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.

New approach methodologies to enhance human health risk assessment of immunotoxic properties of chemicals - a PARC (Partnership for the Assessment of Risk from Chemicals) project.

As a complex system governing and interconnecting numerous functions within the human body, the immune system is unsurprisingly susceptible to the impact of toxic chemicals. Toxicants can influence the immune system through a multitude of mechanisms, resulting in immunosuppression, hypersensitivity, increased risk of autoimmune diseases and cancer development. At present, the regulatory assessment of the immunotoxicity of chemicals relies heavily on rodent models and a limited number of Organisation for Economic Co-operation and Development (OECD) test guidelines, which only capture a fraction of potential toxic properties. Due to this limitation, various authorities, including the World Health Organization and the European Food Safety Authority have highlighted the need for the development of novel approaches without the use of animals for immunotoxicity testing of chemicals. In this paper, we present a concise overview of ongoing efforts dedicated to developing and standardizing methodologies for a comprehensive characterization of the immunotoxic effects of chemicals, which are performed under the EU-funded Partnership for the Assessment of Risk from Chemicals (PARC).

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.

An integrated in silico-in vitro investigation to assess the skin sensitization potential of 4-Octylphenol.

One of the major challenges in chemical toxicity testing is the possibility to protect human health against adverse effects with non-animal methods. In this paper, 4-Octylphenol (OP) was tested for skin sensitization and immunomodulatory effects using an integrated in silico-in vitro test approach. In silico tools (QSAR TOOLBOX 4.5, ToxTree and VEGA) were used together with several in vitro tests including HaCaT cells (quantification of IL-6; IL-8; IL-1α and IL-18 by ELISA and expression of genes TNF, IL1A, IL6 and IL8 by RT- qPCR), RHE model (quantification of IL-6; IL-8; IL-1α and IL-18 by ELISA) and THP-1 activation assay (CD86/CD54 expression and IL-8 release). Additionally, the immunomodulatory effect of OP was investigated using lncRNAs MALAT1 and NEAT1 expression and LPS-induced THP-1 activation (CD86/CD54 expression and IL-8 release). The in silico tools predicted OP as a sensitizer. In vitro tests are also concordant with the in silico prediction. OP increased IL-6 expression (HaCaT cells); IL-18 and IL-8 expressions (RHE model). An irritant potential was also shown by a great expression of IL-1α (RHE model); and increased expression of CD54 marker and IL-8 in THP-1 cells. Immunomodulatory effects of OP were demonstrated by the downregulation of NEAT1, MALAT1 (epigenetic markers), IL6 and IL8; and an increase in LPS-induced CD54 and IL-8 expressions. Overall, results indicate that OP is a skin sensitizer, being positive in three key events of the AOP for skin sensitization, also showing immunomodulatory effects.

New approach methodologies in immunotoxicology: challenges and opportunities.

To maintain the integrity of an organism, a well-functioning immune system is essential. Immunity is dynamic, with constant surveillance needed to determine whether to initiate an immune response or to not respond. Both inappropriate immunostimulation and decreased immune response can be harmful to the host. A reduced immune response can lead to high susceptibility to cancer or infections, whereas an increased immune response can be related to autoimmunity or hypersensitivity reactions. Animal testing has been the gold standard for hazard assessment in immunotoxicity but a lot of efforts are ongoing to develop non-animal-based test systems, and important successes have been achieved. The term "new approach methodologies" (NAMs) refer to the approaches which are not based on animal models. They are applied in hazard and risk assessment of chemicals and include approaches such as defined approaches for data interpretation and integrated approaches to testing and assessment. This review aims to summarize the available NAMs for immunotoxicity assessment, taking into consideration both inappropriate immunostimulation and immunosuppression, including implication for cancer development.

In Vitro Effects of Bisphenol Analogs on Immune Cells Activation and Th Differentiation.

