Hazard characterization of Alternaria toxins to identify data gaps and improve risk assessment for human health.

Fungi of the genus Alternaria are ubiquitous plant pathogens and saprophytes which are able to grow under varying temperature and moisture conditions as well as on a large range of substrates. A spectrum of structurally diverse secondary metabolites with toxic potential has been identified, but occurrence and relative proportion of the different metabolites in complex mixtures depend on strain, substrate, and growth conditions. This review compiles the available knowledge on hazard identification and characterization of Alternaria toxins. Alternariol (AOH), its monomethylether AME and the perylene quinones altertoxin I (ATX-I), ATX-II, ATX-III, alterperylenol (ALP), and stemphyltoxin III (STTX-III) showed in vitro genotoxic and mutagenic properties. Of all identified Alternaria toxins, the epoxide-bearing analogs ATX-II, ATX-III, and STTX-III show the highest cytotoxic, genotoxic, and mutagenic potential in vitro. Under hormone-sensitive conditions, AOH and AME act as moderate xenoestrogens, but in silico modeling predicts further Alternaria toxins as potential estrogenic factors. Recent studies indicate also an immunosuppressive role of AOH and ATX-II; however, no data are available for the majority of Alternaria toxins. Overall, hazard characterization of Alternaria toxins focused, so far, primarily on the commercially available dibenzo-α-pyrones AOH and AME and tenuazonic acid (TeA). Limited data sets are available for altersetin (ALS), altenuene (ALT), and tentoxin (TEN). The occurrence and toxicological relevance of perylene quinone-based Alternaria toxins still remain to be fully elucidated. We identified data gaps on hazard identification and characterization crucial to improve risk assessment of Alternaria mycotoxins for consumers and occupationally exposed workers.

Glioma Cells Expressing High Levels of ALDH5A1 Exhibit Enhanced Migration Transcriptional Signature in Patient Tumors.

Accumulating data shows that altered metabolic activity contributes to glioma development. Recently, modulation of SSADH (succinic semialdehyde dehydrogenase) expression, implicated in the catabolism of GABA neurotransmitter, was shown to impact glioma cell properties, such as proliferation, self-renewal and tumorigenicity. The purpose of this study was to investigate the clinical significance of SSADH expression in human gliomas. Using public single-cell RNA-sequencing data from glioma surgical resections, we initially grouped cancer cells according to ALDH5A1 (Aldehyde dehydrogenase 5 family member A1) expression, which encodes SSADH. Gene ontology enrichment analysis of genes differentially expressed between cancer cells expressing high or low levels of ALDH5A1, highlighted enrichment in genes implicated in cell morphogenesis and motility. In glioblastoma cell lines, ALDH5A1 knockdown inhibited cell proliferation, induced apoptosis and reduced their migratory potential. This was accompanied by a reduction in the mRNA levels of the adherens junction molecule ADAM-15 and deregulation in the expression of EMT biomarkers, with increased CDH1 and decreased vimentin mRNA levels. Evaluation of SSADH expression in a cohort of 95 gliomas using immunohistochemistry showed that SSADH expression was significantly elevated in cancer tissues compared to normal brain tissues, without any significant correlation with clinicopathological characteristics. In summary, our data show that SSADH is upregulated in glioma tissues irrespective of the histological grade and its expression sustains glioma cell motility.

Transcriptional analysis in peripheral blood cells of individuals with elevated phthalate exposure - Results of the EuroMix study.

