Review of dose setting for the extended one-generation reproductive toxicity studies (OECD TG 443): Considerations on ECHA's dose level selection recommendations.

During 2020, The European Chemicals Agency (ECHA) began evaluating the OECD Test Guideline 443: Extended One Generation Reproductive Toxicity Study (EOGRTS) to analyze specific aspects related to study design, conduct and toxicological findings. A significant outcome of this ECHA evaluation focused on adequate dose level selection. Subsequently, ECHA published recommendations for DART studies, however, these recommendations seemingly do not align with the principles of the 3Rs, animal welfare or human safety goals, specifically, regarding three aspects. First, the requirement to segregate testing for sexual function and fertility from the ability to produce normally developing offspring increases the risk of inadequate identification of postnatal hazards for development and sexual function and fertility, therefore failing human health protection goals. Second, the current ECHA high-dose level setting recommendations for EOGRTS exceed the MTD (Maximum Tolerated Dose), and therefore compromise the interpretation of the biological response relative to the intrinsic effect of the chemical under evaluation. Third, the combination of these aspects will result in an increase in the number of animals tested, increasing animal welfare concerns. This paper reflects the consensus of subject matter experts, professional, and scientific societies who have authored and signed on to this statement. The signatories encourage ECHA to adopt a revised science-driven approach to the dose selection criteria that strikes a balance between regulatory vigilance and scientific pragmatism.

Mixed Patterns of Intergenerational DNA Methylation Inheritance in Acropora.

For sessile organisms at high risk from climate change, phenotypic plasticity can be critical to rapid acclimation. Epigenetic markers like DNA methylation are hypothesized as mediators of plasticity; methylation is associated with the regulation of gene expression, can change in response to ecological cues, and is a proposed basis for the inheritance of acquired traits. Within reef-building corals, gene-body methylation (gbM) can change in response to ecological stressors. If coral DNA methylation is transmissible across generations, this could potentially facilitate rapid acclimation to environmental change. We investigated methylation heritability in Acropora, a stony reef-building coral. Two Acropora millepora and two Acropora selago adults were crossed, producing eight offspring crosses (four hybrid, two of each species). We used whole-genome bisulfite sequencing to identify methylated loci and allele-specific alignments to quantify per-locus inheritance. If methylation is heritable, differential methylation (DM) between the parents should equal DM between paired offspring alleles at a given locus. We found a mixture of heritable and nonheritable loci, with heritable portions ranging from 44% to 90% among crosses. gBM was more heritable than intergenic methylation, and most loci had a consistent degree of heritability between crosses (i.e. the deviation between parental and offspring DM were of similar magnitude and direction). Our results provide evidence that coral methylation can be inherited but that heritability is heterogenous throughout the genome. Future investigations into this heterogeneity and its phenotypic implications will be important to understanding the potential capability of intergenerational environmental acclimation in reef building corals.

Towards a science-based testing strategy to identify maternal thyroid hormone imbalance and neurodevelopmental effects in the progeny - Part IV: the ECETOC and CLE Proposal for a Thyroid Function-Related Neurodevelopmental Toxicity Testing and Assessment Scheme (Thyroid-NDT-TAS).

