Calprotectin (S100A8/S100A9) and Myeloperoxidase: Co-Regulators of Formation of Reactive Oxygen Species

Inflammatory mediators trigger polymorphonuclear neutrophils (PMN) to produce reactive oxygen species (ROS: O2-, H2O2, ∙OH). Mediated by myeloperoxidase in PMN, HOCl is formed, detectable in a chemiluminescence (CL) assay. We have shown that the abundant cytosolic PMN protein calprotectin (S100A8/A9) similarly elicits CL in response to H2O2 in a cell-free system. Myeloperoxidase and calprotectin worked synergistically. Calprotectin-induced CL increased, whereas myeloperoxidase-triggered CL decreased with pH > 7.5. Myeloperoxidase needed NaCl for CL, calprotectin did not. 4-hydroxybenzoic acid, binding ∙OH, almost abrogated calprotectin CL, but moderately increased myeloperoxidase activity. The combination of native calprotectin, or recombinant S100A8/A9 proteins, with NaOCl markedly enhanced CL. NaOCl may be the synergistic link between myeloperoxidase and calprotectin. Surprisingly- and unexplained- at higher concentration of S100A9 the stimulation vanished, suggesting a switch from pro-oxidant to anti-oxidant function. We propose that the ∙OH is predominant in ROS production by calprotectin, a function not described before

New approach methods to assess developmental and adult neurotoxicity for regulatory use: a PARC work package 5 project

In the European regulatory context, rodent in vivo studies are the predominant source of neurotoxicity information. Although they form a cornerstone of neurotoxicological assessments, they are costly and the topic of ethical debate. While the public expects chemicals and products to be safe for the developing and mature nervous systems, considerable numbers of chemicals in commerce have not, or only to a limited extent, been assessed for their potential to cause neurotoxicity. As such, there is a societal push toward the replacement of animal models with in vitro or alternative methods. New approach methods (NAMs) can contribute to the regulatory knowledge base, increase chemical safety, and modernize chemical hazard and risk assessment. Provided they reach an acceptable level of regulatory relevance and reliability, NAMs may be considered as replacements for specific in vivo studies. The European Partnership for the Assessment of Risks from Chemicals (PARC) addresses challenges to the development and implementation of NAMs in chemical risk assessment. In collaboration with regulatory agencies, Project 5.2.1e (Neurotoxicity) aims to develop and evaluate NAMs for developmental neurotoxicity (DNT) and adult neurotoxicity (ANT) and to understand the applicability domain of specific NAMs for the detection of endocrine disruption and epigenetic perturbation. To speed up assay time and reduce costs, we identify early indicators of later-onset effects. Ultimately, we will assemble second-generation developmental neurotoxicity and first-generation adult neurotoxicity test batteries, both of which aim to provide regulatory hazard and risk assessors and industry stakeholders with robust, speedy, lower-cost, and informative next-generation hazard and risk assessment tools

Analysis of elimination half-lives in MamTKDB 1.0 related to bioaccumulation: Requirement of repeated administration and blood plasma values underrepresent tissues

