A prospective whole-mixture approach to assess risk of the food and chemical exposome.

Many widely used chemicals result in ubiquitous human exposure from multiple sources, including diet. Legislation mainly deals with the toxicological evaluation of single substances owing to a methodological and conceptual lack of alternatives, and does so within defined silos subject to over 40 distinct regulations in the EU alone. Furthermore, much of the research and many of the initiatives concerned with the assessment and evaluation of chemical mixtures and their potential effects on human health rely on retrospective analysis. Here we propose an approach for the prospective identification, assessment and regulation of mixtures relevant to human health. We address two distinct aspects of toxicology-which chemicals actually do occur together, and how potential mixture-related health hazards can be predicted-with an adapted concept of the exposome and large-scale hazard screens. The proactive use of the likelihood of co-exposure, together with the new approach of methods-based testing, may be a timely and feasible way of identifying those substances and mixtures where hazards may have been overlooked and regulatory action is needed. Ideally, we would generate co-exposure patterns for specific consumer groups, depending on lifestyle and dietary habits, to assess the specific risk of identified mixtures.

Workshop on the validation and regulatory acceptance of innovative 3R approaches in regulatory toxicology - Evolution versus revolution.

At a joint workshop organized by RIVM and BfR, international experts from governmental institutes, regulatory agencies, industry, academia and animal welfare organizations discussed and provided recommendations for the development, validation and implementation of innovative 3R approaches in regulatory toxicology. In particular, an evolutionary improvement of our current approach of test method validation in the context of defined approaches or integrated testing strategies was discussed together with a revolutionary approach based on a comprehensive description of the physiological responses of the human body to chemical exposure and the subsequent definition of relevant and predictive in vitro, in chemico or in silico methods. A more comprehensive evaluation of biological relevance, scientific validity and regulatory purpose of new test methods and assessment strategies together with case studies that provide practical experience with new approaches were discussed as essential steps to build up the necessary confidence to facilitate regulatory acceptance.

Workshop on acceleration of the validation and regulatory acceptance of alternative methods and implementation of testing strategies.

This report describes the proceedings of the BfR-RIVM workshop on validation of alternative methods which was held 23 and 24 March 2017 in Berlin, Germany. Stakeholders from governmental agencies, regulatory authorities, universities, industry and the OECD were invited to discuss current problems concerning the regulatory acceptance and implementation of alternative test methods and testing strategies, with the aim to develop feasible solutions. Classical validation of alternative methods usually involves one to one comparison with the gold standard animal study. This approach suffers from the reductionist nature of an alternative test as compared to the animal study as well as from the animal study being considered as the gold standard. Modern approaches combine individual alternatives into testing strategies, for which integrated and defined approaches are emerging at OECD. Furthermore, progress in mechanistic toxicology, e.g. through the adverse outcome pathway approach, and in computational systems toxicology allows integration of alternative test battery results into toxicity predictions that are more fine-tuned to the human situation. The road towards transition to a mechanistically-based human-focused hazard and risk assessment of chemicals requires an open mind towards stepping away from the animal study as the gold standard and defining human biologically based regulatory requirements for human hazard and risk assessment.

Non-standard structures of the Ni-Fe cofactor in the regulatory and the NAD-reducing hydrogenases from Ralstonia eutropha.

Spectroscopy on two oxygen-insensitive Ni-Fe hydrogenases from Ralstonia eutropha (NAD-reducing, soluble hydrogenase; hydrogen sensor, regulatory hydrogenase) reveals non-standard catalytic behaviour and unique structures of their Ni-Fe cofactors. Possible mechanistic implications are briefly discussed.

A hydrogen-sensing multiprotein complex controls aerobic hydrogen metabolism in Ralstonia eutropha.