Exposure to phthalates is widespread in Europe. Phthalates are considered endocrine disrupting compounds and are classified as toxic for reproduction. However how phthalates affect the transcriptome in humans remains largely unknown. To investigate the effects of phthalate exposure on the transcriptomic profile we conducted RNA sequencing on peripheral blood samples from the Norwegian EuroMix cohort. We compared gene expression changes between participants with high, medium, and low exposure of six phthalates and 1,2-cyclohexane dicarboxylic acid diisononyl ester (DINCH). Comparing high and low exposure groups, DINCH was the compound that showed the highest number of differentially expressed genes (126 genes) followed by mono-n-butyl phthalate (MnBP; 89 genes) and mono-iso-nonyl phthalate (MiBP; 70 genes). Distributions between up- or down-regulated genes were similar across the different phthalates and DINCH. All phthalates including DINCH shared common differentially expressed genes ranging from 3 to 37 overlaps. Enriched Gene Ontology (GO) and biological pathway analysis revealed that most of the differentially expressed genes were associated with general cellular metabolism GO terms. MnBP and DINCH, particularly, showed a marked enrichment in various immunological function pathways including neutrophil degranulation, adaptive immune system and signaling by interleukins. Furthermore, the association between genes involved in the peroxisome proliferator activated receptor (PPAR) signaling pathway and phthalates, including DINCH, was evaluated. In total, 15 genes showed positive or negative associations across 5 phthalates and DINCH. MnBP and MiBP were the phthalate metabolites with the highest number of associations: 8 and 4 PPAR signaling pathway genes, respectively. Overall, we have performed an association study between phthalate exposure levels and modulation of transcriptomic profiles in human peripheral blood cells. DINCH, which is often mentioned as a substitute for phthalates, had comparable effects on differential gene expression in peripheral blood cells as phthalates.

Protocol for designing INVITES-IN, a tool for assessing the internal validity of in vitro studies.

This protocol describes the design and development of a tool for evaluation of the internal validity of in vitro studies, which is needed to include the data as evidence in systematic reviews and chemical risk assessments. The tool will be designed specifically to be applied to cell culture studies, including, but not restricted to, studies meeting the new approach methodology (NAM) definition. The tool is called INVITES-IN (IN VITro Experimental Studies INternal validity). In this protocol, three of the four studies that will be performed to create the release version of INVITES-IN are described. In the first study, evaluation of existing assessment tools will be combined with focus group discussions to identify how characteristics of the design or conduct of an in vitro study can affect its internal validity. Bias domains and items considered to be of relevance for in vitro studies will be identified. In the second study, group agreement on internal validity domains and items of importance for in vitro studies will be identified via a modified Delphi methodology. In the third study, the draft version of the tool will be created, based on the data on relevance and importance of bias domains and items collected in Studies 1 and 2. A separate protocol will be prepared for the fourth study, which includes the user testing and validation of the tool, and collection of users' experience.

Investigating the in vitro steatotic mixture effects of similarly and dissimilarly acting test compounds using an adverse outcome pathway-based approach.

Within the EuroMix project, we have previously developed an adverse outcome pathway (AOP)-based in vitro assay toolbox to investigate the combined effects of liver steatosis-inducing compounds in human HepaRG hepatocarcinoma cells. In this study, we applied the toolbox to further investigate mixture effects of combinations, featuring either similarly acting or dissimilarly acting substances. The valproic acid structural analogs 2-propylheptanoic acid (PHP) and 2-propylhexanoic acid (PHX) were chosen for establishing mixtures of similarly acting substances, while a combination with the pesticidal active substance clothianidin (CTD) was chosen for establishing mixtures of dissimilarly acting compounds. We first determined relative potency factors (RPFs) for each compound based on triglyceride accumulation results. Thereafter, equipotent mixtures were tested for nuclear receptor activation in transfected HepG2 cells, while gene expression and triglyceride accumulation were investigated in HepaRG cells, following the proposed AOP for liver steatosis. Dose addition was observed for all combinations and endpoints tested, indicating the validity of the additivity assumption also in the case of the tested mixtures of dissimilarly acting substances. Gene expression results indicate that the existing steatosis AOP can still be refined with respect to the early key event (KE) of gene expression, in order to reflect the diversity of molecular mechanisms underlying the adverse outcome.

An adverse outcome pathway on the disruption of retinoic acid metabolism leading to developmental craniofacial defects.

