Protecting Human and Animal Health: The Road from Animal Models to New Approach Methods.

Animals and animal models have been invaluable for our current understanding of human and animal biology, including physiology, pharmacology, biochemistry, and disease pathology. However, there are increasing concerns with continued use of animals in basic biomedical, pharmacological, and regulatory research to provide safety assessments for drugs and chemicals. There are concerns that animals do not provide sufficient information on toxicity and/or efficacy to protect the target population, so scientists are utilizing the principles of replacement, reduction, and refinement (the 3Rs) and increasing the development and application of new approach methods (NAMs). NAMs are any technology, methodology, approach, or assay used to understand the effects and mechanisms of drugs or chemicals, with specific focus on applying the 3Rs. Although progress has been made in several areas with NAMs, complete replacement of animal models with NAMs is not yet attainable. The road to NAMs requires additional development, increased use, and, for regulatory decision making, usually formal validation. Moreover, it is likely that replacement of animal models with NAMs will require multiple assays to ensure sufficient biologic coverage. The purpose of this manuscript is to provide a balanced view of the current state of the use of animal models and NAMs as approaches to development, safety, efficacy, and toxicity testing of drugs and chemicals. Animals do not provide all needed information nor do NAMs, but each can elucidate key pieces of the puzzle of human and animal biology and contribute to the goal of protecting human and animal health. SIGNIFICANCE STATEMENT: Data from traditional animal studies have predominantly been used to inform human health safety and efficacy. Although it is unlikely that all animal studies will be able to be replaced, with the continued advancement in new approach methods (NAMs), it is possible that sometime in the future, NAMs will likely be an important component by which the discovery, efficacy, and toxicity testing of drugs and chemicals is conducted and regulatory decisions are made.

Redox-activity and in vitro effects of regional atmospheric aerosol pollution: Seasonal differences and correlation between oxidative potential and in vitro toxicity of PM1.

Particulate Matter (PM) is a complex and heterogeneous mixture of atmospheric particles recognized as a threat to human health. Oxidative Potential (OP) measurement is a promising and integrative method for estimating PM-induced health impacts since it is recognized as more closely associated with adverse health effects than ordinarily used PM mass concentrations. OP measurements could be introduced in the air quality monitoring, along with the parameters currently evaluated. PM deposition in the lungs induces oxidative stress, inflammation, and DNA damage. The study aimed to compare the OP measurements with toxicological effects on BEAS-2B and THP-1 cells of winter and summer PM1 collected in the Po Valley (Italy) during 2021. PM1 was extracted in deionized water by mechanical agitation and tested for OP and, in parallel, used to treat cells. Cytotoxicity, genotoxicity, oxidative stress, and inflammatory responses were assessed by MTT test, DCFH-DA assay, micronucleus, γ-H2AX, comet assay modified with endonucleases, ELISA, and Real-Time PCR. The evaluation of OP was performed by applying three different assays: dithiothreitol (OPDTT), ascorbic acid (OPAA), and 2',7'-dichlorofluorescein (OPDCFH), in addition, the reducing potential was also analysed (RPDPPH). Seasonal differences were detected in all the parameters investigated. The amount of DNA damage detected with the Comet assay and ROS formation highlights the presence of oxidative damage both in winter and in summer samples, while DNA damage (micronucleus) and genes regulation were mainly detected in winter samples. A positive correlation with OPDCFH (Spearman's analysis, p < 0.05) was detected for IL-8 secretion and γ-H2AX. These results provide a biological support to the implementation in air quality monitoring of OP measurements as a useful proxy to estimate PM-induced cellular toxicological responses. In addition, these results provide new insights for the assessment of the ability of secondary aerosol in the background atmosphere to induce oxidative stress and health effects.

The evaluation of skin sensitization potential of the UVCB substance diisopentyl phthalate by in silico and in vitro methods.

