Evolutionarily conserved transcription factors as regulators of longevity and targets for geroprotection.

Aging is the single largest risk factor for many debilitating conditions, including heart diseases, stroke, cancer, diabetes, and neurodegenerative disorders. Although far from understood in its full complexity, it is scientifically well established that aging is influenced by genetic and environmental factors and can be modulated by various interventions. One of aging's early hallmarks is aberrations in transcriptional networks, controlling for example metabolic homeostasis or the response to stress. Evidence in different model organisms abounds that a number of evolutionarily conserved transcription factors, which control such networks, can affect life span and health span across species. These transcription factors thus potentially represent conserved regulators of longevity and are emerging as important targets in the challenging quest to develop treatments to mitigate age-related diseases, and possibly even to slow aging itself. This review provides an overview of evolutionarily conserved transcription factors that impact longevity or age-related diseases in at least one multicellular model organism (nematodes, flies, or mice) and/or are tentatively linked to human aging. Discussed is the general evidence for transcriptional regulation of aging and disease, followed by a more detailed look at selected transcription factor families, the common metabolic pathways involved, and the targeting of transcription factors as a strategy for geroprotective interventions.

The ion channel CALHM6 controls bacterial infection-induced cellular cross-talk at the immunological synapse.

Membrane ion channels of the calcium homeostasis modulator (CALHM) family promote cell-cell crosstalk at neuronal synapses via ATP release, where ATP acts as a neurotransmitter. CALHM6, the only CALHM highly expressed in immune cells, has been linked to the induction of natural killer (NK) cell anti-tumour activity. However, its mechanism of action and broader functions in the immune system remain unclear. Here, we generated Calhm6-/- mice and report that CALHM6 is important for the regulation of the early innate control of Listeria monocytogenes infection in vivo. We find that CALHM6 is upregulated in macrophages by pathogen-derived signals and that it relocates from the intracellular compartment to the macrophage-NK cell synapse, facilitating ATP release and controlling the kinetics of NK cell activation. Anti-inflammatory cytokines terminate CALHM6 expression. CALHM6 forms an ion channel when expressed in the plasma membrane of Xenopus oocytes, where channel opening is controlled by a conserved acidic residue, E119. In mammalian cells, CALHM6 is localised to intracellular compartments. Our results contribute to the understanding of neurotransmitter-like signal exchange between immune cells that fine-tunes the timing of innate immune responses.

Tall Bornean forests experience higher canopy disturbance rates than those in the eastern Amazon or Guiana shield.

The future of tropical forests hinges on the balance between disturbance rates, which are expected to increase with climate change, and tree growth. Whereas tree growth is a slow process, disturbance events occur sporadically and tend to be short-lived. This difference challenges forest monitoring to achieve sufficient resolution to capture tree growth, while covering the necessary scale to characterize disturbance rates. Airborne LiDAR time series can address this challenge by measuring landscape scale changes in canopy height at 1 m resolution. In this study, we present a robust framework for analysing disturbance and recovery processes in LiDAR time series data. We apply this framework to 8000 ha of old-growth tropical forests over a 4-5-year time frame, comparing growth and disturbance rates between Borneo, the eastern Amazon and the Guiana shield. Our findings reveal that disturbance was balanced by growth in eastern Amazonia and the Guiana shield, resulting in a relatively stable mean canopy height. In contrast, tall Bornean forests experienced a decrease in canopy height due to numerous small-scale (<0.1 ha) disturbance events outweighing the gains due to growth. Within sites, we found that disturbance rates were weakly related to topography, but significantly increased with maximum canopy height. This could be because taller trees were particularly vulnerable to disturbance agents such as drought, wind and lightning. Consequently, we anticipate that tall forests, which contain substantial carbon stocks, will be disproportionately affected by the increasing severity of extreme weather events driven by climate change.

Occurrence of Major Perfluorinated Alkylate Substances in Human Blood and Target Organs.