Adverse outcome pathway (AOP) is a conceptual framework that links a molecular initiating event (MIE) via intermediate key events (KEs) with adverse effects (adverse outcomes, AO) relevant for risk assessment, through defined KE relationships (KERs). The aim of the present work is to describe a linear AOP, supported by experimental data, for skeletal craniofacial defects as the AO. This AO was selected in view of its relative high incidence in humans and the suspected relation to chemical exposure. We focused on inhibition of CYP26, a retinoic acid (RA) metabolizing enzyme, as MIE, based on robust previously published data. Conazoles were selected as representative stressors. Intermediate KEs are RA disbalance, aberrant HOX gene expression, disrupted specification, migration, and differentiation of neural crest cells, and branchial arch dysmorphology. We described the biological basis of the postulated events and conducted weight of evidence (WoE) assessments. The biological plausibility and the overall empirical evidence were assessed as high and moderate, respectively, the latter taking into consideration the moderate evidence for concordance of dose-response and temporal relationships. Finally, the essentiality assessment of the KEs, considered as high, supported the robustness of the presented AOP. This AOP, which appears of relevance to humans, thus contributes to mechanistic underpinning of selected test methods, thereby supporting their application in integrated new approach test methodologies and strategies and application in a regulatory context.

Maternal exposure to mixtures of dienestrol, linuron and flutamide. Part II: Endocrine-related gene expression assessment on male offspring rat testes.

Exposure to endocrine-disrupting compounds (EDCs) during pregnancy and early development can lead to adverse developmental outcomes in offspring. One of the endpoints of concern is feminization. The present study aimed to investigate for any possible correlations with endocrine sensitive parameters in the testes of male rat offspring following dam exposure to three EDCs by assessing the expression of endocrine-related genes. Dienestrol (DIES) [0.37-6.25 µg/kg bw/day], linuron (LIN) [1.5-50 mg/kg bw/day], flutamide (FLU) [3.5-50 mg/kg bw/day] as well as their binary mixtures were administered to sexually mature female rats from gestation day (GD) 6 until postnatal day (PND) 21. Gene expression analysis of Star, Cyp11a1, Cyp17a1, Hsd3b2, Pgr and Insl3 was performed by RT-qPCR. Administration of the anti-androgen FLU alone significantly upregulated Cyp11a1 and Cyp17a1 gene expression while administration of LIN and DIES alone did not alter significantly gene expression. The effects of the binary mixtures on gene expression were not as marked as those seen after single compound administrations. Deregulation of Cyp17a1 in rat pup testis, following administration of FLU alone or in mixtures to dams, was significantly correlated with the observed feminization endpoints in male pups.

An adverse outcome pathway-based approach to assess steatotic mixture effects of hepatotoxic pesticides in vitro.

Exposure to complex chemical mixtures requires a tiered strategy for efficient mixture risk assessment. As a part of the EuroMix project we developed an adverse outcome pathway (AOP)-based assay toolbox to investigate the combined effects of the liver steatosis-inducing compounds imazalil, thiacloprid, and clothianidin in human HepaRG hepatocarcinoma cells. Compound-specific relative potency factors were determined using a benchmark dose approach. Equipotent mixtures were tested for nuclear receptor activation, gene and protein expression, and triglyceride accumulation, according to the molecular initiating events and key events proposed in the steatosis AOP. All three compounds affected the activity of nuclear receptors, but not key genes/proteins as proposed. Triglyceride accumulation was observed with three different methods. Mixture effects were in agreement with the assumption of dose additivity for all the combinations and endpoints tested. Compound-specific RPFs remained similar over the different endpoints studied downstream the AOP. Therefore, it might be possible to reduce testing to a smaller battery of key tests. The results demonstrate the suitability of our in vitro assay toolbox, integrated within an AOP framework and combined with the RPF approach, for the analysis of steatotic effects of chemical mixtures. However, mRNA results suggest that the steatosis AOP still needs improvement.

Adverse Outcome Pathway-Driven Analysis of Liver Steatosis in Vitro: A Case Study with Cyproconazole.

