Exploring BPA alternatives - Environmental levels and toxicity review.

Bisphenol A alternatives are manufactured as potentially less harmful substitutes of bisphenol A (BPA) that offer similar functionality. These alternatives are already in the market, entering the environment and thus raising ecological concerns. However, it can be expected that levels of BPA alternatives will dominate in the future, they are limited information on their environmental safety. The EU PARC project highlights BPA alternatives as priority chemicals and consolidates information on BPA alternatives, with a focus on environmental relevance and on the identification of the research gaps. The review highlighted aspects and future perspectives. In brief, an extension of environmental monitoring is crucial, extending it to cover BPA alternatives to track their levels and facilitate the timely implementation of mitigation measures. The biological activity has been studied for BPA alternatives, but in a non-systematic way and prioritized a limited number of chemicals. For several BPA alternatives, the data has already provided substantial evidence regarding their potential harm to the environment. We stress the importance of conducting more comprehensive assessments that go beyond the traditional reproductive studies and focus on overlooked relevant endpoints. Future research should also consider mixture effects, realistic environmental concentrations, and the long-term consequences on biota and ecosystems.

Priorities and Challenges in Methodology for Human Health Risk Assessment from Combined Exposure to Multiple Chemicals.

This paper reviews key elements in the assessment of human health effects from combined exposure to multiple chemicals taking into consideration current knowledge and challenges to identify areas where scientific advancement is mostly needed and proposes a decision-making scheme on the basis of existing methods and tools. The assumption of dose addition and estimation of the hazard index (HI) is considered as a starting point in component-based risk assessments. When, based on the generic HI approach, an unacceptable risk is identified, more specific risk assessment options may be implemented sequentially or in parallel depending on problem formulation, characteristics of the chemical group under assessment, exposure levels, data availability and resources. For prospective risk assessments, the reference point index/margin of exposure (RPI/MOET) (Option 1) or modified RPI/normalized MOET (mRPI/nMOET) (Option 2) approaches may be implemented focusing on the specific mixture effect. Relative potency factors (RPFs) may also be used in the RPI approach since a common uncertainty factor for each mixture component is introduced in the assessment. Increased specificity in the risk assessment may also be achieved when exposure of selected population groups is considered (Option 3/exposure). For retrospective risk assessments, human biomonitoring data available for vulnerable population groups (Option 3/susceptibility) may present more focused scenarios for consideration in human health risk management decisions. In data-poor situations, the option of using the mixture assessment factor (MAF) is proposed (Option 4), where an additional uncertainty factor is applied on each mixture component prior to estimating the HI. The magnitude of the MAF may be determined by the number of mixture components, their individual potencies and their proportions in the mixture, as previously reported. It is acknowledged that implementation of currently available methods and tools for human health risk assessment from combined exposure to multiple chemicals by risk assessors will be enhanced by ongoing scientific developments on new approach methodologies (NAMs), integrated approaches to testing and assessment (IATA), uncertainty analysis tools, data sharing platforms, risk assessment software as well as guideline development to meet legislative requirements.

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.

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.

Comparative UHPLC-HRMS Profiling, Toxicological Assessment, and Protection Against H2O2-Induced Genotoxicity of Different Parts of Opuntia ficus indica.

Opuntia ficus indica has been an important dietary source and a traditionally used medicinal plant. Given the promising health-promoting properties of this plant, a comparative toxicological assessment and antioxidant bioevaluation of extracts from different parts of the plant were carried out in relation to their chemical profile. Toxicity was examined at multiple endpoints using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), Comet and the γH2AX In-Cell Western Assay, while hyphenated ultra-high-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS) analysis was carried out to identify main constituents. None of the extracts showed any cytotoxic and genotoxic effect on cell lines used, apart from the flower extract in HepG2 cells at the highest concentration tested (2.5 mg/mL). Both fruit flesh and seed extracts demonstrated a prominent protective effect against H2O2-induced genotoxicity in almost all concentrations tested, while extracts originated from flowers and cladodes were effective only at the low non-cytotoxic (0.312 and 0.625 mg/mL) and high (1.25 and 2.5 mg/mL) concentrations, respectively. In total, 2 phenolic acids, 12 flavonoids, along with 3 feruloyl derivatives and the plant pigment indicaxanthin, were tentatively identified by UHPLC-HRMS analysis. Phenolic acids (compounds 1 and 2) were mainly distributed in cladodes (64.6%), while flavonoids (3-14) in the flowers (81.8%). Overall, the highest amount of total flavonoids (22.76 ± 0.015 mg of quercetin equivalent [QE]/g) and total phenolics (62.80 ± 0.009 mg gallic acid equivalents [GAE]/g) was found in the flower extract. Flavonoid glycosides have not been detected in the seeds and the flesh, while the fruit seed extract contained mainly feruloyl derivatives. Our data provide convincing evidences for the lack of cytotoxic and genotoxic effects of O. ficus indica aqueous extracts and, in parallel, support the potential for further exploitation of this plant in the food supplement or functional food sector.

Modulation of CYP1A1 and CYP1A2 hepatic enzymes after oral administration of Chios mastic gum to male Wistar rats.