H(2) is an attractive energy source for many microorganisms and is mostly consumed before it enters oxic habitats. Thus aerobic H(2)-oxidizing organisms receive H(2) only occasionally and in limited amounts. Metabolic adaptation requires a robust oxygen-tolerant hydrogenase enzyme system and special regulatory devices that enable the organism to respond rapidly to a changing supply of H(2). The proteobacterium Ralstonia eutropha strain H16 that harbours three [NiFe] hydrogenases perfectly meets these demands. The unusual biochemical and structural properties of the hydrogenases are described, including the strategies that confer O(2) tolerance to the NAD-reducing soluble hydrogenase and the H(2)-sensing regulatory hydrogenase. The regulatory hydrogenase that forms a complex with a histidine protein kinase recognizes H(2) in the environment and transmits the signal to a response regulator, which in turn controls transcription of the hydrogenase genes.

Involvement of an unusual mol operon in molybdopterin cofactor biosynthesis in Ralstonia eutropha.

In contrast to its parent strain, transposon Tn5-Mob insertion mutant HB6 of the facultative chemoautotroph Ralstonia eutropha was unable to grow organoautotrophically on formate and exhibited no activity of Mo-dependent, membrane-bound formate dehydrogenase (M-FDH) when cultivated mixotrophically on fructose plus formate. The activity of another molybdoenzyme, the soluble, NAD+-linked formate dehydrogenase which is the key enzyme of formate utilization in R. eutropha, was greatly diminished in the mutant. HB6 also lacked the W-dependent M-FDH activities that were newly discovered in organoautotrophically, lithoautotrophically, or mixotrophically grown wildtype cells. However, an additional W-dependent M-FDH activity, observed in heterotrophically grown stationary-phase cells, was present in the mutant although at a considerably reduced level. Sequence analyses of the complementing chromosomal wildtype and the corresponding mutant DNA fragment revealed the transposon insertion to be located in moeA, a gene involved in the biosynthesis of the molybdopterin cofactor (MoCo). Nevertheless, mutant HB6 was able to grow on xanthine as carbon and energy source and with nitrate as nitrogen source. The utilization of these substrates requires the function of the MoCo-containing enzymes xanthine dehydrogenase and assimilatory nitrate reductase, respectively, that were still active in the mutant. A moeA deletion mutant exhibited the same phenotype as that of HB6. The moeA gene belongs to an unusual mol operon consisting of four genes (moeA, moaD, moaE, and moaF) and being constitutively expressed at low level. Unlike MoeA, the large subunit of molybdopterin synthase encoded by moaE is essential for molybdopterin biosynthesis as was evident by the phenotype of a moaE deletion mutant. MoaF is a novel gene product which showed no similarity to proteins with known function but was indispensable for reconstituting organoautotrophic growth in HB6. The findings suggest that MoeA of R. eutropha is differentially involved in the biosynthesis or incorporation of pterin cofactors of/into the various molybdo- and tungstoenzymes.

The open conformation of a Pseudomonas lipase.

BACKGROUND: . The interfacial activation of lipases results primarily from conformational changes in the enzymes which expose the active site and provide a hydrophobic surface for interaction with the lipid substrate. Comparison of the crystallization conditions used and the structures observed for a variety of lipases suggests that the enzyme conformation is dependent on solution conditions. Pseudomonas cepacia lipase (PCL) was crystallized in conditions from which the open, active conformation of the enzyme was expected. Its three-dimensional structure was determined independently in three different laboratories and was compared with the previously reported closed conformations of the closely related lipases from Pseudomonas glumae (PGL) and Chromobacterium viscosum (CVL). These structures provide new insights into the function of this commercially important family of lipases. RESULTS: . The three independent structures of PCL superimpose with only small differences in the mainchain conformations. As expected, the observed conformation reveals a catalytic site exposed to the solvent. Superposition of PCL with the PGL and CVL structures indicates that the rearrangement from the closed to the open conformation involves three loops. The largest movement involves a 40 residue stretch, within which a helical segment moves to afford access to the catalytic site. A hydrophobic cleft that is presumed to be the lipid binding site is formed around the active site. CONCLUSIONS: . The interfacial activation of Pseudomonas lipases involves conformational rearrangements of surface loops and appears to conform to models of activation deduced from the structures of fungal and mammalian lipases. Factors controlling the conformational rearrangement are not understood, but a comparison of crystallization conditions and observed conformation suggests that the conformation of the protein is determined by the solution conditions, perhaps by the dielectric constant.