Following the European Commission Endocrine Disruptor Criteria, substances shall be considered as having endocrine disrupting properties if they (a) elicit adverse effects, (b) have endocrine activity, and (c) the two are linked by an endocrine mode-of-action (MoA) unless the MoA is not relevant for humans. A comprehensive, structured approach to assess whether substances meet the Endocrine Disruptor Criteria for the thyroid modality (EDC-T) is currently unavailable. Here, the European Centre for Ecotoxicology and Toxicology of Chemicals Thyroxine Task Force and CropLife Europe propose a Thyroid Function-Related Neurodevelopmental Toxicity Testing and Assessment Scheme (Thyroid-NDT-TAS). In Tier 0, before entering the Thyroid-NDT-TAS, all available in vivo, in vitro and in silico data are submitted to weight-of-evidence (WoE) evaluations to determine whether the substance of interest poses a concern for thyroid disruption. If so, Tier 1 of the Thyroid-NDT-TAS includes an initial MoA and human relevance assessment (structured by the key events of possibly relevant adverse outcome pathways) and the generation of supportive in vitro/in silico data, if relevant. Only if Tier 1 is inconclusive, Tier 2 involves higher-tier testing to generate further thyroid- and/or neurodevelopment-related data. Tier 3 includes the final MoA and human relevance assessment and an overarching WoE evaluation to draw a conclusion on whether, or not, the substance meets the EDC-T. The Thyroid-NDT-TAS is based on the state-of-the-science, and it has been developed to minimise animal testing. To make human safety assessments more accurate, it is recommended to apply the Thyroid-NDT-TAS during future regulatory assessments.

Towards a science-based testing strategy to identify maternal thyroid hormone imbalance and neurodevelopmental effects in the progeny-part III: how is substance-mediated thyroid hormone imbalance in pregnant/lactating rats or their progeny related to neurodevelopmental effects?

This review investigated which patterns of thyroid- and brain-related effects are seen in rats upon gestational/lactational exposure to 14 substances causing thyroid hormone imbalance by four different modes-of-action (inhibition of thyroid peroxidase, sodium-iodide symporter and deiodinase activities, enhancement of thyroid hormone clearance) or to dietary iodine deficiency. Brain-related parameters included motor activity, cognitive function, acoustic startle response, hearing function, periventricular heterotopia, electrophysiology and brain gene expression. Specific modes-of-action were not related to specific patterns of brain-related effects. Based upon the rat data reviewed, maternal serum thyroid hormone levels do not show a causal relationship with statistically significant neurodevelopmental effects. Offspring serum thyroxine together with offspring serum triiodothyronine and thyroid stimulating hormone appear relevant to predict the likelihood for neurodevelopmental effects. Based upon the collated database, thresholds of ≥60%/≥50% offspring serum thyroxine reduction and ≥20% and statistically significant offspring serum triiodothyronine reduction indicate an increased likelihood for statistically significant neurodevelopmental effects; accuracies: 83% and 67% when excluding electrophysiology (and gene expression). Measurements of brain thyroid hormone levels are likely relevant, too. The extent of substance-mediated thyroid hormone imbalance appears more important than substance mode-of-action to predict neurodevelopmental impairment in rats. Pertinent research needs were identified, e.g. to determine whether the phenomenological offspring thyroid hormone thresholds are relevant for regulatory toxicity testing. The insight from this review shall be used to suggest a tiered testing strategy to determine whether gestational/lactational substance exposure may elicit thyroid hormone imbalance and potentially also neurodevelopmental effects.

Copper transporter 1 (CTR1) expression by mouse testicular germ cells, but not Sertoli cells, is essential for functional spermatogenesis.

An imbalance in copper (Cu) tissue homeostasis has a degenerative effect on spermatogenesis and male fertility. The high-affinity Cu transporter 1 (CTR1; SLC31A1) is the major protein responsible for Cu acquisition in eukaryotes and is highly expressed in mouse testes. Studies on yeast and Drosophila have demonstrated the conserved essential function of Cu and CTR1 for meiosis and fertility, implying that CTR1 may play an essential function in mammalian spermatogenesis. In mice, spermatogenesis takes place within the seminiferous epithelium, where tight junctions between somatic Sertoli cells (SCs) create a specialized microenvironment for the development of meiotic germ cells (GCs) by tightly regulating the free transport of metabolites and ions to reach these cells. Here, it is demonstrated that within the seminiferous epithelium, CTR1 is expressed on the membrane of primary pachytene spermatocytes and SCs. To examine the physiological significance of CTR1 in spermatogenesis, mice with a GC-specific (Ctr1ΔGC) and SC-specific (Ctr1ΔSC) disruption of the Ctr1 gene were generated. The testis of Ctr1ΔGC mice exhibits a severe progressive loss of GCs starting at postnatal day (PND) 28 leading to testis hypoplasia by adulthood. No spermatogenic recovery was observed in Ctr1ΔGC testis beyond PND 41, despite the presence of FOXO-1 expressing undifferentiated spermatogonial cells. However, Ctr1ΔSC mice displayed functional spermatogenesis and were fertile, even though testicular Cu levels and Cu-dependent cellular activities were significantly reduced. These results reveal, for the first time, the importance of CTR1 expression by GCs for maintaining functional spermatogenesis.

