Basal MET phosphorylation is an indicator of hepatocyte dysregulation in liver disease.

Chronic liver diseases are worldwide on the rise. Due to the rapidly increasing incidence, in particular in Western countries, metabolic dysfunction-associated steatotic liver disease (MASLD) is gaining importance as the disease can develop into hepatocellular carcinoma. Lipid accumulation in hepatocytes has been identified as the characteristic structural change in MASLD development, but molecular mechanisms responsible for disease progression remained unresolved. Here, we uncover in primary hepatocytes from a preclinical model fed with a Western diet (WD) an increased basal MET phosphorylation and a strong downregulation of the PI3K-AKT pathway. Dynamic pathway modeling of hepatocyte growth factor (HGF) signal transduction combined with global proteomics identifies that an elevated basal MET phosphorylation rate is the main driver of altered signaling leading to increased proliferation of WD-hepatocytes. Model-adaptation to patient-derived hepatocytes reveal patient-specific variability in basal MET phosphorylation, which correlates with patient outcome after liver surgery. Thus, dysregulated basal MET phosphorylation could be an indicator for the health status of the liver and thereby inform on the risk of a patient to suffer from liver failure after surgery.

Immuno-inflammatory in vitro hepatotoxicity models to assess side effects of biologicals exemplified by aldesleukin.

Introduction: Hepatotoxicity induced by immunotherapeutics is an appearing cause for immune-mediated drug-induced liver injury. Such immuno-toxic mechanisms are difficult to assess using current preclinical models and the incidence is too low to detect in clinical trials. As hepatotoxicity is a frequent reason for post-authorisation drug withdrawal, there is an urgent need for immuno-inflammatory in vitro models to assess the hepatotoxic potential of immuno-modulatory drug candidates. We developed several immuno-inflammatory hepatotoxicity test systems based on recombinant human interleukin-2 (aldesleukin). Methods: Co-culture models of primary human CD8+ T cells or NK cells with the hepatocyte cell line HepaRG were established and validated with primary human hepatocytes (PHHs). Subsequently, the HepaRG model was refined by increasing complexity by inclusion of monocyte-derived macrophages (MdMs). The main readouts were cytotoxicity, inflammatory mediator release, surface marker expression and specific hepatocyte functions. Results: We identified CD8+ T cells as possible mediators of aldesleukin-mediated hepatotoxicity, with MdMs being implicated in increased aldesleukin-induced inflammatory effects. In co-cultures of CD8+ T cells with MdMs and HepaRG cells, cytotoxicity was induced at intermediate/high aldesleukin concentrations and perforin was upregulated. A pro-inflammatory milieu was created measured by interleukin-6 (IL-6), c-reactive protein (CRP), interferon gamma (IFN-γ), and monocyte chemoattractant protein-1 (MCP-1) increase. NK cells responded to aldesleukin, however, only minor aldesleukin-induced cytotoxic effects were measured in co-cultures. Results obtained with HepaRG cells and with PHHs were comparable, especially regarding cytotoxicity, but high inter-donor variations limited meaningfulness of the PHH model. Discussion: The in vitro test systems developed contribute to the understanding of potential key mechanisms in aldesleukin-mediated hepatotoxicity. In addition, they may aid assessment of immune-mediated hepatotoxicity during the development of novel immunotherapeutics.

PFASs-restriction proposal commentary on ECHA's Annex XV restriction report, proposal for a restriction, March 2023.