Human exposure to perfluorinated alkylate substances (PFASs) is usually assessed from the concentrations in serum or plasma, assuming one-compartment toxicokinetics. To characterize body distributions of major PFASs, we obtained and extracted tissue samples from 19 forensic autopsies of healthy adult subjects who had died suddenly and were not known to have elevated levels of PFAS exposure. As target organs of toxicological importance, we selected the liver, kidneys, lungs, spleen, and brain, as well as whole blood. Samples weighing about 0.1 g were analyzed by liquid chromatography coupled to triple mass spectrometers. Minor variations in PFAS concentrations were found between the kidney cortex and medulla and between lung lobes. Organ concentrations of perfluorooctanoic sulfonate (PFOS) and perfluorononanoate (PFNA) correlated well with blood concentrations, while perfluorooctanoate (PFOA) and perfluorohexanoic sulfonate (PFHxS) showed more variable associations. Likewise, the liver concentrations correlated well with those of other organs. Calculations of relative distributions were carried out to assess the interdependence of organ retentions. Equilibrium model predictions largely explained the observed PFAS distributions, except for the brain. Although the samples were small and affected by a possible lack of homogeneity, these findings support the use of blood-PFAS concentrations as a measure of PFAS exposure, with the liver possibly acting as the main organ of retention.

Binding of Per- and Polyfluoroalkyl Substances (PFAS) to Serum Proteins: Implications for Toxicokinetics in Humans.

Per- and polyfluoroalkyl substances (PFAS) are a diverse class of highly persistent anthropogenic chemicals that are detectable in the serum of most humans. PFAS exposure has been associated with many adverse effects on human health including immunotoxicity, increased risk of certain cancers, and metabolic disruption. PFAS binding to the most abundant blood serum proteins (human serum albumin [HSA] and globulins) is thought to affect transport to active sites, toxicity, and elimination half-lives. However, few studies have investigated the competitive binding of PFAS to these proteins in human serum. Here, we use C18 solid-phase microextraction fibers to measure HSA-water and globulin-water distribution coefficients (DHSA/w, Dglob/w) for PFAS with carbon chains containing 4 to 13 perfluorinated carbons (ηpfc = 4-13) and several functional head-groups. PFAS with ηpfc < 7 were highly bound to HSA relative to globulins, whereas PFAS with ηpfc ≥ 7 showed a greater propensity for binding to globulins. Experimentally measured DHSA/w and Dglob/w and concentrations of serum proteins successfully predicted the variability in PFAS binding in human serum. We estimated that the unbound fraction of serum PFAS varied by up to a factor of 2.5 among individuals participating in the 2017-2018 U.S. National Health and Nutrition Examination Survey. These results suggest that serum HSA and globulins are important covariates for epidemiological studies aimed at understanding the effects of PFAS exposure.

Unbound Fractions of PFAS in Human and Rodent Tissues: Rat Liver a Suitable Proxy for Evaluating Emerging PFAS?

Adverse health effects associated with exposures to perfluoroalkyl and polyfluoroalkyl substances (PFAS) are a concern for public health and are driven by their elimination half-lives and accumulation in specific tissues. However, data on PFAS binding in human tissues are limited. Accumulation of PFAS in human tissues has been linked to interactions with specific proteins and lipids in target organs. Additional data on PFAS binding and unbound fractions (funbound) in whole human tissues are urgently needed. Here, we address this gap by using rapid equilibrium dialysis to measure the binding and funbound of 16 PFAS with 3 to 13 perfluorinated carbon atoms (ηpfc = 3-13) and several functional headgroups in human liver, lung, kidney, heart, and brain tissue. We compare results to mouse (C57BL/6 and CD-1) and rat tissues. Results show that funbound decreases with increasing fluorinated carbon chain length and hydrophobicity. Among human tissues, PFAS binding was generally greatest in brain > liver ≈ kidneys ≈ heart > lungs. A correlation analysis among human and rodent tissues identified rat liver as a suitable surrogate for predicting funbound for PFAS in human tissues (R2 ≥ 0.98). The funbound data resulting from this work and the rat liver prediction method offer input parameters and tools for toxicokinetic models for legacy and emerging PFAS.