Diisopentyl phthalate (DiPeP) is primarily used as a plasticizer or additive within the production of polyvinyl chloride (PVC), and has many additional industrial applications. Its metabolites were recently found in urinary samples of pregnant women; thus, this substance is of concern as relates to human exposure. Depending upon the nature of the alcohol used in its synthesis, DiPeP may exist either as a mixture consisting of several branched positional isomers, or as a single defined structure. This article investigates the skin sensitization potential and immunomodulatory effects of DiPeP CAS No. 84777-06-0, which is currently marketed and classified as a UVCB substance, by in silico and in vitro methods. Our findings showed an immunomodulatory effect for DiPeP in LPS-induced THP-1 activation assay (increased CD54 expression). In silico predictions using QSAR TOOLBOX 4.5, ToxTree, and VEGA did not identify DiPeP, in the form of a discrete compound, as a skin sensitizer. The keratinocyte activation (Key Event 2 (KE2) of the adverse outcome pathway (AOP) for skin sensitization) was evaluated by two different test methods (HaCaT assay and RHE assay), and results were discordant. While the HaCaT assay showed that DiPeP can activate keratinocytes (increased levels of IL-6, IL-8, IL-1α, and ILA gene expression), in the RHE assay, DiPeP slightly increased IL-6 release. Although inconclusive for KE2, the role of DiPeP in KE3 (dendritic cell activation) was demonstrated by the increased levels of CD54 and IL-8 and TNF-α in THP-1 cells (THP-1 activation assay). Altogether, findings were inconclusive regarding the skin sensitization potential of the UVCB DiPeP-disagreeing with the results of DiPeP in the form of discrete compound (skin sensitizer by the LLNA assay). Additional studies are needed to elucidate the differences between DiPeP isomer forms, and to better understand the applicability domains of non-animal methods in identifying skin sensitization hazards of UVCB substances.

In Vitro Immunoreactivity Evaluation of H-Ferritin-Based Nanodrugs.

Biological nanoparticles, such as proteins and extracellular vesicles, are rapidly growing as nanobased drug-delivery agents due to their biocompatibility, high loading efficiency, and bioavailability. However, most of the candidates emerging preclinically hardly confirm their potential when entering clinical trials. Among other reasons, this is due to the low control of synthesis processes and the limited characterization of their potential immunoreactivity profiles. Here, we propose a combined method that allow us to fully characterize H-ferritin nanoparticles' immunoreactivity during their production, purification, endotoxin removal, and drug loading. H-Ferritin is an extremely interesting nanocage that is being under evaluation for cancer therapy due to its innate cancer tropism, favorable size, and high stability. However, being a recombinant protein, its immunoreactivity should be carefully evaluated preclinically to enable further clinical translation. Surprisingly, this aspect is often underestimated by the scientific community. By measuring proinflammatory cytokine release as a function of endotoxin content, we found that even removing all pyrogenic contaminants from the nanocage, a mild immunoreactivity was still left. When we further purified H-ferritin by loading doxorubicin through a highly standardized loading method, proinflammatory cytokine release was eliminated. This confirmed the safety of H-ferritin nanocages to be used for drug delivery in cancer therapy. Our approach demonstrated that when evaluating the safety of nanodrugs, a combined analysis of acute toxicity and immunoreactivity is necessary to guarantee the safety of newly developed products and to unveil their real translational potential.

Impact of endocrine disruptors on peripheral blood mononuclear cells in vitro: role of gender.

Humans can be exposed to endocrine disruptors (EDs) in numerous ways. EDs can interfere with endogenous hormones at different levels, resulting in numerous adverse human health outcomes, including immunotoxicity. In this regard, this study aimed to investigate in vitro the possible effects of EDs on immune cells and possible gender differences. Peripheral blood mononuclear cells from healthy humans, both males and females, were exposed to 6 different EDs, namely atrazine (herbicide), cypermethrin (insecticide), diethyl phthalate (plasticizer), 17α-ethynylestradiol (contraceptive drug), perfluorooctanesulfonic acid (persistent organic pollutant), and vinclozolin (fungicide). We evaluated the effect of EDs on RACK1 (receptor for activated C kinase 1) expression, considering it as a bridge between the endocrine and the immune system, and putatively used as screening tool of immunotoxic effects of EDs. The exposure to EDs resulted at different extent in alteration in RACK1 expression, pro-inflammatory activity, natural killer lytic ability, and lymphocyte differentiation, with sex-related differences. In particular, diethyl phthalate and perfluorooctanesulfonic acid resulted the most active EDs tested, with gender differences in terms of effects and magnitude. The results from our study evidenced the ability of EDs to directly affect immune cells.

