Basic concepts of mixture toxicity and relevance for risk evaluation and regulation.

Exposure to multiple substances is a challenge for risk evaluation. Currently, there is an ongoing debate if generic "mixture assessment/allocation factors" (MAF) should be introduced to increase public health protection. Here, we explore concepts of mixture toxicity and the potential influence of mixture regulation concepts for human health protection. Based on this analysis, we provide recommendations for research and risk assessment. One of the concepts of mixture toxicity is additivity. Substances may act additively by affecting the same molecular mechanism within a common target cell, for example, dioxin-like substances. In a second concept, an "enhancer substance" may act by increasing the target site concentration and aggravating the adverse effect of a "driver substance". For both concepts, adequate risk management of individual substances can reliably prevent adverse effects to humans. Furthermore, we discuss the hypothesis that the large number of substances to which humans are exposed at very low and individually safe doses may interact to cause adverse effects. This commentary identifies knowledge gaps, such as the lack of a comprehensive overview of substances regulated under different silos, including food, environmentally and occupationally relevant substances, the absence of reliable human exposure data and the missing accessibility of ratios of current human exposure to threshold values, which are considered safe for individual substances. Moreover, a comprehensive overview of the molecular mechanisms and most susceptible target cells is required. We conclude that, currently, there is no scientific evidence supporting the need for a generic MAF. Rather, we recommend taking more specific measures, which focus on compounds with relatively small ratios between human exposure and doses, at which adverse effects can be expected.

New approach methodologies in human regulatory toxicology - Not if, but how and when!

The predominantly animal-centric approach of chemical safety assessment has increasingly come under pressure. Society is questioning overall performance, sustainability, continued relevance for human health risk assessment and ethics of this system, demanding a change of paradigm. At the same time, the scientific toolbox used for risk assessment is continuously enriched by the development of "New Approach Methodologies" (NAMs). While this term does not define the age or the state of readiness of the innovation, it covers a wide range of methods, including quantitative structure-activity relationship (QSAR) predictions, high-throughput screening (HTS) bioassays, omics applications, cell cultures, organoids, microphysiological systems (MPS), machine learning models and artificial intelligence (AI). In addition to promising faster and more efficient toxicity testing, NAMs have the potential to fundamentally transform today's regulatory work by allowing more human-relevant decision-making in terms of both hazard and exposure assessment. Yet, several obstacles hamper a broader application of NAMs in current regulatory risk assessment. Constraints in addressing repeated-dose toxicity, with particular reference to the chronic toxicity, and hesitance from relevant stakeholders, are major challenges for the implementation of NAMs in a broader context. Moreover, issues regarding predictivity, reproducibility and quantification need to be addressed and regulatory and legislative frameworks need to be adapted to NAMs. The conceptual perspective presented here has its focus on hazard assessment and is grounded on the main findings and conclusions from a symposium and workshop held in Berlin in November 2021. It intends to provide further insights into how NAMs can be gradually integrated into chemical risk assessment aimed at protection of human health, until eventually the current paradigm is replaced by an animal-free "Next Generation Risk Assessment" (NGRA).

Genotoxicity assessment: opportunities, challenges and perspectives for quantitative evaluations of dose-response data.

Genotoxicity data are mainly interpreted in a qualitative way, which typically results in a binary classification of chemical entities. For more than a decade, there has been a discussion about the need for a paradigm shift in this regard. Here, we review current opportunities, challenges and perspectives for a more quantitative approach to genotoxicity assessment. Currently discussed opportunities mainly include the determination of a reference point (e.g., a benchmark dose) from genetic toxicity dose-response data, followed by calculation of a margin of exposure (MOE) or derivation of a health-based guidance value (HBGV). In addition to new opportunities, major challenges emerge with the quantitative interpretation of genotoxicity data. These are mainly rooted in the limited capability of standard in vivo genotoxicity testing methods to detect different types of genetic damage in multiple target tissues and the unknown quantitative relationships between measurable genotoxic effects and the probability of experiencing an adverse health outcome. In addition, with respect to DNA-reactive mutagens, the question arises whether the widely accepted assumption of a non-threshold dose-response relationship is at all compatible with the derivation of a HBGV. Therefore, at present, any quantitative genotoxicity assessment approach remains to be evaluated case-by-case. The quantitative interpretation of in vivo genotoxicity data for prioritization purposes, e.g., in connection with the MOE approach, could be seen as a promising opportunity for routine application. However, additional research is needed to assess whether it is possible to define a genotoxicity-derived MOE that can be considered indicative of a low level of concern. To further advance quantitative genotoxicity assessment, priority should be given to the development of new experimental methods to provide a deeper mechanistic understanding and a more comprehensive basis for the analysis of dose-response relationships.

Mixture Risk Assessment of Complex Real-Life Mixtures-The PANORAMIX Project.

