Personal exposure assessment of pesticides in residents: The association between hand wipes and urinary biomarkers.

BACKGROUND: Residential exposure to pesticides may occur via inhalation of airborne pesticides, direct skin contacts with pesticide-contaminated surfaces, and consumption of food containing pesticide residues. The aim was to study the association of dermal exposure to pesticides between the use and non-use periods, between farmer and non-farmer families and between dermal exposure and the excretion of metabolites from urine in residents living close to treated agricultural fields. METHODS: In total, 112 hand wipes and 206 spot urine samples were collected from 16 farmer and 38 non-farmer participants living within 50 m from an agricultural field in the Netherlands. The study took place from May 2016 to December 2017 during the use as well as the non-use periods of pesticides. Hand wipes were analysed for the parent compound and urines samples for the corresponding urinary metabolite of five applied pesticides: asulam, carbendazim (applied as thiophanate-methyl), chlorpropham, prochloraz and tebuconazole. Questionnaire data was used to study potential determinants of occurrence and levels of pesticides in hand wipes according to univariate and multivariate analysis. RESULTS: Carbendazim and tebuconazole concentrations in hand wipes were statistically significantly higher in the pesticide-use period compared to the non-use period. In addition, especially during the use periods, concentrations were statistically significantly higher in farmer families compared to non-farmer families. For asulam, chlorpropham and prochloraz, the frequency of non-detects was too high (57-85%) to be included in this analysis. The carbendazim contents in urine samples and hand wipes were correlated on the first and second day after taking the hand wipe, whereas chlorpropham was only observed to be related on the second day following the spray event. CONCLUSIONS: Concentrations in hand wipes were overall higher in pesticide use periods compared to non-use periods and higher in farmer families compared to non-farmer families. Only for carbendazim a strong correlation between concentrations in hand wipes and its main metabolite in urine was observed, indicating dermal exposure via contaminated indoor surfaces. We expect this to be related to the lower vapour pressure and longer environmental lifetime of carbendazim compared to the other pesticides studies.

Interaction of tetanus toxin derived hybrid proteins with neuronal cells.

The non-toxic ganglioside binding domain of tetanus toxin (Hc fragment C or TTC) has been studied as a vector for delivering therapeutic proteins to neurons. There is little information on the cellular processing of proteins delivered by linkage to TTC. We have evaluated the cellular handling of a multi-domain hybrid protein containing TTC and both the human enzyme superoxide dismutase and the maltose binding protein from E. coli. Binding, internalization, and cleavage of this protein during prolonged incubation with fetal cortical neurons or cells of the N18-RE-105 line was evaluated by immunoblot analysis, ELISA, and immunocytochemistry. Hybrid proteins were bound and internalized in a manner very similar to TTC. Internalized proteins showed long-term stability within cells, and were degraded into predictable large protein fragments in both cell types. Fragments that were cleaved away from the TTC domain were released into extracellular fluid after internalization. Proteins coupled to TTC share its long-term stability after cellular internalization. After internalization, dissociation of proteins linked to TTC facilitates their release from the cell, but not into other cellular compartments such as the cytosol. TTC linked proteins are probably enclosed within a stable endosomal compartment throughout their cellular lifetime.

Protective effect of supplemental superoxide dismutase on survival of neuronal cells during starvation. Requirement for cytosolic distribution.

There is evidence that raising cellular levels of Cu2+/Zn2+ superoxide dismutase (SOD1) can protect neurons from oxidative injury. We compared a novel method of elevating neuronal SOD activity using a recombinant hybrid protein composed of the atoxic neuronal binding domain of tetanus toxin (C fragment or TTC) and human SOD1 (hSOD1) with increasing cellular SOD levels through overexpression. Fetal murine cortical neurons or N18-RE-105 cells were incubated with the TTC-hSOD1 hybrid protein and compared to cells constitutively expressing hSOD1 for level of SOD activity, cellular localization of hSOD1, and capacity to survive glucose and pyruvate starvation. Cells incubated with TTC-hSOD1 showed a threefold increase in cellular SOD activity over control cells. This level of increase was comparable to fetal cortical neurons from transgenic mice constitutively expressing hSOD1 and transfected N18-RE-105 cells expressing a green fluorescent protein-hSOD1 fusion protein (GFP-hSOD1). Human SOD1 was distributed diffusely throughout the cytoplasm of the transgenic murine neurons and transfected N18-RE-105 cells. In contrast, cells incubated with TTC-hSOD1 showed hSOD1 localized to the cell surface and intra-cytoplasmic vesicles. The cells expressing hSOD1 showed enhanced survival in glucose- and pyruvate-free medium. Neither cortical neurons nor N18-RE-105 cells incubated in TTC-hSOD1 showed increased survival during starvation. Access to the site where toxic superoxides are generated or their targets may be necessary for the protective function of SOD1.

Human colostrum and serum contain antibodies reactive to the intimin-binding region of the enteropathogenic Escherichia coli translocated intimin receptor.

BACKGROUND: In Brazil, enteropathogenic Escherichia coli (EPEC) diarrhoea is endemic in young infants. A characteristic feature of EPEC adhesion to host cells is intimate attachment leading to the formation of distinctive "attaching and effacing" (A/E) lesions on mammalian cells. Two genes directly involved in intimate adhesion, eae and tir, encode the adhesion molecule intimin and its translocated receptor Tir, respectively. The intimin-binding domain of Tir was recently mapped to the middle part of the polypeptide (Tir-M), and the amino (Tir-N) and carboxy (Tir-C) termini were found to be located within infected host cells. Recently, it was shown that colostrum samples from mothers living in Sao Paulo contain IgA-class antibodies reactive with a number of proteins associated with EPEC virulence. It has also been shown that patients infected with verocytotoxin-producing E. coli O157 can produce antibodies to Tir. In the current study antibody responses to the different Tir domains were analyzed in sera and colostrum samples collected in an EPEC-endemic area of Brazil. METHODS: Recombinant Tir, Tir-N, Tir-M, and Tir-C were expressed as His-tagged protein in E. coli BL21a and purified on nickel columns. Western blot analysis was used to investigate colostrum IgA- and serum IgG-class antibodies reactive with the Tir fragments. RESULTS: Anti-Tir IgG antibodies were detected in the serum of children, with (63%) or without (50%) diarrhoea. Anti-Tir IgA-class antibodies were detected in all the colostrum pools tested. With the use of both serum IgG- and colostrum IgA-class antibodies, an immunodominant domain of the Tir-polypeptide, Tir M, was identified. CONCLUSION: The intimin-binding region of Tir (Tir-M) is the immunodominant region of the polypeptide in humans. Both serum IgG-class and colostrum IgA-class antibodies reacted predominantly with the Tir-M domain.

Delivery of recombinant tetanus-superoxide dismutase proteins to central nervous system neurons by retrograde axonal transport.

The nontoxic C fragment of tetanus toxin (TC) can transport other proteins from the circulation to central nervous system (CNS) motor neurons. Increased levels of CuZn superoxide dismutase (SOD) are protective in experimental models of stroke and Parkinson's disease, whereas mutations in SOD can cause motor neuron disease. We have linked TC to SOD and purified the active recombinant proteins in both the TC-SOD and SOD-TC orientations. Light microscopic immunohistochemistry and quantitative enzyme-linked immunosorbant assays (ELISA) of mouse brainstem, after intramuscular injection, demonstrate that the fusion proteins undergo retrograde axonal transport and transsynaptic transfer as efficiently as TC alone.