Adverse outcome pathways (AOPs) describe causal relationships between molecular perturbation and adverse cellular effects and are being increasingly adopted for linking in vitro mechanistic toxicology to in vivo data from regulatory toxicity studies. In this work, a case study was performed by developing a bioassay toolbox to assess key events in the recently proposed AOP for chemically induced liver steatosis. The toolbox is comprised of in vitro assays to measure nuclear receptor activation, gene and protein expression, lipid accumulation, mitochondrial respiration, and formation of fatty liver cells. Assay evaluation was performed in human HepaRG hepatocarcinoma cells exposed to the model compound cyproconazole, a fungicide inducing steatosis in rodents. Cyproconazole dose-dependently activated RARα and PXR, two molecular initiating events in the steatosis AOP. Moreover, cyproconazole provoked a disruption of mitochondrial functions and induced triglyceride accumulation and the formation of fatty liver cells as described in the AOP. Gene and protein expression analysis, however, showed expression changes different from those proposed in the AOP, thus suggesting that the current version of the AOP might not fully reflect the complex mechanisms linking nuclear receptor activation and liver steatosis. Our study shows that cyproconazole induces steatosis in human liver cells in vitro and demonstrates the utility of systems-based approaches in the mechanistic assessment of molecular and cellular key events in an AOP. AOP-driven in vitro testing as demonstrated can further improve existing AOPs, provide insight regarding molecular mechanisms of toxicity, and inform predictive risk assessment.

Continuous hydrostatic pressure induces differentiation phenomena in chondrocytes mediated by changes in polycystins, SOX9, and RUNX2.

PURPOSE: The present study aimed to investigate the long-term effects of hydrostatic pressure on chondrocyte differentiation, as indicated by protein levels of transcription factors SOX9 and RUNX2, on transcriptional activity of SOX9, as determined by pSOX9 levels, and on the expression of polycystin-encoding genes Pkd1 and Pkd2. MATERIALS AND METHODS: ATDC5 cells were cultured in insulin-supplemented differentiation medium (ITS) and/or exposed to 14.7 kPa of hydrostatic pressure for 12, 24, 48, and 96 h. Cell extracts were assessed for SOX9, pSOX9, and RUNX2 using western immunoblotting. The Pkd1 and Pkd2 mRNA levels were detected by real-time PCR. RESULTS: Hydrostatic pressure resulted in an early drop in SOX9 and pSOX9 protein levels at 12 h followed by an increase from 24 h onwards. A reverse pattern was followed by RUNX2, which reached peak levels at 24 h of hydrostatic pressure-treated chondrocytes in ITS culture. Pkd1 and Pkd2 mRNA levels increased at 24 h of combined hydrostatic pressure and ITS treatment, with the latter remaining elevated up to 96 h. CONCLUSIONS: Our data indicate that long periods of continuous hydrostatic pressure stimulate chondrocyte differentiation through a series of molecular events involving SOX9, RUNX2, and polycystins-1, 2, providing a theoretical background for functional orthopedic mechanotherapies.

Mechanosensor polycystin-1 potentiates differentiation of human osteoblastic cells by upregulating Runx2 expression via induction of JAK2/STAT3 signaling axis.

Polycystin-1 (PC1) has been proposed as a chief mechanosensing molecule implicated in skeletogenesis and bone remodeling. Mechanotransduction via PC1 involves proteolytic cleavage of its cytoplasmic tail (CT) and interaction with intracellular pathways and transcription factors to regulate cell function. Here we demonstrate the interaction of PC1-CT with JAK2/STAT3 signaling axis in mechanically stimulated human osteoblastic cells, leading to transcriptional induction of Runx2 gene, a master regulator of osteoblastic differentiation. Primary osteoblast-like PC1-expressing cells subjected to mechanical-stretching exhibited a PC1-dependent increase of the phosphorylated(p)/active form of JAK2. Specific interaction of PC1-CT with pJAK2 was observed after stretching while pre-treatment of cells with PC1 (anti-IgPKD1) and JAK2 inhibitors abolished JAK2 activation. Consistently, mechanostimulation triggered PC1-mediated phosphorylation and nuclear translocation of STAT3. The nuclear phosphorylated(p)/DNA-binding competent pSTAT3 levels were augmented after stretching followed by elevated DNA-binding activity. Pre-treatment with a STAT3 inhibitor either alone or in combination with anti-IgPKD1 abrogated this effect. Moreover, PC1-mediated mechanostimulation induced elevation of Runx2 mRNA levels. ChIP assays revealed direct regulation of Runx2 promoter activity by STAT3/Runx2 after mechanical-stretching that was PC1-dependent. Our findings show that mechanical load upregulates expression of Runx2 gene via potentiation of PC1-JAK2/STAT3 signaling axis, culminating to possibly control osteoblastic differentiation and ultimately bone formation.