TBK1 and IKKε act like an OFF switch to limit NLRP3 inflammasome pathway activation.

NACHT, LRR, and PYD domains-containing protein 3 (NLRP3) inflammasome activation is beneficial during infection and vaccination but, when uncontrolled, is detrimental and contributes to inflammation-driven pathologies. Hence, discovering endogenous mechanisms that regulate NLRP3 activation is important for disease interventions. Activation of NLRP3 is regulated at the transcriptional level and by posttranslational modifications. Here, we describe a posttranslational phospho-switch that licenses NLRP3 activation in macrophages. The ON switch is controlled by the protein phosphatase 2A (PP2A) downstream of a variety of NLRP3 activators in vitro and in lipopolysaccharide-induced peritonitis in vivo. The OFF switch is regulated by two closely related kinases, TANK-binding kinase 1 (TBK1) and I-kappa-B kinase epsilon (IKKε). Pharmacological inhibition of TBK1 and IKKε, as well as simultaneous deletion of TBK1 and IKKε, but not of either kinase alone, increases NLRP3 activation. In addition, TBK1/IKKε inhibitors counteract the effects of PP2A inhibition on inflammasome activity. We find that, mechanistically, TBK1 interacts with NLRP3 and controls the pathway activity at a site distinct from NLRP3-serine 3, previously reported to be under PP2A control. Mutagenesis of NLRP3 confirms serine 3 as an important phospho-switch site but, surprisingly, reveals that this is not the sole site regulated by either TBK1/IKKε or PP2A, because all retain the control over the NLRP3 pathway even when serine 3 is mutated. Altogether, a model emerges whereby TLR-activated TBK1 and IKKε act like a "parking brake" for NLRP3 activation at the time of priming, while PP2A helps remove this parking brake in the presence of NLRP3 activating signals, such as bacterial pore-forming toxins or endogenous danger signals.

Ingestion of single guide RNAs induces gene overexpression and extends lifespan in Caenorhabditis elegans via CRISPR activation.

Inhibition of gene expression in Caenorhabditis elegans, a versatile model organism for studying the genetics of development and aging, is achievable by feeding nematodes with bacteria expressing specific dsRNAs. Overexpression of hypoxia-inducible factor 1 (hif-1) or heat-shock factor 1 (hsf-1) by conventional transgenesis has previously been shown to promote nematodal longevity. However, it is unclear whether other methods of gene overexpression are feasible, particularly with the advent of CRISPR-based techniques. Here, we show that feeding C. elegans engineered to stably express a Cas9-derived synthetic transcription factor with bacteria expressing promoter-specific single guide RNAs (sgRNAs) also allows activation of gene expression. We demonstrate that CRISPR activation via ingested sgRNAs specific for the respective promoter regions of hif-1 or hsf-1 increases gene expression and extends lifespan of C. elegans. Furthermore, and as an in silico resource for future studies aiming to use CRISPR activation in C. elegans, we provide predicted promoter-specific sgRNA target sequences for >13,000 C. elegans genes with experimentally defined transcription start sites. We anticipate that the approach and components described herein will help to facilitate genome-wide gene overexpression studies, for example, to identify modulators of aging or other phenotypes of interest, by enabling induction of transcription by feeding of sgRNA-expressing bacteria to nematodes.

Paired Liver:Plasma PFAS Concentration Ratios from Adolescents in the Teen-LABS Study and Derivation of Empirical and Mass Balance Models to Predict and Explain Liver PFAS Accumulation.