Role of miR-24-3p and miR-146a-5p in dendritic cells' maturation process induced by contact sensitizers.

MiRNAs are non-coding RNA molecules that regulate gene expression at the post-transcriptional level. Although allergic contact dermatitis has been studied extensively, few studies addressed miRNA expression and their role in dendritic cell activation. The main aim of this work was to investigate the role of miRNAs in the underlying mechanism of dendritic cell maturation induced by contact sensitizers of different potency. Experiments were conducted using THP-1-derived immature DCs (iDCs). Contact allergens of different potency were used: p-benzoquinone, Bandrowski's base, and 2,4-dinitrochlorobenzene as extreme; nickel sulfate hexahydrate, diethyl maleate and 2-mercaptobenzothiazole as moderate; and α-hexyl cinnamaldehyde, eugenol, and imidazolidinyl urea as weak. Selective inhibitor and mimic miRNAs were then used and several cell surface markers was evaluated as targets. Also, patients patch tested with nickel were analyzed to determine miRNAs expression. Results indicate an important role of miR-24-3p and miR-146a-5p in DCs activation. miR-24-3p was up-regulated by extreme and weak contact allergens, while miR-146a-5p was up-regulated by weak and moderate contact allergens and down-regulated only by the extreme ones. Also, the involvement of PKCß in contact allergen-induced miR-24-3p and miR-146a-5p expression was demonstrated. Furthermore, the expression of the two miRNAs maintains the same trend of expression in both in vitro and in human conditions after nickel exposure. Results obtained suggest the involvement of miR-24 and miR-146a in DCs maturation process in the proposed in vitro model, supported also by human evidences.

Role of Protein Kinase C in Immune Cell Activation and Its Implication Chemical-Induced Immunotoxicity.

Protein kinase C (PKCs) isoforms play a key regulatory role in a variety of cellular functions, including cell growth and differentiation, gene expression, hormone secretion, etc. Patterns of expression for each PKC isoform differ among tissues, and it is also clear that different PKCs are often not functionally redundant, for example specific PKCs mediate specific cellular signals required for activation, proliferation, differentiation and survival of immune cells. In the last 20 years, we have been studying the role of PKCs, mainly PKCß and its anchoring protein RACK1 (Receptor for Activated C Kinase 1), in immune cell activation, and their implication in immunosenescence and immunotoxicity. We could demonstrate that PKCß and RACK1 are central in dendritic cell maturation and activation by chemical allergens, and their expressions can be targeted by EDCs and anti-inflammatory drugs. In this chapter, current knowledge on the role of PKC in immune cell activation and possible implication in immunotoxicity will be described.

Effect of estrogen-active compounds on the expression of RACK1 and immunological implications.

We previously demonstrated the existence of a balance among steroid hormones, i.e. glucocorticoids and androgens, in RACK1 (receptor for activated C kinase 1) expression and innate immunity activation, which may offer the opportunity to use RACK1 expression as marker to evaluate immunotoxicity of hormone-active substances. Because of the existence of close interconnections between the different steroid hormone receptors with overlapping ligand specificities and signaling pathways, in this study, we wanted to investigate a possible effect of estrogenic active compounds, namely 17ß-estradiol, diethylstilbestrol, and zearalenone, on RACK-1 expression and innate immune responses using THP-1 cells as experimental model. All compounds increased RACK1 transcriptional activity as evaluated by reporter luciferase activity, mRNA expression as assessed by real time-PCR and protein expression by western blot analysis, which paralleled an increase in LPS-induced IL-8, TNF-α production, and CD86 expression, which we previously demonstrated to be dependent on RACK1/PKCß activation. As the induction of RACK1 expression can be blocked by the antagonist G15, induced by the agonist G1 and by the non-cell permeable 17ß-estradiol conjugated with BSA, a role of GPER (previously named GPR30) activation in estrogen-induced RACK1 expression could be demonstrated. In addition, a role of androgen receptor (AR) in RACK1 transcription was also demonstrated by the ability of flutamide, a nonsteroidal antiandrogen, to completely prevent diethylstilbestrol-induced RACK1 transcriptional activity and protein expression. Altogether, our data suggest that RACK1 may represent an interesting target of steroid-active compounds, and its evaluation may offer the opportunity to screen the immunotoxic potential of hormone-active substances.