Mechanotransduction pathways in bone pathobiology.

The skeleton is subject to dynamic changes throughout life and bone remodeling is essential for maintenance of bone functionality. The cell populations which predominantly participate in bone and cartilage remodeling, namely osteocytes, osteoblasts, osteoclasts and chondrocytes sense and respond to external mechanical signals and via a series of molecular cascades control bone metabolism and turnover rate. The aforementioned process, known as mechanotransduction, is the underlying mechanism that controls bone homeostasis and function. A wide array of cross-talking signaling pathways has been found to play an important role in the preservation of bone and cartilage tissue health. Moreover, alterations in bone mechanotransduction pathways, due to genetic, hormonal and biomechanical factors, are considered responsible for the pathogenesis of bone and cartilage diseases. Extensive research has been conducted and demonstrated that aberrations in mechanotransduction pathways result in disease-like effects, however only few signaling pathways have actually been engaged in the development of bone disease. The aim of the present review is to present these signaling molecules and cascades that have been found to be mechano-responsive and implicated in bone disease development, as revealed by research in the last five years. In addition, the role of these molecules as prognostic or diagnostic disease markers and their potential as therapeutic targets are also discussed.

Emerging role of linker histone variant H1x as a biomarker with prognostic value in astrocytic gliomas. A multivariate analysis including trimethylation of H3K9 and H4K20.

Although epigenetic alterations play an essential role in gliomagenesis, the relevance of aberrant histone modifications and the respective enzymes has not been clarified. Experimental data implicates histone H3 lysine (K) methyltransferases SETDB1 and SUV39H1 into glioma pathobiology, whereas linker histone variant H1.0 and H4K20me3 reportedly affect prognosis. We investigated the expression of H3K9me3 and its methyltransferases along with H4K20me3 and H1x in 101 astrocytic tumors with regard to clinicopathological characteristics and survival. The effect of SUV39H1 inhibition by chaetocin on the proliferation, colony formation and migration of T98G cells was also examined. SETDB1 and cytoplasmic SUV39H1 levels increased from normal brain through low-grade to high-grade tumors, nuclear SUV39H1 correlating inversely with grade. H3K9me3 immunoreactivity was higher in normal brain showing no association with grade, whereas H1x and H4K20me3 expression was higher in grade 2 than in normal brain or high grades. These expression patterns of H1x, H4K20me3 and H3K9me3 were verified by Western immunoblotting. Chaetocin treatment significantly reduced proliferation, clonogenic potential and migratory ability of T98G cells. H1x was an independent favorable prognosticator in glioblastomas, this effect being validated in an independent set of 66 patients. Diminished nuclear SUV39H1 expression adversely affected survival in univariate analysis. In conclusion, H4K20me3 and H3K9 methyltransferases are differentially implicated in astroglial tumor progression. Deregulation of H1x emerges as a prognostic biomarker.

Complex interactions between the components of the PI3K/AKT/mTOR pathway, and with components of MAPK, JAK/STAT and Notch-1 pathways, indicate their involvement in meningioma development.