Mice with a Sertoli cell-specific knockout of the Ctr1 gene exhibit a reduced sensitivity to cisplatin-induced testicular germ cell apoptosis.

Exposure to the chemotherapeutic agent cis-diamminedichloroplatinum(ii) (cDDP) is well known to instigate acute and prolonged testicular injury in male patients. Many investigators have hypothesized that cDDP-induced dysfunction of Sertoli cells (SCs) may, in part, account for the cDDP-induced lasting testicular injury. Nevertheless, the relative contribution of cDDP-induced SC injury versus direct effects on germ cells (GCs) to the pathogenesis of GC loss remains to be elucidated. The expression of the copper transporter 1 (CTR1) protein in cells directly corresponds with cDDP uptake and its cellular toxicity. Therefore, to discern the role of SCs in the pathogenic mechanism, mice were developed with a SC-specific disruption of the Ctr1 gene (SC ΔCtr1 ) as a strategy to prevent their exposure to cDDP. Adult mice at postnatal day (PND) 60 were treated with 5 mg kg-1 cDDP and then testis collected at 48 hours. A two-fold increase in GC-apoptosis occurred in the testis of cDDP-treated wildtype (WT) mice as compared to saline-treated WT mice. In contrast, cDDP-treated SC ΔCtr1 mice exhibited only a half-fold increase in GC-apoptosis as compared to the saline-treated SC ΔCtr1 mice. This reduced incidence of GC apoptosis in the SC ΔCtr1 mice corresponded to a significantly lower level of platinum within the testis. Taken together, these findings reveal that the uptake of cDDP by CTR1 in SCs accounts for the accumulation of cDDP in the testis and plays a pivotal role in the pathogenic sequence of events leading to the loss of germ cells via apoptosis.

MEHP-induced rat testicular inflammation does not exacerbate germ cell apoptosis.

The testis is an organ that maintains an immune suppressive environment. We previously revealed that exposure of pre-pubertal rats to an acute dose of a well-described Sertoli cell toxicant, mono-(2-ethylhexyl) phthalate (MEHP), leads to an accumulation of CD11b+ immune cells in the testicular interstitial space that closely correlates with a robust incidence of germ cell (GC) apoptosis. Here, we test the hypothesis that the infiltrating immune cells contribute to GC apoptosis. Postnatal day 28 Fischer rats that received an oral dose of 700 mg/kg MEHP showed a significant infiltration of both CD11bc+/CD68+/CD163- macrophages and neutrophils. The infiltration peaked at 12 h, but had reduced by 48 h. Testicular macrophages from MEHP-treated rats showed significantly upregulated expression of Tnfa and Il6, and the Arg1/Nos2 ratio was reduced compared to controls. However, small increases in anti-inflammatory genes Il10 and Tgfb1 were also observed. Depletion of circulating monocytes with clodronate liposomes prior to MEHP treatment reduced the macrophage influx into the testis, but did not lower GC apoptosis. Additionally, depletion of neutrophils using an anti-polymorphonuclear cell antibody prevented both macrophage and neutrophil infiltration into the testis, and also did not affect GC apoptosis. Together, these results show that exposure to MEHP leads to a rapid and temporary influx of pro-inflammatory monocytes and neutrophils in the interstitium of the testis. However, with this acute dosing paradigm, these infiltrating leukocytes do not appear to contribute to MEHP-induced testicular GC apoptosis leaving the functional significance of these infiltrating cells in the pathogenesis of MEHP-induced testicular injury unresolved.