PFASs are defined as substances that contain at least one fully fluorinated methyl (CF3-) or methylene (-CF2-) carbon atom. The excellent technical properties of members of the PFAS group have led to their use in a wide range of applications. The substance group comprises more than 10,000 individual compounds. A variety of adverse effects has been described for single substances. For the majority of the PFASs, neither toxicokinetic data nor effect data is available. Hence, because of the small number of PFASs for which a full toxicological profile is available, grouping based on the existing data is not feasible. A critical problem of PFASs and their degradation products is the very high persistence, which clearly fulfils the criterion for the substance property Very Persistent (vP) according to Annex XIII of the REACH Regulation. Because of this property the European Commission is planning to take action. Defining suitable subgroups appears to be a scientifically based approach. However, to reach this goal, large data gaps would have to be closed which would take up to centuries, a time-frame, which is not defendable with respect to potential irreversible harm. Because of the time pressure resulting from the potential irreversible harm, the precautionary principle has been selected as an appropriate tool to handle PFASs and in the restriction proposal PFASs are treated as one group. This approach is justified in the view of the advisory committee of the German Society for Toxicology. ECHA's proposal received a lot of attention in the public. However, communication so far has obviously led to the misunderstanding of a proven health hazard for all PFASs. Communication should explain the justification of the broad inclusion of substances as being based on the precautionary principle. Data gaps versus current knowledge need to be clearly communicated; communication should also include the possibility for derogation of essential use. It should address the issue of suitable substitutes to avoid unintended health consequences; and it should mention that existing persistent environmental contamination calls for developing innovation in remediation techniques.

SARS-CoV-2 mRNA vaccination-induced immunological memory in human nonlymphoid and lymphoid tissues.

Tissue-resident lymphocytes provide organ-adapted protection against invading pathogens. Whereas their biology has been examined in great detail in various infection models, their generation and functionality in response to vaccination have not been comprehensively analyzed in humans. We therefore studied SARS-CoV-2 mRNA vaccine-specific T cells in surgery specimens of kidney, liver, lung, bone marrow, and spleen compared with paired blood samples from largely virus-naive individuals. As opposed to lymphoid tissues, nonlymphoid organs harbored significantly elevated frequencies of spike-specific CD4+ T cells compared with blood showing hallmarks of tissue residency and an expanded memory pool. Organ-derived CD4+ T cells further exhibited increased polyfunctionality over those detected in blood. Single-cell RNA-Seq together with T cell receptor repertoire analysis indicated that the clonotype rather than organ origin is a major determinant of transcriptomic state in vaccine-specific CD4+ T cells. In summary, our data demonstrate that SARS-CoV-2 vaccination entails acquisition of tissue memory and residency features in organs distant from the inoculation site, thereby contributing to our understanding of how local tissue protection might be accomplished.

Optimization of extracellular matrix for primary human hepatocyte cultures using mixed collagen-Matrigel matrices.

Loss of differentiation of primary human hepatocytes (PHHs) ex vivo is a known problem of in vitro liver models. Culture optimizations using collagen type I and Matrigel reduce the dedifferentiation process but are not able to prevent it. While neither of these extracellular matrices (ECMs) on their own correspond to the authentic hepatic ECM, a combination of them could more closely resemble the in vivo situation. Our study aimed to systematically analyze the influence of mixed matrices composed of collagen type I and Matrigel on the maintenance and reestablishment of hepatic functions. Therefore, PHHs were cultured on mixed collagen-Matrigel matrices in monolayer and sandwich cultures and viability, metabolic capacity, differentiation markers, cellular arrangement and the cells' ability to repolarize and form functional bile canaliculi were assessed by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), functional assays and immunofluorescence microscopy. Our results show that mixed matrices were superior to pure matrices in maintaining metabolic capacity and hepatic differentiation. In contrast, Matrigel supplementation can impair the development of a proper hepatocytic polarization. Our systematic study helps to compose an optimized ECM to maintain and reestablish hepatic differentiation on cellular and multicellular levels in human liver models.

Critical Investigation of the Usability of Hepatoma Cell Lines HepG2 and Huh7 as Models for the Metabolic Representation of Resectable Hepatocellular Carcinoma.

