RHE) was used as an in vitro model to discriminate the effects of Tween 80, Triton X100 and benzalkonium chloride (BC) as irritants and 1-chloro-2,4-dinitrobenzene (DNCB) as sensitizing agent. The extent of epidermal irritation and sensitization was evaluated morphologically and on the basis of intracellular and extracellular levels of interleukin-1alpha (IL-1alpha) and interleukin-8 (IL-8). The corresponding constitutive mRNA levels were quantified and the cytotoxic response was assessed by MTT assay. The RHE resembled normal human epidermis with all typical layers. The keratin 10 (K10) was typically confined in the suprabasal layers of the tissue, suggesting normal epidermal terminal differentiation. Topical application of the three surfactants resulted in significant changes of tissue morphology and was coupled with different dose-dependent decreases in cell viability corresponding to their in vivo irritant potency. The IL-1alpha release increased concomitantly with cell viability decrease, but surprisingly, the surfactants did not elicit elevated IL-8 levels. In contrast, DNCB did not induce elevated IL-1alpha release although it induced a rapid dose-dependent decrease in cell viability. On the contrary, it increased the IL-8 release. RT-PCR demonstrated the presence of mRNA for IL-1alpha as well as for IL-8. IL-1alpha was the most abundant cytokine transcript. BC, Triton X100 and DNCB upregulated IL-8 mRNA expression while only BC induced a significant increase in IL-1alpha mRNA expression. Our results showed that the production of IL-1alpha and its release into the extracellular medium was ascribed not only to direct cytotoxicity but also to the extent of tissue stimulation. Conversely, the production of IL-8 did not directly correlate with cytotoxicity but seems to be linked to the type of product applied either irritant or sensitizer. Our data demonstrate that divergent IL-1alpha and IL-8 release profiles and corresponding mRNA upregulation characterize the response to the tested irritants and DNCB. These results currently need confirmation by the introduction of more numerous known irritants and sensitizers in the tests used in the present study. However, they already suggest that it may be possible in a single assay to classify and to discriminate between irritant and sensitizing agents as a function of induced cytokine production patterns and of cell viability measurements.

Comparison of the protein adsorption selectivity of salt-promoted agarose-based adsorbents. Hydrophobic, thiophilic and electron donor-acceptor adsorbents.

Protein adsorption of human serum onto six different agarose-based chromatographic gels that were representative of the salt-promoted adsorbent family [octyl- and phenyl-Sepharose, mercaptoethanol-divinyl sulfone agarose (T gel), mercaptomethylene pyridine-derivatized agarose gel (MP gel), tricyanoaminopropene-divinyl sulfone agarose (DVS-TCP gel), tricyanoamino-propene-bisoxirane agarose (bisoxirane-TCP gel)] was studied in the presence of moderate or high concentrations of the water structuring salt, sodium sulfate. Study of the protein adsorption selectivity by two-dimensional gel electrophoresis revealed an opposed selectivity for hydrophobic interaction adsorbents and electron donor-acceptor adsorbents. The T gel, MP gel and TCP gels belonged to the electron donor-acceptor adsorbents, displaying a main selectivity for immunoglobulins, whereas octyl-Sepharose belonged to the hydrophobic adsorbents, displaying a main selectivity for 'hydrophobic' proteins. Phenyl-Sepharose for its part was described as an example of a composite selectivity of both families. The conclusion of this work is two-fold: (1) hydrophobic interaction chromatography (HIC) and electron donor-acceptor chromatography (EDAC) have opposed protein selectivities and are both salt-promoted. As a main consequence, it means that high concentrations of a water-structuring salt can promote different types of weak molecular interactions, resulting in different protein adsorption selectivities: (2) thiophilic adsorption chromatography (TAC) should be renamed EDAC as similar protein selectivity is demonstrated for electron donor-acceptor ligand devoid of sulfur atoms.

Effect of Ginkor Fort on hypoxia-induced neutrophil adherence to human saphenous vein endothelium.

This study was performed to evaluate the effects of Ginkor Fort, a venotropic drug composed of Ginkgo biloba extract, troxerutine, and heptaminol, on neutrophil adherence to the endothelium of saphenous veins. When saphenous veins were incubated 2 h in hypoxic conditions, they showed a five- to sixfold increase in neutrophil adherence to the endothelium. Ginkor Fort at 0.3 mg/ml was able to inhibit this increase by 69%. These results were confirmed by observations in scanning electron microscopy. Ginkor Fort also inhibited the subsequent activation of these neutrophils, as evidenced by the inhibition of superoxide anion release. The biochemical mechanism of this inhibition of neutrophil adherence was studied on endothelial cells in culture. We observed that Ginkor Fort was able to inhibit the different steps of the activation of endothelial cells by hypoxia: the activation of phospholipase A2 and the decrease in adenosine triphosphate (ATP) content. By preventing the first step of the activation cascade, the decrease in ATP content, Ginkor Fort blocks the subsequent increase in neutrophil adherence as well as neutrophil activation. The biochemical mechanism evidenced in this work might explain the beneficial effect of this drug in the treatment of patients with chronic venous insufficiency.

Polyol-promoted adsorption of serum proteins to amphiphilic agarose-based adsorbents.

We tested the promotion of protein adsorption onto amphiphilic agarose-based adsorbents by addition of high concentrations of polyols during the adsorption phase. C3- to C5-polyols were inefficient in promoting protein adsorption, whereas some of the C6-polyols studied (sorbitol, dulcitol and mannitol) could promote serum protein adsorption onto mercaptomethylene pyridine-derivatized agarose, octyl- and phenyl-Sepharose. Sorbitol was the most potent protein adsorption promoter, with a direct relation between the amount of protein adsorbed and the concentration of sorbitol. For each chromatographic gel, the effects of increasing concentrations of sorbitol or sodium sulfate on protein adsorption were similar and two-dimensional electrophoresis revealed the preservation of the protein adsorption specificity whether sorbitol or sodium sulfate was used. These results show that a water-structuring salt or a polyol can promote protein adsorption in the same manner, presumably by a related mechanism.

Cosolvent-induced adsorption and desorption of serum proteins on an amphiphilic mercaptomethylene pyridine-derivatized agarose gel.

We studied the effects of the following cosolvents on the adsorption and desorption of serum proteins from an amphiphilic mercaptomethylene pyridine-derivatized agarose gel: glucose, sucrose, polyethylene glycol (PEG), 2-methyl-2,4-pentanediol (MFD), sorbitol, pentaerythritol, glycerol, and Na2SO4. The water-structuring salt 0.4 M Na2SO4 was the most potent promoter of protein adsorption, followed by 5 M sorbitol and, to a lesser extent, 0.2 M PEG 1000 and 2.25 M MPD. The other cosolvents (4 M glucose, 1.5 M sucrose, 0.3 M pentaerythritol, and 7.6 M glycerol) were unable to promote protein adsorption to the gel. Attempts to modulate the salt-promotion effect of Na2SO4 with different cosolvents demonstrated the occurrence of synergistic effects for pentaerythritol, sorbitol, and glucose and antagonistic effects for the other cosolvents. Sorbitol and glycerol were found to be the most interesting co-solvents studied, as the first promoted protein adsorption, whereas the other disrupted protein interaction. As a consequence of these novel findings we propose sorbitol and glycerol, both well-known protein stabilizers, as possible alternatives to water-structuring salts during the adsorption phase and to deleterious organic solvents during the desorption phase on amphiphilic gels.