We investigated the significance of PI3K/AKT/mTOR pathway and its interactions with MAPK, JAK/STAT and Notch pathways in meningioma progression. Paraffin-embedded tissue from 108 meningioma patients was analysed for the presence of mutations in PIK3CA and AKT1. These were correlated with the expression status of components of the PI3K/AKT/mTOR pathway, including p85α and p110γ subunits of PI3K, phosphorylated (p)-AKT, p-mTOR, p-p70S6K and p-4E-BP1, as well as of p-ERK1/2, p-STAT3 and Notch-1, clinicopathological data and patient survival. A mutation in PIK3CA or AKT1 was found in around 9 % of the cases. Higher grade meningiomas displayed higher nuclear expression of p-p70S6K; higher nuclear and cytoplasmic expression of p-4E-BP1 and of Notch-1; lower cytoplasmic expression of p85αPI3K, p-p70S6K and p-ERK1/2; and lower PTEN Histo-scores (H-scores). PTEN H-score was inversely correlated with recurrence probability. In univariate survival analysis, nuclear expression of p-4E-BP1 and absence of p-ERK1/2 expression portended adverse prognosis, whereas in multivariate survival analysis, p-ERK1/2 expression emerged as an independent favourable prognostic factor. Treatment of the human meningioma cell line HBL-52 with the PI3K inhibitor LY294002 resulted in reduction of p-AKT, p-p70S6K and p-ERK1/2 protein levels. The complex interactions established between components of the PI3K/AKT/mTOR pathway, or with components of the MAPK, JAK/STAT and Notch-1 pathways, appear to be essential for facilitating and fuelling meningioma progression.

Role of histone lysine methyltransferases SUV39H1 and SETDB1 in gliomagenesis: modulation of cell proliferation, migration, and colony formation.

Posttranslational modifications of histones are considered as critical regulators of gene expression, playing significant role in the pathogenesis and progression of tumors. Trimethylation of histone 3 lysine 9 (H3K9me3), a repressed transcription mark, is mainly regulated by the histone lysine N-methyltransferases (HKMTs), SUV39H1 and SETDB1. The present study investigated the implication of these HKMTs in glioma progression. SUV39H1 and SETDB1 expression was upregulated in glioma cell lines (GOS-3, 1321N1, T98G, U87MG) and in glioma tissues compared to normal brain being positively correlated with grade and histological malignancy. Suppression by siRNA of the two HKMTs for 24 and 48 h resulted in significantly reduced proliferation of GOS-3 and T98G glioma cells with siSUV39H1 effects been most prominent. Furthermore, HKMTs knockdown-induced apoptosis with a high rate of apoptotic cells have been observed after siSUV39H1 and siSETDB1 for both cell lines. Additionally, suppression of the two HKMTs reduced cell migration and clonogenic ability of both glioma cell lines. Our results indicate overexpression of SETDB1 and SUV39H1 in gliomas. Treatments that alter HKMT expression affect the proliferative and apoptotic rates in glioma cells as well as their migratory and colony formation capacity. These data suggest that both HKMTs and especially SUV39H1 may serve as novel biomarkers for future therapeutic targeting of these tumors.

Deregulated chromatin remodeling in the pathobiology of brain tumors.

Brain tumors encompass a heterogeneous group of malignant tumors with variable histopathology, aggressiveness, clinical outcome and prognosis. Current gene expression profiling studies indicate interplay of genetic and epigenetic alterations in their pathobiology. A central molecular event underlying epigenetics is the alteration of chromatin structure by post-translational modifications of DNA and histones as well as nucleosome repositioning. Dynamic remodeling of the fundamental nucleosomal structure of chromatin or covalent histone marks located in core histones regulate main cellular processes including DNA methylation, replication, DNA-damage repair as well as gene expression. Deregulation of these processes has been linked to tumor suppressor gene silencing, cancer initiation and progression. The reversible nature of deregulated chromatin structure by DNA methylation and histone deacetylation inhibitors, leading to re-expression of tumor suppressor genes, makes chromatin-remodeling pathways as promising therapeutic targets. In fact, a considerable number of these inhibitors are being tested today either alone or in combination with other agents or conventional treatments in the management of brain tumors with considerable success. In this review, we focus on the mechanisms underpinning deregulated chromatin remodeling in brain tumors, discuss their potential clinical implications and highlight the advances toward new therapeutic strategies.