Metabolic alterations in hepatocellular carcinoma (HCC) are fundamental for the development of diagnostic screening and therapeutic intervention since energy metabolism plays a central role in differentiated hepatocytes. In HCC research, hepatoma cell lines (HCLs) like HepG2 and Huh7 cells are still the gold standard. In this study, we characterized the metabolic profiles of primary human hepatoma cells (PHCs), HCLs and primary human hepatocytes (PHHs) to determine their differentiation states. PHCs and PHHs (HCC-PHHs) were isolated from surgical specimens of HCC patients and their energy metabolism was compared to PHHs from non-HCC patients and the HepG2 and Huh7 cells at different levels (transcript, protein, function). Our analyses showed successful isolation of PHCs with a purity of 50-73% (CK18+). The transcript data revealed that changes in mRNA expression levels had already occurred in HCC-PHHs. While many genes were overexpressed in PHCs and HCC-PHHs, the changes were mostly not translated to the protein level. Downregulated metabolic key players of PHCs revealed a correlation with malign transformation and were predominantly pronounced in multilocular HCC. Therefore, HCLs failed to reflect these expression patterns of PHCs at the transcript and protein levels. The metabolic characteristics of PHCs are closer to those of HCC-PHHs than to HCLs. This should be taken into account for future optimized tumor metabolism research.

In vitro/in silico prediction of drug induced steatosis in relation to oral doses and blood concentrations by the Nile Red assay.

The accumulation of lipid droplets in hepatocytes is a key feature of drug-induced liver injury (DILI) and can be induced by a subset of hepatotoxic compounds. In the present study, we optimized and evaluated an in vitro technique based on the fluorescent dye Nile Red, further named Nile Red assay to quantify lipid droplets induced by the exposure to chemicals. The Nile Red assay and a cytotoxicity test (CTB assay) were then performed on cells exposed concentration-dependently to 60 different compounds. Of these, 31 were known to induce hepatotoxicity in humans, and 13 were reported to also cause steatosis. In order to compare in vivo relevant blood concentrations, pharmacokinetic models were established for all compounds to simulate the maximal blood concentrations (Cmax) at therapeutic doses. The results showed that several hepatotoxic compounds induced an increase in lipid droplets at sub-cytotoxic concentrations. To compare how well (1) the cytotoxicity test alone, (2) the Nile Red assay alone, and (3) the combination of the cytotoxicity test and the Nile Red assay (based on the lower EC10 of both assays) allow the differentiation between hepatotoxic and non-hepatotoxic compounds, a previously established performance metric, the Toxicity Separation Index (TSI) was calculated. In addition, the Toxicity Estimation Index (TEI) was calculated to determine how well blood concentrations that cause an increased DILI risk can be estimated for hepatotoxic compounds. Our findings indicate that the combination of both assays improved the TSI and TEI compared to each assay alone. In conclusion, the study demonstrates that inclusion of the Nile Red assay into in vitro test batteries may improve the prediction of DILI compounds.

Primary-like Human Hepatocytes Genetically Engineered to Obtain Proliferation Competence as a Capable Application for Energy Metabolism Experiments in In Vitro Oncologic Liver Models.

Non-alcoholic fatty liver disease (NAFLD), characterized by lipid accumulation in the liver, is the most common cause of liver diseases in Western countries. NAFLD is a major risk factor for developing hepatocellular carcinoma (HCC); however, in vitro evaluation of hepatic cancerogenesis fails due to a lack of liver models displaying a proliferation of hepatocytes. Originally designed to overcome primary human hepatocyte (PHH) shortages, upcyte hepatocytes were engineered to obtain continuous proliferation and, therefore, could be a suitable tool for HCC research. We generated upcyte hepatocytes, termed HepaFH3 cells, and compared their metabolic characteristics to HepG2 hepatoma cells and PHHs isolated from resected livers. For displaying NAFLD-related HCCs, we induced steatosis in all liver models. Lipid accumulation, lipotoxicity and energy metabolism were characterized using biochemical assays and Western blot analysis. We showed that proliferating HepaFH3 cells resemble HepG2, both showing a higher glucose uptake rate, lactate levels and metabolic rate compared to PHHs. Confluent HepaFH3 cells displayed some similarities to PHHs, including higher levels of the transaminases AST and ALT compared to proliferating HepaFH3 cells. We recommend proliferating HepaFH3 cells as a pre-malignant cellular model for HCC research, while confluent HepaFH3 cells could serve as PHH surrogates for energy metabolism studies.

Diploid hepatocytes drive physiological liver renewal in adult humans.