Antiproliferative effects of chalcones on T cell acute lymphoblastic leukemia-derived cells: Role of PKCß.

In this study, a series of 20 chalcone derivatives was synthesized, and their antiproliferative activity was tested against the human T cell acute lymphoblastic leukemia-derived cell line, CCRF-CEM. On the basis of the structural features of the most active compounds, a new library of chalcone derivatives, according to the structure-activity relationship design, was synthesized, and their antiproliferative activity was tested against the same cancer cell line. Furthermore, four of these derivatives (compounds 3, 4, 8, 28), based on lower IC50 values (between 6.1 and 8.9 µM), were selected for further investigation regarding the modulation of the protein expression of RACK1 (receptor for activated C kinase), protein kinase C (PKC)α and PKCß, and their action on the cell cycle level. The cell cycle analysis indicated a block in the G0/G1 phase for all four compounds, with a statistically significant decrease in the percentage of cells in the S phase, with no indication of apoptosis (sub-G0/G1 phase). Compounds 4 and 8 showed a statistically significant reduction in the expression of PKCα and an increase in PKCß, which together with the demonstration of an antiproliferative role of PKCß, as assessed by treating cells with a selective PKCß activator, indicated that the observed antiproliferative effect is likely to be mediated through PKCß induction.

Endocrine-Disrupting Chemicals' (EDCs) Effects on Tumour Microenvironment and Cancer Progression: Emerging Contribution of RACK1.

Endocrine disruptors (EDCs) can display estrogenic and androgenic effects, and their exposure has been linked to increased cancer risk. EDCs have been shown to directly affect cancer cell regulation and progression, but their influence on tumour microenvironment is still not completely elucidated. In this context, the signalling hub protein RACK1 (Receptor for Activated C Kinase 1) could represent a nexus between cancer and the immune system due to its roles in cancer progression and innate immune activation. Since RACK1 is a relevant EDCs target that responds to steroid-active compounds, it could be considered a molecular bridge between the endocrine-regulated tumour microenvironment and the innate immune system. We provide an analysis of immunomodulatory and cancer-promoting effects of different EDCs in shaping tumour microenvironment, with a final focus on the scaffold protein RACK1 as a pivotal molecular player due to its dual role in immune and cancer contexts.

Contact allergen (PPD and DNCB)-induced keratinocyte sensitization is partly mediated through a low molecular weight hyaluronan (LMWHA)/TLR4/NF-κB signaling axis.

Allergic contact dermatitis (ACD) is caused by topical exposure to chemical allergens. Keratinocytes play a key role in innate immunity, as well as in ACD progression. The transmembrane Toll-like receptor 4 (TLR4), strongly implicated in skin inflammation, has the ability to bind Damage Associated Molecular Patterns (DAMPs), like Low Molecular Weight Hyaluronan (LMWHA). Previously, we had determined that p-phenylenediamine (PPD) and 2,4-dinitrochlorobenzene (DNCB) modulate keratinocyte HA deposition in a manner correlated to their sensitization. In the present study, we aimed to investigate putative co-operation of HA and TLR4 in the process of PPD and DNCB-induced keratinocyte activation. Contact sensitizers were shown to significantly increase the expression of Hyaluronan Synthases (HAS) and TLR4 in NCTC2544 human keratinocytes, as demonstrated by western blot and Real-Time PCR. These data, in correlation to earlier shown enhanced HA degradation suggest that the contact sensitizers facilitate HA turnover of keratinocytes and increase the release of pro-inflammatory, LMWHA fragments. Treatment with exogenous LMWHA enhanced TLR4, HAS levels and Nuclear factor-kappa beta (NF-κΒ) activation. PPD, DNCB and LMWHA-effects were shown to be partly executed through TLR4 downstream signaling as shown by Real-Time, western blot, siRNA and confocal microscopy approaches. Specifically, PPD and DNCB stimulated the activation of the TLR4 downstream mediator NF-κB. Therefore, the shown upregulation of TLR4 expression is suggested to further facilitate the release of endogenous, bioactive HA fragments and sustain keratinocyte activation. In conclusion, keratinocyte contact allergen-dependent sensitization is partly mediated through a LMWHA/TLR4/ NF-κB signaling axis.