Humans are involuntarily exposed to hundreds of chemicals that either contaminate our environment and food or are added intentionally to our daily products. These complex mixtures of chemicals may pose a risk to human health. One of the goals of the European Union's Green Deal and zero-pollution ambition for a toxic-free environment is to tackle the existent gaps in chemical mixture risk assessment by providing scientific grounds that support the implementation of adequate regulatory measures within the EU. We suggest dealing with this challenge by: (1) characterising 'real-life' chemical mixtures and determining to what extent they are transferred from the environment to humans via food and water, and from the mother to the foetus; (2) establishing a high-throughput whole-mixture-based in vitro strategy for screening of real-life complex mixtures of organic chemicals extracted from humans using integrated chemical profiling (suspect screening) together with effect-directed analysis; (3) evaluating which human blood levels of chemical mixtures might be of concern for children's development; and (4) developing a web-based, ready-to-use interface that integrates hazard and exposure data to enable component-based mixture risk estimation. These concepts form the basis of the Green Deal project PANORAMIX, whose ultimate goal is to progress mixture risk assessment of chemicals.

Muscle-Specific Lipid Hydrolysis Prolongs Lifespan through Global Lipidomic Remodeling.

A growing body of evidence suggests that changes in fat metabolism may have a significant effect on lifespan. Accumulation of lipid deposits in non-adipose tissue appears to be critical for age-related pathologies and may also contribute to the aging process itself. We established a model of lipid storage in muscle cells of C. elegans to reveal a mechanism that promotes longevity non-cell-autonomously. Here, we describe how muscle-specific activation of adipose triglyceride lipase (ATGL) and the phospholipase A2 (PLA2) ortholog IPLA-7 collectively affect inter-tissular communication and systemic adaptation that requires the activity of AMP-dependent protein kinase (AMPK) and a highly conserved nuclear receptor outside of the muscle. Our data suggest that muscle-specific bioactive lipid signals, or "lipokines," are generated following triglyceride breakdown and that these signals impinge on a complex network of genes that modify the global lipidome, consequently extending the lifespan.

Branched-chain amino acid catabolism is a conserved regulator of physiological ageing.

Ageing has been defined as a global decline in physiological function depending on both environmental and genetic factors. Here we identify gene transcripts that are similarly regulated during physiological ageing in nematodes, zebrafish and mice. We observe the strongest extension of lifespan when impairing expression of the branched-chain amino acid transferase-1 (bcat-1) gene in C. elegans, which leads to excessive levels of branched-chain amino acids (BCAAs). We further show that BCAAs reduce a LET-363/mTOR-dependent neuro-endocrine signal, which we identify as DAF-7/TGFß, and that impacts lifespan depending on its related receptors, DAF-1 and DAF-4, as well as ultimately on DAF-16/FoxO and HSF-1 in a cell-non-autonomous manner. The transcription factor HLH-15 controls and epistatically synergizes with BCAT-1 to modulate physiological ageing. Lastly and consistent with previous findings in rodents, nutritional supplementation of BCAAs extends nematodal lifespan. Taken together, BCAAs act as periphery-derived metabokines that induce a central neuro-endocrine response, culminating in extended healthspan.

Neuronal ROS signaling rather than AMPK/sirtuin-mediated energy sensing links dietary restriction to lifespan extension.

Dietary restriction (DR) extends lifespan and promotes metabolic health in evolutionary distinct species. DR is widely believed to promote longevity by causing an energy deficit leading to increased mitochondrial respiration. We here show that inhibitors of mitochondrial complex I promote physical activity, stress resistance as well as lifespan of Caenorhabditis elegans despite normal food uptake, i.e. in the absence of DR. However, complex I inhibition does not further extend lifespan in dietarily restricted nematodes, indicating that impaired complex I activity mimics DR. Promotion of longevity due to complex I inhibition occurs independently of known energy sensors, including DAF-16/FoxO, as well as AAK-2/AMPK and SIR-2.1/sirtuins, or both. Consistent with the concept of mitohormesis, complex I inhibition transiently increases mitochondrial formation of reactive oxygen species (ROS) that activate PMK-1/p38 MAP kinase and SKN-1/NRF-2. Interference with this retrograde redox signal as well as ablation of two redox-sensitive neurons in the head of the worm similarly prevents extension of lifespan. These findings unexpectedly indicate that DR extends organismal lifespan through transient neuronal ROS signaling rather than sensing of energy depletion, providing unexpected pharmacological options to promote exercise capacity and healthspan despite unaltered eating habits.

Role of sirtuins in lifespan regulation is linked to methylation of nicotinamide.

Sirtuins, a family of histone deacetylases, have a fiercely debated role in regulating lifespan. In contrast with recent observations, here we find that overexpression of sir-2.1, the ortholog of mammalian SirT1, does extend Caenorhabditis elegans lifespan. Sirtuins mandatorily convert NAD(+) into nicotinamide (NAM). We here find that NAM and its metabolite, 1-methylnicotinamide (MNA), extend C. elegans lifespan, even in the absence of sir-2.1. We identify a previously unknown C. elegans nicotinamide-N-methyltransferase, encoded by a gene now named anmt-1, to generate MNA from NAM. Disruption and overexpression of anmt-1 have opposing effects on lifespan independent of sirtuins, with loss of anmt-1 fully inhibiting sir-2.1-mediated lifespan extension. MNA serves as a substrate for a newly identified aldehyde oxidase, GAD-3, to generate hydrogen peroxide, which acts as a mitohormetic reactive oxygen species signal to promote C. elegans longevity. Taken together, sirtuin-mediated lifespan extension depends on methylation of NAM, providing an unexpected mechanistic role for sirtuins beyond histone deacetylation.