Physiological liver cell replacement is central to maintaining the organ's high metabolic activity, although its characteristics are difficult to study in humans. Using retrospective radiocarbon (14C) birth dating of cells, we report that human hepatocytes show continuous and lifelong turnover, allowing the liver to remain a young organ (average age <3 years). Hepatocyte renewal is highly dependent on the ploidy level. Diploid hepatocytes show more than 7-fold higher annual birth rates than polyploid hepatocytes. These observations support the view that physiological liver cell renewal in humans is mainly dependent on diploid hepatocytes, whereas polyploid cells are compromised in their ability to divide. Moreover, cellular transitions between diploid and polyploid hepatocytes are limited under homeostatic conditions. With these findings, we present an integrated model of homeostatic liver cell generation in humans that provides fundamental insights into liver cell turnover dynamics.

Epigenomic and transcriptional profiling identifies impaired glyoxylate detoxification in NAFLD as a risk factor for hyperoxaluria.

Epigenetic modifications (e.g. DNA methylation) in NAFLD and their contribution to disease progression and extrahepatic complications are poorly explored. Here, we use an integrated epigenome and transcriptome analysis of mouse NAFLD hepatocytes and identify alterations in glyoxylate metabolism, a pathway relevant in kidney damage via oxalate release-a harmful waste product and kidney stone-promoting factor. Downregulation and hypermethylation of alanine-glyoxylate aminotransferase (Agxt), which detoxifies glyoxylate, preventing excessive oxalate accumulation, is accompanied by increased oxalate formation after metabolism of the precursor hydroxyproline. Viral-mediated Agxt transfer or inhibiting hydroxyproline catabolism rescues excessive oxalate release. In human steatotic hepatocytes, AGXT is also downregulated and hypermethylated, and in NAFLD adolescents, steatosis severity correlates with urinary oxalate excretion. Thus, this work identifies a reduced capacity of the steatotic liver to detoxify glyoxylate, triggering elevated oxalate, and provides a mechanistic explanation for the increased risk of kidney stones and chronic kidney disease in NAFLD patients.

In Vivo and In Vitro Characterization of Primary Human Liver Macrophages and Their Inflammatory State.

Liver macrophages (LMs) play a central role in acute and chronic liver pathologies. Investigation of these processes in humans as well as the development of diagnostic tools and new therapeutic strategies require in vitro models that closely resemble the in vivo situation. In our study, we sought to gain further insight into the role of LMs in different liver pathologies and into their characteristics after isolation from liver tissue. For this purpose, LMs were characterized in human liver tissue sections using immunohistochemistry and bioinformatic image analysis. Isolated cells were characterized in suspension using FACS analyses and in culture using immunofluorescence staining and laser scanning microscopy as well as functional assays. The majority of our investigated liver tissues were characterized by anti-inflammatory LMs which showed a homogeneous distribution and increased cell numbers in correlation with chronic liver injuries. In contrast, pro-inflammatory LMs appeared as temporary and locally restricted reactions. Detailed characterization of isolated macrophages revealed a complex disease dependent pattern of LMs consisting of pro- and anti-inflammatory macrophages of different origins, regulatory macrophages and monocytes. Our study showed that in most cases the macrophage pattern can be transferred in adherent cultures. The observed exceptions were restricted to LMs with pro-inflammatory characteristics.

Influence of Genistein on Hepatic Lipid Metabolism in an In Vitro Model of Hepatic Steatosis.

Nonalcoholic fatty liver disease (NAFLD) is among the leading causes of end-stage liver disease. The impaired hepatic lipid metabolism in NAFLD is exhibited by dysregulated PPARα and SREBP-1c signaling pathways, which are central transcription factors associated with lipid degradation and de novo lipogenesis. Despite the growing prevalence of this disease, current pharmacological treatment options are unsatisfactory. Genistein, a soy isoflavone, has beneficial effects on lipid metabolism and may be a candidate for NAFLD treatment. In an in vitro model of hepatic steatosis, primary human hepatocytes (PHHs) were incubated with free fatty acids (FFAs) and different doses of genistein. Lipid accumulation and the cytotoxic effects of FFAs and genistein treatment were evaluated by colorimetric and enzymatic assays. Changes in lipid homeostasis were examined by RT-qPCR and Western blot analyses. PPARα protein expression was induced in steatotic PHHs, accompanied by an increase in CPT1L and ACSL1 mRNA. Genistein treatment increased PPARα protein expression only in control PHHs, while CPTL1 and ACSL1 were unchanged and PPARα mRNA was reduced. In steatotic PHHs, genistein reversed the increase in activated SREBP-1c protein. The model realistically reflected the molecular changes in hepatic steatosis. Genistein suppressed the activation of SREBP-1c in steatotic hepatocytes, but the genistein-mediated effects on PPARα were abolished by high hepatic lipid levels.