Cortisol-induced SRSF3 expression promotes GR splicing, RACK1 expression and breast cancer cells migration.

Recent data have demonstrated that triple negative breast cancer (TNBC) with high glucocorticoid receptor (GR) expression are associated to therapy resistance and increased mortality. Given that GR alternative splicing generates mainly GRα, responsible of glucocorticoids action, we investigated its role in the regulation of RACK1 (Receptor for Activated C Kinase 1), a scaffolding protein with a GRE (Glucocorticoid Response Element) site on its promoter and involved in breast cancer cells migration and invasion. We provide the first evidence that GRα transcriptionally regulates RACK1 by a mechanism connected to SRSF3 splicing factor, which promotes GRα, essential for RACK1 transcriptional regulation and consequently for cells migration. We also establish that this mechanism can be positively regulated by cortisol. Hence, our data elucidate RACK1 transcriptional regulation and demonstrate that SRSF3 involvement in cells migration implies its role in controlling different pathways thus highlighting that new players have to be considered in GR-positive TNBC.

THP-1 Cells and Pro-inflammatory Cytokine Production: An in Vitro Tool for Functional Characterization of NOD1/NOD2 Antagonists.

THP-1 cells express high levels of native functional nucleotide-binding oligomerization domain 1 (NOD1), NOD2, and Toll-like receptor 4 (TLR4) receptors, and have often been used for investigating the immunomodulatory effects of small molecules. We postulated that they would represent an ideal cell-based model for our study, the aim of which was to develop a new in vitro tool for functional characterization of NOD antagonists. NOD antagonists were initially screened for their effect on NOD agonist-induced interleukin-8 (IL-8) release. Next, we examined the extent to which the selected NOD antagonists block the NOD-TLR4 synergistic crosstalk by measuring the effect of NOD antagonism on tumor necrosis factor-α (TNF-α) secretion from doubly activated THP-1 cells. Overall, the results obtained indicate that pro-inflammatory cytokine secretion from THP-1 provides a valuable, simple and reproducible in vitro tool for functional characterization of NOD antagonists.

Ultrafine Particles from Residential Biomass Combustion: A Review on Experimental Data and Toxicological Response.

Biomass burning is considered an important source of indoor and outdoor air pollutants worldwide. Due to competitive costs and climate change sustainability compared to fossil fuels, biomass combustion for residential heating is increasing and expected to become the major source of primary particulate matter emission over the next 5-15 years. The understanding of health effects and measures necessary to reduce biomass emissions of harmful compounds is mandatory to protect public health. The intent of this review is to report available data on ultrafine particles (UFPs, i.e., particles with diameter smaller than 100 nm) emitted by residential biomass combustion and their effects on human health (in vitro and in vivo studies). Indeed, as far as we know, papers focusing specifically on UFPs originating from residential biomass combustion and their impact on human health are still lacking.

Chemical-induced contact allergy: from mechanistic understanding to risk prevention.