Mono-(2-ethylhexyl) phthalate-induced Sertoli cell injury stimulates the production of pro-inflammatory cytokines in Fischer 344 rats.

Exposure of rodents to the Sertoli cell (SC) toxicant mono-(2-ethylhexyl) phthalate (MEHP) has been reported to trigger an infiltration of macrophages into the testis in an age- and species-dependent manner. Here we challenge the hypothesis that the peripubertal rat-specific infiltration of macrophages after MEHP exposure is due, in part, to an increase in SC-specific inflammatory cytokine expression. To rule out that germ cell(GC) apoptosis itself is responsible for macrophage recruitment, rats were exposed to a direct GC toxicant, methoxyacetic acid (MAA), but no infiltration of macrophages was observed. Next, mRNA levels of inflammatory cytokines were evaluated after MEHP exposure. IL-1α, IL-6, and MCP-1 expression were increased in vivo and correlated with macrophage infiltration in a species-specific manner. Additionally, IL-6 and MCP-1 expression was increased in SC-GC co-cultures and ASC-17D SCs. These results indicate that MEHP-injury in pubertal rats specifically stimulates secretion of pro-inflammatory cytokines and alters the immune microenvironment.

Maize chromosomal knobs are located in gene-dense areas and suppress local recombination.

Knobs are conspicuous heterochromatic regions found on the chromosomes of maize and its relatives. The number, locations, and sizes of knobs vary dramatically, with most lines containing between four and eight knobs in mid-arm positions. Prior data suggest that some knobs may reduce recombination. However, comprehensive tests have not been carried out, primarily because most knobs have not been placed on the genetic map. We used fluorescent in situ hybridization and two recombinant inbred populations to map seven knobs and to accurately place three knobs from the B73 inbred on the genomic sequence assembly. The data show that knobs lie in gene-dense regions of the maize genome. Comparisons to 23 other recombinant inbred populations segregating for knobs at the same sites confirm that large knobs can locally reduce crossing over by as much as twofold on a cM/Mb scale. These effects do not extend beyond regions ~10 cM to either side of knobs and do not appear to affect linkage disequilibrium among genes within and near knob repeat regions of the B73 RefGen_v2 assembly.

Properties of tryptophan indole-lyase from a piezophilic bacterium, Photobacterium profundum SS9.

Tryptophan indole-lyase (Trpase), PBPRA2532, from Photobacterium profundum SS9, a piezophilic marine bacterium, has been cloned, expressed in Escherichia coli, and purified. The P. profundum Trpase (PpTrpase) exhibits similar substrate specificity as the enzyme from E. coli (EcTrpase). PpTrpase has an optimum temperature for activity at about 30°C, compared with 53°C for EcTrpase, and loses activity rapidly (t(1/2)∼30min) when incubated at 50°C, while EcTrpase is stable up to 65°C. PpTrpase retains complete activity when incubated more than 3h at 0°C, while EcTrpase has only about 20% remaining activity. Under hydrostatic pressure, PpTrpase remains fully active up to 100MPa (986atm), while EcTrpase exhibits only about 10% activity at 100MPa. PpTrpase forms external aldimine and quinonoid intermediates in stopped-flow experiments with l-Trp, S-Et-l-Cys, S-benzyl-l-Cys, oxindolyl-l-Ala, l-Ala and l-Met, similar to EcTrpase. However, with l-Trp a gem-diamine is observed that decays to a quinonoid complex. An aminoacrylate is observed with l-Trp in the presence of benzimidazole, as was seen previously with EcTrpase [28] but not with S-Et-l-Cys. The results show that PpTrpase is adapted for optimal activity in the low temperature, high pressure marine environment.