Hepatocyte pyroptosis and release of inflammasome particles induce stellate cell activation and liver fibrosis.

BACKGROUND & AIMS: Increased hepatocyte death contributes to the pathology of acute and chronic liver diseases. However, the role of hepatocyte pyroptosis and extracellular inflammasome release in liver disease is unknown. METHODS: We used primary mouse and human hepatocytes, hepatocyte-specific leucine 351 to proline Nlrp3KICreA mice, and GsdmdKO mice to investigate pyroptotic cell death in hepatocytes and its impact on liver inflammation and damage. Extracellular NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasomes were isolated from mutant NLRP3-YFP HEK cells and internalisation was studied in LX2 and primary human hepatic stellate cells. We also examined a cohort of 154 adult patients with biopsy-proven non-alcoholic fatty liver disease (Sir Charles Gairdner Hospital, Nedlands, Western Australia). RESULTS: We demonstrated that primary mouse and human hepatocytes can undergo pyroptosis upon NLRP3 inflammasome activation with subsequent release of NLRP3 inflammasome proteins that amplify and perpetuate inflammasome-driven fibrogenesis. Pyroptosis was inhibited by blocking caspase-1 and gasdermin D activation. The activated form of caspase-1 was detected in the livers and in serum from patients with non-alcoholic steatohepatitis and correlated with disease severity. Nlrp3KICreA mice showed spontaneous liver fibrosis under normal chow diet, and increased sensitivity to liver damage and inflammation after treatment with low dose lipopolysaccharide. Mechanistically, hepatic stellate cells engulfed extracellular NLRP3 inflammasome particles leading to increased IL-1ß secretion and α-smooth muscle actin expression. This effect was abrogated when cells were pre-treated with the endocytosis inhibitor cytochalasin B. CONCLUSIONS: These results identify hepatocyte pyroptosis and release of inflammasome components as a novel mechanism to propagate liver injury and liver fibrosis development. LAY SUMMARY: Our findings identify a novel mechanism of inflammation in the liver. Experiments in cell cultures, mice, and human samples show that a specific form of cell death, called pyroptosis, leads to the release of complex inflammatory particles, the NLRP3 inflammasome, from inside hepatocytes into the extracellular space. From there they are taken up by other cells and thereby mediate inflammatory and pro-fibrogenic stress signals. The discovery of this mechanism may lead to novel treatments for chronic liver diseases in the future.

Identification of Interleukin1ß as an Amplifier of Interferon alpha-induced Antiviral Responses.

The induction of an interferon-mediated response is the first line of defense against pathogens such as viruses. Yet, the dynamics and extent of interferon alpha (IFNα)-induced antiviral genes vary remarkably and comprise three expression clusters: early, intermediate and late. By mathematical modeling based on time-resolved quantitative data, we identified mRNA stability as well as a negative regulatory loop as key mechanisms endogenously controlling the expression dynamics of IFNα-induced antiviral genes in hepatocytes. Guided by the mathematical model, we uncovered that this regulatory loop is mediated by the transcription factor IRF2 and showed that knock-down of IRF2 results in enhanced expression of early, intermediate and late IFNα-induced antiviral genes. Co-stimulation experiments with different pro-inflammatory cytokines revealed that this amplified expression dynamics of the early, intermediate and late IFNα-induced antiviral genes can also be achieved by co-application of IFNα and interleukin1 beta (IL1ß). Consistently, we found that IL1ß enhances IFNα-mediated repression of viral replication. Conversely, we observed that in IL1ß receptor knock-out mice replication of viruses sensitive to IFNα is increased. Thus, IL1ß is capable to potentiate IFNα-induced antiviral responses and could be exploited to improve antiviral therapies.