Chemical allergens are small molecules able to form a sensitizing complex once they bound to proteins. One of the most frequent manifestations of chemical allergy is contact hypersensitivity, which can have serious impact on quality of life. Allergic contact dermatitis is a predominantly CD8 + T cell-mediated immune disease, resulting in erythema and eczema. Chemical allergy is of considerable importance to the toxicologist, who has the responsibility of identifying and characterizing the allergenic potential of chemicals, and estimating the risk they pose to human health. This review aimed at exploring the phenomena of chemical-induced contact allergy starting from a mechanistic understanding, immunoregulatory mechanisms, passing through the potency of contract allergen until the hazard identification, pointing out the in vitro models for assessing contact allergen-induced cell activation and the risk prevention.

Immunomodulators Inspired by Nature: A Review on Curcumin and Echinacea.

The immune system is an efficient integrated network of cellular elements and chemicals developed to preserve the integrity of the organism against external insults and its correct functioning and balance are essential to avoid the occurrence of a great variety of disorders. To date, evidence from literature highlights an increase in immunological diseases and a great attention has been focused on the development of molecules able to modulate the immune response. There is an enormous global demand for new effective therapies and researchers are investigating new fields. One promising strategy is the use of herbal medicines as integrative, complementary and preventive therapy. The active components in medical plants have always been an important source of clinical therapeutics and the study of their molecular pharmacology is an enormous challenge since they offer a great chemical diversity with often multi-pharmacological activity. In this review, we mainly analysed the immunomodulatory/antinflammatory activity of Echinacea spp. and Curcuma longa, focusing on some issues of the phytochemical research and on new possible strategies to obtain novel agents to supplement the present therapies.

The scaffold protein RACK1 is a target of endocrine disrupting chemicals (EDCs) with important implication in immunity.

We recently demonstrated the existence of a complex hormonal balance between steroid hormones in the control of RACK1 (Receptor for Activated C Kinase 1) expression and immune activation, suggesting that this scaffold protein may also be targeted by endocrine disrupting chemicals (EDCs). As a proof of concept, we investigated the effect of the doping agent nandrolone, an androgen receptor (AR) agonist, and of p,p'DDT (dichlorodiphenyltrichloroethane) and its main metabolite p,p'DDE (dichlorodiphenyldichloroethylene), a weak and strong AR antagonist, respectively, on RACK1 expression and innate immune response. In analogy to endogenous androgens, nandrolone induced a dose-related increase in RACK1 transcriptional activity and protein expression, resulting in increased LPS-induced IL-8 and TNF-α production and proliferation in THP-1 cells. Conversely, p,p'DDT and p,p'DDE significantly decrease RACK1 expression, LPS-induced cytokine production and CD86 expression; with p,p'DDE exerting a stronger repressor effect than p,p'DDT, consistent with its stronger AR antagonistic effect. These results indicate that RACK1 could be a relevant target of EDCs, responding in opposite ways to agonist or antagonist of AR, representing a bridge between the endocrine system and the innate immune system.

Role of spliceosome proteins in the regulation of glucocorticoid receptor isoforms by cortisol and dehydroepiandrosterone.

Dehydroepiandrosterone (DHEA) can counteract the activity of cortisol by modulating the glucocorticoid receptor ß (GRß) expression and antagonizing the binding of GRα to the glucocorticoid responsive element (GRE) in RACK1 (Receptor for Activated C Kinase 1) promoter. These observations are important in the context of immunosenescence and can be extended to recognize a complex hormonal balance in the control of GR isoform expression and consequently in the expression of GR responsive genes. To elucidate the mechanism of DHEA on GR alternative splicing, we investigated its possible involvement in the expression of proteins such as the Serine/arginine (SR)-Rich Splicing Factors (SRSF) regulating GR splicing, specifically SRSF9 and SRSF3 also known as SRp30c and SRp20 respectively. We demonstrated that DHEA can induce the up-regulation of GR mRNA which is preferentially directed toward the ß isoform. The effect is due to an increase in expression of the splicing factor SRSF9. On the other hand cortisol up-regulated SRSF3, the splicing factor promoting GRα isoform. We demonstrated that DHEA and cortisol modulate SRSF9 and SRSF3 in a different way and our data suggest that the anti-glucocorticoid effect of DHEA, among other mechanisms, is also exerted by modulating the expression of proteins involved in the splicing of the GR pre-mRNA.