Chios mastic gum (CMG), a resin derived from Pistacia lentiscus var. chia, is known since ancient times for its pharmacological activities. CYP1A1 and CYP1A2 enzymes are among the most involved in the biotransformation of chemicals and the metabolic activation of pro-carcinogens. Previous studies referring to the modulation of these enzymes by CMG have revealed findings of unclear biological and toxicological significance. For this purpose, the modulation of CYP1A1 and CYP1A2 enzymes in the liver of male Wistar rats following oral administration of CMG extract (CMGE), at the levels of mRNA and CYP1A1 enzyme activity, was compared to respective enzyme modulation following oral administration of a well-known bioactive natural product, caffeine, as control compound known to involve hepatic enzymes in its metabolism. mRNA levels of Cyp1a1 and Cyp1a2 were measured by reverse transcription real-time polymerase chain reaction and their relative quantification was calculated. CYP1A1 enzyme induction was measured through the activity of ethoxyresorufin-O-deethylase (EROD). The results indicated that administration of CMGE at the recommended pharmaceutical dose does not induce significant transcriptional modulation of Cyp1a1/2 and subsequent enzyme activity induction of CYP1A1 while effects of the same order of magnitude were observed in the same test system following the administration of caffeine at the mean daily consumed levels. The outcome of this study further confirms the lack of any toxicological or biological significance of the specific findings on liver following the administration of CMGE.

Monitoring of systemic exposure to plant protection products and DNA damage in orchard workers.

The systemic exposure of plum tree growers and operators to plant protection products (PPPs) and effects on DNA were assessed. Specifically, a GC-MS/MS method was developed and validated for the analysis of serum samples for the presence of seven active substances of PPPs. The analytical results verified the presence of myclobutanil, propargite, cypermethrin and deltamethrin in 7 out of 19 serum samples. The incidence of DNA damage was monitored using the single cell electrophoresis assay (comet assay). A paired Student's t-test revealed a statistically significant increase of SSBs in the blood samples collected at the end of the cropping period as compared to the samples collected from the same subjects before the start of PPPs application period. Moreover, the group of seven subjects with detectable serum pesticides levels revealed statistically significant increase of SSBs as compared to the group of subjects with no detectable PPP levels. The results of the present study demonstrate that the agriculture workers may exhibit detectable level of systemic exposure to the applied PPPs which are correlated to increased DNA damage during the cultivation period.

The adhesion molecule TAG-1 is required for proper migration of the superficial migratory stream in the medulla but not of cortical interneurons.

The neural cell adhesion molecule TAG-1 has been implicated in the tangential migration of neurons of the caudal medulla and of cortical interneurons. In the former case, protein is expressed by the neurons as they migrate, and blocking its function results in altered and reduced migration in vitro. In the latter case, protein is expressed, in part, by the pathway the interneurons use to reach the cortex, and in vitro experiments propose a role for TAG-1 in this system, as well. However, the in vivo requirement of TAG-1 in these migrations has not been investigated. In this report, we analyze the developmental phenotype of TAG-1-deficient animals in these two migratory systems. We show that mutant mice have smaller lateral reticular nuclei as a result of increased cell death in the superficial migratory stream of the caudal medulla. On the other hand, the absence of TAG-1 does not affect the number, morphology, timing and routes of GABAergic interneurons that migrate from the ganglionic eminences to the cortex. Therefore, TAG-1 function is required for the survival of the neurons of some precerebellar nuclei, while it is not required for cortical interneuron migration in vivo.

A combination of chain and neurophilic migration involving the adhesion molecule TAG-1 in the caudal medulla.

Neuronal populations destined to form several precerebellar nuclei are generated by the rhombic lip in the caudal hindbrain. These immature neurons gather into the olivary and the superficial migratory streams and migrate tangentially around the hindbrain to reach their final position. We focus on the cells of the superficial stream that migrate ventrally, cross the midline and form the lateral reticular (LRN) and external cuneate (ECN) nuclei. The cells of the superficial steam are preceded by long leading processes; in the dorsal neural tube, they migrate in close apposition to each other and form distinct chains, whereas they disperse and follow Tuj-1 immunoreactive axons on reaching the ventral hindbrain. This suggests that, in the superficial stream, neuronal migration combines both homotypic and heterotypic mechanisms. We also show that the adhesion molecule TAG-1 is expressed by the migrating cells. Blocking TAG-1 function results in alterations in the superficial migration, indicating that TAG-1 is involved in the superficial migration. Other members of the immunoglobulin superfamily and known ligands of TAG-1 are also expressed in the region of the migration but are not involved in the migration. These findings provide evidence that the TAG-1 protein is involved as a contact-dependent signal guiding not only axonal outgrowth but also cell migration.

Novel sites of expression of the bHLH gene NSCL1 in the developing nervous system.

We report on novel sites of expression of the bHLH transcription factor NSCL1 in the developing forebrain, hindbrain and spinal cord in chick and mouse. In the hindbrain in particular, NSCL1 is the first bHLH transcription factor detected so far in rhombomere boundaries and its expression is coincident with boundary formation and maintenance. Novel sites of expression of this gene include the hippocampus, septum, tectum and hypothalamic nuclei. NSCL1 is thus expressed in various neuronal populations that are either not actively proliferating or postmitotic.

Novel sites of expression of the bHLH gene NSCL1 in the developing nervous system.

We report on novel sites of expression of the bHLH transcription factor NSCL1 in the developing forebrain, hindbrain and spinal cord in chick and mouse. In the hindbrain in particular, NSCL1 is the first bHLH transcription factor detected so far in rhombomere boundaries and its expression is coincident with boundary formation and maintenance. Novel sites of expression of this gene include the hippocampus, septum, tectum and hypothalamic nuclei. NSCL1 is thus expressed in various neuronal populations that are either not actively proliferating or postmitotic.