When building the novel public mammalian toxicokinetic database (MamTKDB) we collected and included 3927 elimination half-lives (elimt1/2) for 1407 xenobiotics in various species (rat, human, mouse, dog, monkey, rabbit, cattle, pig, sheep, guinea pig, hamster, horse and goat) with specification of compartment (whole body, organ/tissue, cell type, medium) studied. Here we describe and analyse the collected data in MamTKDB 1.0. Most elimt1/2 are for humans and rats and their data differ in some ways: whereas the rat data are mainly for pesticides, the human data are mainly for pharmaceuticals and environmental contaminants. There are also differences in types of compartments studied and in metabolites followed: human elimt1/2 are mainly whole body based (i.e. based on blood plasma or excretion), animal data are additionally for various organs/tissues, cells or media. Contrary to human studies, animal studies regularly administrate radiolabeled (e.g. 14C) substances and distribution of both parent and eventual metabolites are followed, measuring the radioactivity. In rats, substances had been given through single, preconditioning or repeated administration. Single administration studies dominated, but repeated studies generally had longer elimt1/2 than single or preconditioning studies for which elimt1/2 were similar. Repeated administration studies should better ascertain steady state conditions throughout the body, a process involving time-dependent tissue loading, and the data show that for most substances, repeated studies are required to address bioaccumulation potential. About 65% of the substances in MamTKDB 1.0 fulfilled the octanol–water and octanol–air partitioning-based screening criteria (log Kow > 2 and log Koa > 5) for further bioaccumulation assessment and/or testing, and most of the substances with long elimt1/2 in both humans and rats fulfill these criteria. Of note, however, there are also many chemicals with log Kow > 2 with intermediate or short elimt1/2. Per- and polyfluoroalkyl substances (PFAS) stand out in that they often have log Koa < 5. Rats are poor toxicokinetic test models for perfluoroalkyl acids (PFAAs) for which pigs (and possibly mice) elimt1/2 data resemble those of humans better. Perfluorinated carboxylic acids (PFCAs) and perfluorinated sulfonic acids (PFSAs) of similar molecular weight had similar elimt1/2 in the species tested. For polychlorinated biphenyls (PCBs), elimt1/2 increases with the degree of chlorination in humans. In relation to other compartments, blood plasma/serum had among the shortest elimt1/2 in rats and often underrepresent elimt1/2 in tissues. Rat data were divided into 38 compartment (tissue or media) types out of which 20 had sufficient data for correlational tests. In general, there was a strong degree of correlation of rat elimt1/2 in-between most compartments, but there were also exceptions. Surprisingly, the correlation between brain and white fat was relatively weak. Interestingly, several substances or their metabolites bound to haemoglobin in red blood cells. MamTKDB 1.0 allows investigation on how certain chemical characteristics influence elimt1/2 and is a promising database for assessment of bioaccumulation potential

Metal Dyshomeostasis and Inflammation in Alzheimer’s and Parkinson’s Diseases: Possible Impact of Environmental Exposures

A dysregulated metal homeostasis is associated with both Alzheimer’s (AD) and Parkinson’s (PD) diseases; AD patients have decreased cortex and elevated serum copper levels along with extracellular amyloid-beta plaques containing copper, iron, and zinc. For AD, a putative hepcidin-mediated lowering of cortex copper mechanism is suggested. An age-related mild chronic inflammation and/or elevated intracellular iron can trigger hepcidin production followed by its binding to ferroportin which is the only neuronal iron exporter, thereby subjecting it to lysosomal degradation. Subsequently raised neuronal iron levels can induce translation of the ferroportin assisting and copper binding amyloid precursor protein (APP); constitutive APP transmembrane passage lowers the copper pool which is important for many enzymes. Using in silico gene expression analyses, we here show significantly decreased expression of copper-dependent enzymes in AD brain and metallothioneins were upregulated in both diseases. Although few AD exposure risk factors are known, AD-related tauopathies can result from cyanobacterial microcystin and β-methylamino-L-alanine (BMAA) intake. Several environmental exposures may represent risk factors for PD; for this disease neurodegeneration is likely to involve mitochondrial dysfunction, microglial activation, and neuroinflammation. Administration of metal chelators and anti-inflammatory agents could affect disease outcomes

Repeated five-day administration of L-BMAA, microcystin-LR, or as mixture, in adult C57BL/6 mice - lack of adverse cognitive effects

The cyanobacterial toxins β-methylamino-L-alanine (L-BMAA) and microcystin-LR (MC-LR; a potent liver toxin) are suspected to cause neurological disorders. Adult male C57BL/6JOlaHsd mice aged approximately 11 months were subcutaneously injected for five consecutive days with L-BMAA and microcystin-LR alone, or as a mixture. A dose-range study determined a tolerable daily dose to be ~31 µg MC-LR/kg BW/day based on survival, serum liver status enzymes, and relative liver and kidney weight. Mice tolerating the first one-two doses also tolerated the subsequent three-four doses indicating adaptation. The LD50 was 43–50 μg MC-LR/kg BW. Long-term effects (up to 10 weeks) on spatial learning and memory performance was investigated using a Barnes maze, were mice were given 30 µg MC-LR/kg BW and/or 30 mg L-BMAA/kg BW either alone or in mixture for five consecutive days. Anxiety, general locomotor activity, willingness to explore, hippocampal and peri-postrhinal cortex dependent memory was investigated after eight weeks using Open field combined with Novel location/Novel object recognition tests. Toxin exposed animals did not perform worse than controls, and MC-LR exposed animals performed somewhat better during the first Barnes maze re-test session. MC-LR exposed mice rapidly lost up to ~5% body weight, but regained weight from day eight