Animal studies have pointed at the liver as a hotspot for per- and polyfluoroalkyl substances (PFAS) accumulation and toxicity; however, these findings have not been replicated in human populations. We measured concentrations of seven PFAS in matched liver and plasma samples collected at the time of bariatric surgery from 64 adolescents in the Teen-Longitudinal Assessment of Bariatric Surgery (Teen-LABS) study. Liver:plasma concentration ratios were perfectly explained (r2 > 0.99) in a multilinear regression (MLR) model based on toxicokinetic (TK) descriptors consisting of binding to tissue constituents and membrane permeabilities. Of the seven matched plasma and liver PFAS concentrations compared in this study, the liver:plasma concentration ratio of perfluoroheptanoic acid (PFHpA) was considerably higher than the liver:plasma concentration ratio of other PFAS congeners. Comparing the MLR model with an equilibrium mass balance model (MBM) suggested that complex kinetic transport processes are driving the unexpectedly high liver:plasma concentration ratio of PFHpA. Intratissue MBM modeling pointed to membrane lipids as the tissue constituents that drive the liver accumulation of long-chain, hydrophobic PFAS, whereas albumin binding of hydrophobic PFAS dominated PFAS distribution in plasma. The liver:plasma concentration data set, empirical MLR model, and mechanistic MBM modeling allow the prediction of liver from plasma concentrations measured in human cohort studies. Our study demonstrates that combining biomonitoring data with mechanistic modeling can identify underlying mechanisms of internal distribution and specific target organ toxicity of PFAS in humans.

Tallo: A global tree allometry and crown architecture database.

Data capturing multiple axes of tree size and shape, such as a tree's stem diameter, height and crown size, underpin a wide range of ecological research-from developing and testing theory on forest structure and dynamics, to estimating forest carbon stocks and their uncertainties, and integrating remote sensing imagery into forest monitoring programmes. However, these data can be surprisingly hard to come by, particularly for certain regions of the world and for specific taxonomic groups, posing a real barrier to progress in these fields. To overcome this challenge, we developed the Tallo database, a collection of 498,838 georeferenced and taxonomically standardized records of individual trees for which stem diameter, height and/or crown radius have been measured. These data were collected at 61,856 globally distributed sites, spanning all major forested and non-forested biomes. The majority of trees in the database are identified to species (88%), and collectively Tallo includes data for 5163 species distributed across 1453 genera and 187 plant families. The database is publicly archived under a CC-BY 4.0 licence and can be access from: https://doi.org/10.5281/zenodo.6637599. To demonstrate its value, here we present three case studies that highlight how the Tallo database can be used to address a range of theoretical and applied questions in ecology-from testing the predictions of metabolic scaling theory, to exploring the limits of tree allometric plasticity along environmental gradients and modelling global variation in maximum attainable tree height. In doing so, we provide a key resource for field ecologists, remote sensing researchers and the modelling community working together to better understand the role that trees play in regulating the terrestrial carbon cycle.

Endogenous metabolites promote stress resistance through induction of mitohormesis.

Interventions and small molecules, which promote formation of reactive oxygen species (ROS), have repeatedly been shown to increase stress resistance and lifespan of different model organisms. These phenotypes occur only in response to low concentrations of ROS, while higher concentrations exert opposing effects. This non-linear or hormetic dose-response relationship has been termed mitohormesis, since ROS are mainly generated within the mitochondrial compartment. A report by Matsumura et al in this issue of EMBO Reports now demonstrates that an endogenously formed metabolite, namely N-acetyl-L-tyrosine (NAT), is instrumental in promoting cellular and organismal resilience by inducing mitohormetic mechanisms, likely in an evolutionarily conserved manner [1].

Monkeypox in a Patient with Controlled HIV Infection Initially Presenting with Fever, Painful Pharyngitis, and Tonsillitis.