Prolonged Lipid Accumulation in Cultured Primary Human Hepatocytes Rather Leads to ER Stress than Oxidative Stress.

Overweight has become a major health care problem in Western societies and is accompanied by an increasing incidence and prevalence of non-alcoholic fatty liver disease (NAFLD). The progression from NAFLD to non-alcoholic steatohepatitis (NASH) marks a crucial tipping point in the progression of severe and irreversible liver diseases. This study aims to gain further insight into the molecular processes leading to the evolution from steatosis to steatohepatitis. Steatosis was induced in cultures of primary human hepatocytes by continuous five-day exposure to free fatty acids (FFAs). The kinetics of lipid accumulation, lipotoxicity, and oxidative stress were measured. Additionally, ER stress was evaluated by analyzing the protein expression profiles of its key players: PERK, IRE1a, and ATF6a. Our data revealed that hepatocytes are capable of storing enormous amounts of lipids without showing signs of lipotoxicity. Prolonged lipid accumulation did not create an imbalance in hepatocyte redox homeostasis or a reduction in antioxidative capacity. However, we observed an FFA-dependent increase in ER stress, revealing thresholds for triggering the activation of pathways associated with lipid stress, inhibition of protein translation, and apoptosis. Our study clearly showed that even severe lipid accumulation can be attenuated by cellular defenses, but regenerative capacities may be reduced.

Disentangling molecular mechanisms regulating sensitization of interferon alpha signal transduction.

Tightly interlinked feedback regulators control the dynamics of intracellular responses elicited by the activation of signal transduction pathways. Interferon alpha (IFNα) orchestrates antiviral responses in hepatocytes, yet mechanisms that define pathway sensitization in response to prestimulation with different IFNα doses remained unresolved. We establish, based on quantitative measurements obtained for the hepatoma cell line Huh7.5, an ordinary differential equation model for IFNα signal transduction that comprises the feedback regulators STAT1, STAT2, IRF9, USP18, SOCS1, SOCS3, and IRF2. The model-based analysis shows that, mediated by the signaling proteins STAT2 and IRF9, prestimulation with a low IFNα dose hypersensitizes the pathway. In contrast, prestimulation with a high dose of IFNα leads to a dose-dependent desensitization, mediated by the negative regulators USP18 and SOCS1 that act at the receptor. The analysis of basal protein abundance in primary human hepatocytes reveals high heterogeneity in patient-specific amounts of STAT1, STAT2, IRF9, and USP18. The mathematical modeling approach shows that the basal amount of USP18 determines patient-specific pathway desensitization, while the abundance of STAT2 predicts the patient-specific IFNα signal response.

HepaChip-MP - a twenty-four chamber microplate for a continuously perfused liver coculture model.

HepaChip microplate (HepaChip-MP) is a microfluidic platform comprised of 24 independent culture chambers with continuous, unidirectional perfusion. In the HepaChip-MP, an automated dielectrophoresis process selectively assembles viable cells into elongated micro tissues. Freshly isolated primary human hepatocytes (PHH) and primary human liver endothelial cells (HuLEC) were successfully assembled as cocultures aiming to mimic the liver sinusoid. Minimal quantities of primary human cells are required to establish micro tissues in the HepaChip-MP. Metabolic function including induction of CYP enzymes in PHH was successfully measured demonstrating a high degree of metabolic activity of cells in HepaChip-MP cultures and sufficient sensitivity of LC-MS analysis even for the relatively small number of cells per chamber. Further, parallelization realized in HepaChip-MP enabled the acquisition of dose-response toxicity data of diclofenac with a single device. Several unique technical features should enable a widespread application of this in vitro model. We have demonstrated fully automated preparation of cell cultures in HepaChip-MP using a pipetting robot. The tubeless unidirectional perfusion system based on gravity-driven flow can be operated within a standard incubator system. Overall, the system readily integrates in workflows common in cell culture labs. Further research will be directed towards optimization of media composition to further extend culture lifetime and study oxygen gradients and their effect on zonation within the sinusoid-like microorgans. In summary, we have established a novel parallelized and scalable microfluidic in vitro liver model showing hepatocyte function and anticipate future in-depth studies of liver biology and applications in pre-clinical drug development.