Mitochondrial hormesis links low-dose arsenite exposure to lifespan extension.

Arsenite is one of the most toxic chemical substances known and is assumed to exert detrimental effects on viability even at lowest concentrations. By contrast and unlike higher concentrations, we here find that exposure to low-dose arsenite promotes growth of cultured mammalian cells. In the nematode C. elegans, low-dose arsenite promotes resistance against thermal and chemical stressors and extends lifespan of this metazoan, whereas higher concentrations reduce longevity. While arsenite causes a transient increase in reactive oxygen species (ROS) levels in C. elegans, co-exposure to ROS scavengers prevents the lifespan-extending capabilities of arsenite, indicating that transiently increased ROS levels act as transducers of arsenite effects on lifespan, a process known as mitohormesis. This requires two transcription factors, namely DAF-16 and SKN-1, which employ the metallothionein MTL-2 as well as the mitochondrial transporter TIN-9.1 to extend lifespan. Taken together, low-dose arsenite extends lifespan, providing evidence for nonlinear dose-response characteristics of toxin-mediated stress resistance and longevity in a multicellular organism.

Impaired insulin/IGF1 signaling extends life span by promoting mitochondrial L-proline catabolism to induce a transient ROS signal.

Impaired insulin and IGF-1 signaling (iIIS) in C. elegans daf-2 mutants extends life span more than 2-fold. Constitutively, iIIS increases mitochondrial activity and reduces reactive oxygen species (ROS) levels. By contrast, acute impairment of daf-2 in adult C. elegans reduces glucose uptake and transiently increases ROS. Consistent with the concept of mitohormesis, this ROS signal causes an adaptive response by inducing ROS defense enzymes (SOD, catalase), culminating in ultimately reduced ROS levels despite increased mitochondrial activity. Inhibition of this ROS signal by antioxidants reduces iIIS-mediated longevity by up to 60%. Induction of the ROS signal requires AAK-2 (AMPK), while PMK-1 (p38) and SKN-1 (NRF-2) are needed for the retrograde response. IIIS upregulates mitochondrial L-proline catabolism, and impairment of the latter impairs the life span-extending capacity of iIIS while L-proline supplementation extends C. elegans life span. Taken together, iIIS promotes L-proline metabolism to generate a ROS signal for the adaptive induction of endogenous stress defense to extend life span.

Mitochondrial DNA copy number and function decrease with age in the short-lived fish Nothobranchius furzeri.

Among vertebrates that can be kept in captivity, the annual fish Nothobranchius furzeri possesses the shortest known lifespan. It also shows typical signs of aging and is therefore an ideal model to assess the role of different physiological and environmental parameters on aging and lifespan determination. Here, we used Nothobranchius furzeri to study whether aging is associated with mitochondrial DNA (mtDNA) alterations and changes in mitochondrial function. We sequenced the complete mitochondrial genome of N. furzeri and found an extended control region. Large-scale mtDNA deletions have been frequently described to accumulate in other organisms with age, but there was no evidence for the presence of detectable age-related mtDNA deletions in N. furzeri. However, mtDNA copy number significantly decreased with age in skeletal muscle, brain, liver, skin and dorsal fin. Consistent with this finding, expression of Pgc-1α that encodes a transcriptional coactivator of mitochondrial biogenesis and expression of Tfam and mtSsbp both encoding mtDNA binding factors was downregulated with age. The investigation of possible changes in mitochondrial function revealed that the content of respiratory chain complexes III and IV was reduced in skeletal muscle with age. In addition, ADP-stimulated and succinate-dependent respiration was decreased in mitochondria of old fish. These findings suggest that despite the short lifespan, aging in N. furzeri is associated with a decline in mtDNA copy number, the downregulation of mtDNA-associated genes and an impairment of mitochondrial function.

Extending life span by increasing oxidative stress.

Various nutritional, behavioral, and pharmacological interventions have been previously shown to extend life span in diverse model organisms, including Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, mice, and rats, as well as possibly monkeys and humans. This review aims to summarize published evidence that several longevity-promoting interventions may converge by causing an activation of mitochondrial oxygen consumption to promote increased formation of reactive oxygen species (ROS). These serve as molecular signals to exert downstream effects to ultimately induce endogenous defense mechanisms culminating in increased stress resistance and longevity, an adaptive response more specifically named mitochondrial hormesis or mitohormesis. Consistently, we here summarize findings that antioxidant supplements that prevent these ROS signals interfere with the health-promoting and life-span-extending capabilities of calorie restriction and physical exercise. Taken together and consistent with ample published evidence, the findings summarized here question Harman's Free Radical Theory of Aging and rather suggest that ROS act as essential signaling molecules to promote metabolic health and longevity.