Background and Objectives: With more and more cases emerging outside central and west African countries, where the disease is endemic, the World Health Organization (WHO) has recently declared human monkeypox a Public Health Emergency of International Concern. Typical symptoms of the disease include fever, myalgia, and lymphadenopathy followed by a rash, but other symptoms may occur. Immunocompromised patients, including patients with uncontrolled Human Immunodeficiency Virus (HIV) infection, may be at risk for more severe courses. Case presentation: We present the case of a 30-year-old male patient of Brazilian descent with monkeypox. Initial symptoms were fever and general discomfort, with painful pharyngitis and tonsillitis and finally a papular rash of the anogenital area as the disease progressed. The presumed date of infection was a sexual contact with an unknown male eight days before the first symptoms occurred. The patient had a known and controlled HIV infection. The main reason for the initial presentation at the hospital was painful pharyngitis and tonsillitis, limiting food intake. Monkeypox infection was confirmed via PCR testing from a swab sample of cutaneous lesions. Adequate systemic and local analgesia enabled oral food uptake again. Antiviral therapy with Tecovirimat was not administered due to the stable immune status of the patient and the mild clinical symptoms. To cover a possible bacterial superinfection or Syphilis infection of the tonsil, antibiotic therapy with Ceftriaxone was added. Several days after presentation, the inflammation of the pharynx resolved and was followed by non-painful mucosal peeling. The patient was followed up with telephone calls and reported a complete recovery. The skin lesions were completely dried out 18 days after the first symptoms. Conclusions: Painful pharyngitis and tonsillitis can be rare early symptoms of monkeypox, which is highly relevant in everyday clinical practice. Particularly in patients with risk factors for monkeypox infection, further clinical and microbiologic testing for monkeypox should be performed if there is a clinical presentation with pharyngitis and tonsillitis.

PHOrming the inflammasome: phosphorylation is a critical switch in inflammasome signalling.

Inflammasomes are protein complexes in the innate immune system that regulate the production of pro-inflammatory cytokines and inflammatory cell death. Inflammasome activation and subsequent cell death often occur within minutes to an hour, so the pathway must be dynamically controlled to prevent excessive inflammation and the development of inflammatory diseases. Phosphorylation is a fundamental post-translational modification that allows rapid control over protein function and the phosphorylation of inflammasome proteins has emerged as a key regulatory step in inflammasome activation. Phosphorylation of inflammasome sensor and adapter proteins regulates their inter- and intra-molecular interactions, subcellular localisation, and function. The control of inflammasome phosphorylation may thus provide a new strategy for the development of anti-inflammatory therapeutics. Herein we describe the current knowledge of how phosphorylation operates as a critical switch for inflammasome signalling.

Mind the Exposure Gaps-Modeling Chemical Transport in Sediment Toxicity Tests.

Chemical exposure in flow-through sediment toxicity tests can vary in time, between pore and overlying water, and amid free and bound states, complicating the link between toxicity and observable concentrations such as free pore (Cfree,pore), free overlying (Cfree,over), or the corresponding dissolved concentrations (Cdiss, free + bound to dissolved organic carbon, DOC). We introduce a numerical model that describes the desorption from sediments to pore water, diffusion through pores and the sediment-water boundary, DOC-mediated transport, and mixing in and outflow from overlying water. The model explained both the experimentally measured gap between Cfree,over and Cfree,pore and the continuous decrease in overlying Cdiss. Spatially resolved modeling suggested a steep concentration gradient present in the upper millimeter of the sediment due to slow chemical diffusion in sediment pores and fast outflux from the overlying water. In contrast to continuous decrease in overlying Cdiss expected for any chemical, Cfree,over of highly hydrophobic chemicals was kept relatively constant following desorption from DOC, a mechanism comparable to passive dosing. Our mechanistic analyses emphasize that exposure will depend on the chemical's hydrophobicity, the test organism habitat and uptake of bound chemicals, and the properties of sediment components, including DOC. The model can help to re-evaluate existing toxicity data, optimize experimental setups, and extrapolate laboratory toxicity data to field exposure.

Three Small Molecule Entities (MPK18, MPK334 and YAK308) with Activity against Haemonchus contortus In Vitro.

Due to widespread multi-drug resistance in parasitic nematodes of livestock animals, there is an urgent need to discover new anthelmintics with distinct mechanisms of action. Extending previous work, here we screened a panel of 245 chemically-diverse small molecules for anti-parasitic activity against Haemonchus contortus-an economically important parasitic nematode of livestock. This panel was screened in vitro against exsheathed third-stage larvae (xL3) of H. contortus using an established phenotypic assay, and the potency of select compounds to inhibit larval motility and development assessed in dose-response assays. Of the 245 compounds screened, three-designated MPK18, MPK334 and YAK308-induced non-wildtype larval phenotypes and repeatedly inhibited xL3-motility, with IC50 values of 45.2 µM, 17.1 µM and 52.7 µM, respectively; two also inhibited larval development, with IC50 values of 12.3 µM (MPK334) and 6.5 µM (YAK308), and none of the three was toxic to human liver cells (HepG2). These findings suggest that these compounds deserve further evaluation as nematocidal candidates. Future work should focus on structure-activity relationship (SAR) studies of these chemical scaffolds, and assess the in vitro and in vivo efficacies and safety of optimised compounds against adults of H. contortus.