Long-term simulation of lead concentrations in agricultural soils in relation to human adverse health effects.

Lead (Pb) exposure of consumers and the environment has been reduced over the past decades. Despite all measures taken, immission of Pb onto agricultural soils still occurs, with fertilizer application, lead shot from hunting activities, and Pb from air deposition representing major sources. Little is known about the intermediate and long-term consequences of these emissions. To gain more insight, we established a mathematical model that considers input from fertilizer, ammunition, deposition from air, uptake of Pb by crops, and wash-out to simulate the resulting Pb concentrations in soil over extended periods. In a further step, human oral exposure by crop-based food was simulated and blood concentrations were derived to estimate the margin of exposure to Pb-induced toxic effects. Simulating current farming scenarios, a new equilibrium concentration of Pb in soil would be established after several centuries. Developmental neurotoxicity represents the most critical toxicological effect of Pb for humans. According to our model, a Pb concentration of ~ 5 mg/kg in agricultural soil leads to an intake of approximately 10 µg Pb per person per day by the consumption of agricultural products, the dose corresponding to the tolerable daily intake (TDI). Therefore, 5 mg Pb/kg represents a critical concentration in soil that should not be exceeded. Starting with a soil concentration of 0.1 mg/kg, the current control level for crop fields, our simulation predicts periods of ~ 50 and ~ 175 years for two Pb immission scenarios for mass of Pb per area and year [scenario 1: ~ 400 g Pb/(ha × a); scenario 2: ~ 175 g Pb/(ha × a)], until the critical concentration of ~ 5 mg/kg Pb in soil would be reached. The two scenarios, which differ in their Pb input via fertilizer, represent relatively high but not unrealistic Pb immissions. From these scenarios, we calculated that the annual deposition of Pb onto soil should remain below ~ 100 g/(ha × a) in order not to exceed the critical soil level of 5 mg/kg. We propose as efficient measures to reduce Pb input into agricultural soil to lower the Pb content of compost and to use alternatives to Pb ammunition for hunting.

Effect of glucose and insulin supplementation on the isolation of primary human hepatocytes.

Primary human hepatocytes (PHHs) remain the gold standard for in vitro investigations of xenobiotic metabolism and hepatotoxicity. However, scarcity of liver tissue and novel developments in liver surgery has limited the availability and quality of tissue samples. In particular, warm ischemia shifts the intracellular metabolism from aerobic to anaerobic conditions, which increases glycogenolysis, glucose depletion and energy deficiency. Therefore, the aim of the present study was to investigate whether supplementation with glucose and insulin during PHH isolation could reconstitute intracellular glycogen storage and beneficially affect viability and functionality. Furthermore, the study elucidated whether the susceptibility of the tissue's energy status correlates with body mass index (BMI). PHHs from 12 donors were isolated from human liver tissue obtained from partial liver resections using a two-step EDTA/collagenase perfusion technique. For a direct comparison of the influence of glucose/insulin supplementation, we modified the setup, enabling the parallel isolation of two pieces of one tissue sample with varying perfusate. Independent of the BMI of the patient, the glycogen content in liver tissue was notably low in the majority of samples. Furthermore, supplementation with glucose and insulin had no beneficial effect on the glycogen concentration of isolated PHHs. However, an indirect improvement of the availability of energy was shown by increased viability, plating efficiency and partial cellular activity after supplementation. The plating efficiency showed a striking inverse correlation with increasing lipid content of PHHs. However, 60 h of cultivation time revealed no significant impact on the maintenance of albumin and urea synthesis or xenobiotic metabolism after supplementation. In conclusion, surgical procedures and tissue handling may decrease hepatic energy resources and lead to cell stress and death. Consequently, PHHs with low energy resources die during the isolation process without supplementation of glucose/insulin or early cell culture, while their survival rates are improved with glucose/insulin supplementation.