Brain region-specific expression of proteolytic enzymes can control the biological activity of endogenous neuropeptides and has recently been targeted for the development of novel drugs, for neuropathic pain, cancer, and Parkinson's disease. Rapid and sensitive analytical methods to profile modulators of enzymatic activity are important for finding effective inhibitors with high therapeutic value. Combination of in situ enzyme histochemistry with MALDI imaging mass spectrometry allowed developing a highly sensitive method for analysis of brain-area specific neuropeptide conversion of synthetic and endogenous neuropeptides, and for selection of peptidase inhibitors that differentially target conversion enzymes at specific anatomical sites. Conversion and degradation products of Dynorphin B as model neuropeptide and effects of peptidase inhibitors applied to native brain tissue sections were analyzed at different brain locations. Synthetic dynorphin B (2pmol) was found to be converted to the N-terminal fragments on brain sections whereas fewer C-terminal fragments were detected. N-ethylmaleimide (NEM), a non-selective inhibitor of cysteine peptidases, almost completely blocked the conversion of dynorphin B to dynorphin B(1-6; Leu-Enk-Arg), (1-9), (2-13), and (7-13). Proteinase inhibitor cocktail, and also incubation with acetic acid displayed similar results. Bioconversion of synthetic dynorphin B was region-specific producing dynorphin B(1-7) in the cortex and dynorphin B (2-13) in the striatum. Enzyme inhibitors showed region- and enzyme-specific inhibition of dynorphin bioconversion. Both phosphoramidon (inhibitor of the known dynorphin converting enzyme neprilysin) and opiorphin (inhibitor of neprilysin and aminopeptidase N) blocked cortical bioconversion to dynorphin B(1-7), wheras only opiorphin blocked striatal bioconversion to dynorphin B(2-13). This method may impact the development of novel therapies with aim to strengthen the effects of endogenous neuropeptides under pathological conditions such as chronic pain. Combining histochemistry and MALDI imaging MS is a powerful and sensitive tool for the study of inhibition of enzyme activity directly in native tissue sections.

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Even if the two neonicotinoids nitenpyram and imidacloprid have been considered safe for humans, their potential genotoxicity still remains a matter of discussion. The DNA-damaging effects of these two compounds were therefore evaluated in a lymphoma cell line of human origin (U-937) using the comet assay after 3-h exposure to up to 50 μM, with or without metabolic activation using S9 from human liver. The comet data were analysed using a traditional one-way ANOVA after pooling the data on cellular level, and a new alternative approach we have called Uppsala Comet Data Analysis Strategy (UCDAS). UCDAS is a proportional odds model tailored to continuous outcomes, taking the number of pooled cultures, slides and cells into consideration in the same analysis. To the best of our knowledge, the UCDAS approach when analysing comet data has never been presented before. Without metabolic activation, no increase in DNA damage was observed in the neonicotinoide-exposed cells. Nitenpyram was also without DNA-damaging effects when S9 was added. However, in the presence of S9, imidacloprid was found to increase the level of DNA damage. Whereas the ANOVA showed an increase (P < 0.001) both at 5 and 50 μM, UCDAS showed an increase only at the lowest concentration (P < 0.001). Based on these findings, the two neonicotinoids seem to be of little concern when it comes to their potential genotoxicity. However, since the U-937 cells were rather resistant to our positive controls, they may not be the best cells to use when evaluating potential genotoxicity of chemicals.

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