Cellular Metabolism in High-Throughput In Vitro Reporter Gene Assays and Implications for the Quantitative In Vitro-In Vivo Extrapolation.

High-throughput in vitro reporter gene assays are increasingly applied to assess the potency of chemicals to alter specific cellular signaling pathways. Genetically modified reporter gene cell lines provide stable readouts of the activation of cellular receptors or transcription factors of interest, but such reporter gene assays have been criticized for not capturing cellular metabolism. We characterized the metabolic activity of the widely applied AREc32 (human breast cancer MCF-7), ARE-bla (human liver cancer HepG2), and GR-bla (human embryonic kidney HEK293) reporter gene cells in the absence and in the presence of benzo[a]pyrene (BaP), an AhR ligand known to upregulate cytochrome P450 in vitro and in vivo. We combined fluorescence microscopy with chemical analysis, real-time PCR, and ethoxyresorufin-O-deethylase activity measurements to track temporal changes in BaP and its metabolites in the cells and surrounding medium over time in relation to the expression and activity of metabolic enzymes. Decreasing BaP concentrations and formation of metabolites agreed with the high basal CYP1 activity of ARE-bla and the strong CYP1A1 mRNA induction in AREc32, whereas BaP concentrations were constant in GR-bla, in which neither metabolites nor CYP1 induction was detected. The study emphasizes that differences in sensitivity between reporter gene assays may be caused not only by different reporter constructs but also by a varying biotransformation rate of the evaluated parent chemical. The basal metabolic capacity of reporter gene cells in the absence of chemicals is not a clear indication because we demonstrated that the metabolic activity can be upregulated by AhR ligands during the assay. The combination of methods presented here is suitable to characterize the metabolic activity of cells in vitro and can improve the interpretation of in vitro reporter gene effect data and extrapolation to in vivo human exposure.

Slow rate of secondary forest carbon accumulation in the Guianas compared with the rest of the Neotropics.

Secondary forests are a prominent component of tropical landscapes, and they constitute a major atmospheric carbon sink. Rates of carbon accumulation are usually inferred from chronosequence studies, but direct estimates of carbon accumulation based on long-term monitoring of stands are rarely reported. Recent compilations on secondary forest carbon accumulation in the Neotropics are heavily biased geographically as they do not include estimates from the Guiana Shield. We analysed the temporal trajectory of aboveground carbon accumulation and floristic composition at one 25-ha secondary forest site in French Guiana. The site was clear-cut in 1976, abandoned thereafter, and one large plot (6.25 ha) has been monitored continuously since. We used Bayesian modeling to assimilate inventory data and simulate the long-term carbon accumulation trajectory. Canopy change was monitored using two aerial lidar surveys conducted in 2009 and 2017. We compared the dynamics of this site with that of a surrounding old-growth forest. Finally, we compared our results with that from secondary forests in Costa Rica, which is one of the rare long-term monitoring programs reaching a duration comparable to our study. Twenty years after abandonment, aboveground carbon stock was 64.2 (95% credibility interval 46.4, 89.0) Mg C/ha, and this stock increased to 101.3 (78.7, 128.5) Mg C/ha 20 yr later. The time to accumulate one-half of the mean aboveground carbon stored in the nearby old-growth forest (185.6 [155.9, 200.2] Mg C/ha) was estimated at 35.0 [20.9, 55.9] yr. During the first 40 yr, the contribution of the long-lived pioneer species Xylopia nitida, Goupia glabra, and Laetia procera to the aboveground carbon stock increased continuously. Secondary forest mean-canopy height measured by lidar increased by 1.14 m in 8 yr, a canopy-height increase consistent with an aboveground carbon accumulation of 7.1 Mg C/ha (or 0.89 Mg C·ha-1 ·yr-1 ) during this period. Long-term AGC accumulation rate in Costa Rica was almost twice as fast as at our site in French Guiana. This may reflect higher fertility of Central American forest communities or a better adaptation of the forest tree community to intense and frequent disturbances. This finding may have important consequences for scaling-up carbon uptake estimates to continental scales.

Improving plant allometry by fusing forest models and remote sensing.

Allometry determines how tree shape and function scale with each other, related through size. Allometric relationships help scale processes from the individual to the global scale and constitute a core component of vegetation models. Allometric relationships have been expected to emerge from optimisation theory, yet this does not suitably predict empirical data. Here we argue that the fusion of high-resolution data, such as those derived from airborne laser scanning, with individual-based forest modelling offers insight into how plant size contributes to large-scale biogeochemical processes. We review the challenges in allometric scaling, how they can be tackled by advances in data-model fusion, and how individual-based models can serve as data integrators for dynamic global vegetation models.

How To Improve the Dosing of Chemicals in High-Throughput in Vitro Mammalian Cell Assays.

Controlling the exposure of chemicals in in vitro mammalian cell assays is an important prerequisite for the application of in vitro methods in risk and hazard assessment of chemicals. Existing models require numerous physicochemical and system parameters to quantify the effective concentration in the assay. Synthesizing these studies, this article briefly communicates how the protein-rich supplement in the medium can be utilized to adjust constant and quantifiable exposure concentrations without the need for measurements and complex modeling. We present a simplified mass balance equation based on chemical properties and system parameters from openly accessible databases, which can be used to adjust the dose of chemicals in the exposure medium, leading to defined and stable freely dissolved concentrations (Cfree). The proposed framework prevents experimental artifacts associated with the use of cosolvents and medium oversaturation and enables the conversion of in vitro effect data to freely dissolved effect concentrations (ECfree), which can directly be applied in quantitative in vitro to in vivo extrapolation models and compared to other exposure scenarios.

C18-Coated Solid-Phase Microextraction Fibers for the Quantification of Partitioning of Organic Acids to Proteins, Lipids, and Cells.

The effects measured with in vitro cell-based bioassays are typically reported as nominal effect concentrations ( Cnom), but the freely dissolved concentration in the exposure medium ( Cw) and the total cellular concentration ( Ccell) are considered more quantitative dose metrics that allow extrapolation to the whole-organism level. To predict Cw and Ccell, the partitioning of the test chemicals to medium proteins and lipids and cells has to be known. In this study, we developed a solid-phase microextraction (SPME) method based on C18-coated fibers to quantify the partitioning of diclofenac, 2,4-dichlorophenoxyacetic acid (2,4-D), ibuprofen, naproxen, torasemide, warfarin, and genistein to bovine serum albumin (BSA), phospholipid liposomes, fetal bovine serum (FBS), and cells. For ibuprofen, 2,4-D, naproxen, and warfarin, the partitioning to the SPME fibers was found to be concentration dependent, which had to be considered for the calculation of distribution ratios to biological materials. The sorption isotherms to FBS were nonlinear for diclofenac, 2,4-D, ibuprofen, naproxen, and warfarin. The FBS isotherms could be described by assuming that the total amount of chemical bound to FBS is the sum of the amount specifically bound to the binding sites of albumin and nonspecifically bound to all medium proteins and lipids. The determined cell-water distribution ratios ( Dcell/w) differed considerably between four different cell lines (up to 1.83 log-units) and also between different batches of the same cell line (up to 0.48 log-units). The relative importance of protein and lipid content for Dcell/w was evaluated with a mass balance model and different types of cellular proteins and lipids as input parameters. Existing in vitro mass balance models may underestimate Cw because they do not account for saturable protein binding and overestimate Ccell for organic acids, if BSA is used as surrogate for cellular proteins.