AIMS: Investigate the immunomodulatory effects of bisphenols in the THP-1 cell line and peripheral blood mononuclear cells in response to lipopolysaccharide (LPS) activation or to phorbol 12-myristate 13-acetate (PMA) and ionomycin. BACKGROUND: We have previously demonstrated the usefulness of the evaluation of RACK1 expression as a link between endocrine disrupting activity and the immunotoxic effect of xenobiotics. We demonstrated that while BPA and BPAF reduced RACK1 expression, BPS was able to increase it. OBJECTIVE: Bisphenol A (BPA) is one of the most commonly used chemicals in the manufacturing of polycarbonate plastics and plastic consumer products. Its endocrine disrupting (ED) potential and changes in European regulations have led to replacing BPA in many uses with structurally similar chemicals, like bisphenol AF (BPAF) and bisphenol S (BPS). However, emerging data indicated that bisphenol analogues may not be safer than BPA both in toxic effects and ED potential. METHODS: THP-1 cell line and peripheral blood mononuclear cells were activated with lipopolysaccharide (LPS) or with phorbol 12-myristate 13-acetate (PMA) and ionomycin. RESULTS: BPA and BPAF decreased LPS-induced expression of surface markers and the release of pro-inflammatory cytokines, while BPS increased LPS-induced expression of CD86 and cytokines. BPA, BPAF, and BPS affected PMA/ionomycin-induced T helper differentiation and cytokine release with gender-related alterations in some parameters investigated. CONCLUSION: Data confirm that bisphenols can modulate immune cell differentiation and activation, further supporting their immunotoxic effects.

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.

In Vitro Effects of Cypermethrin and Glyphosate on LPS-Induced Immune Cell Activation.

(1) Background: The insecticide cypermethrin (Cypm) and the herbicide glyphosate (Glyp) are among the most widely used pesticides. While the two pesticides have been considered to have low toxicity in mammals, some indication of potential immunotoxicity has emerged. The aim of this work was to investigate in vitro the effects of Cypm and Glyp on bacteria lipopolysaccharide (LPS)-induced immune cell activation and of Cypm on 2-mercaptobenzothiazole (MBT)-induced maturation of dendritic cells (DCs). (2) Methods: The release of the inflammatory cytokines TNF-α and IL-8, the expression of the surface markers CD54 and CD86 in human primary peripheral blood mononuclear cells (PBMC), and THP-1 cells were investigated together with CD83, HLA-DR, IL-6, and IL-18 in DCs. (3) Results: While no significant modulation on LPS-induced immune cell activation was observed following Glyp exposure, with only a trend toward an increase at the highest concentration tested, Cypm reduced the responses to LPS and to MBT, supporting a direct immunosuppressive effect. Overall, the present study contributes to our understanding of pesticide-induced immunotoxicity, and the results obtained support evidence showing the immunosuppressive effects of Cypm.

Air Pollution: Possible Interaction between the Immune and Nervous System?

Exposure to environmental pollutants is a serious and common public health concern associated with growing morbidity and mortality worldwide, as well as economic burden. In recent years, the toxic effects associated with air pollution have been intensively studied, with a particular focus on the lung and cardiovascular system, mainly associated with particulate matter exposure. However, epidemiological and mechanistic studies suggest that air pollution can also influence skin integrity and may have a significant adverse impact on the immune and nervous system. Air pollution exposure already starts in utero before birth, potentially causing delayed chronic diseases arising later in life. There are, indeed, time windows during the life of individuals who are more susceptible to air pollution exposure, which may result in more severe outcomes. In this review paper, we provide an overview of findings that have established the effects of air pollutants on the immune and nervous system, and speculate on the possible interaction between them, based on mechanistic data.

Extracellular vesicles from seminal plasma to improve fertilizing capacity of bulls.

Seminal plasma contains extracellular vesicles (EVs) that vehicle RNA, proteins, and other molecules able to influence the biological function of sperm. The aim of this study was to improve the fertilizing capacity of male gametes of low-fertility bulls using EVs isolated by ultracentrifugation from the seminal plasma of a bull of proven fertility. After dose-response curve, 10×106 sperm of low-fertility bulls were co-incubated for an hour with 400×106 EVs/ml. In addition, it has been verified that the incorporation of EVs, which takes place in the sperm midpiece, is maintained for 5 hours and even after cryopreservation. Subsequently, the spermatozoa of low-fertility bulls, with EVs incorporated, were used for the in vitro production of embryos. The rate of blastocyst at seventh day yield in vitro, with the use of sperm with EVs incorporated, increased by about twice the yield obtained with the same sperm in the absence of EVs: bulls having an average embryonic yield of 6.41±1.48%, 10.32±4.34% and 10.92±0.95% improved their yield to 21.21±1.99%, 22.17±6.09% and 19.99±5.78%, respectively (P<0.05). These encouraging results suggest that it might be possible to keep breeding bulls with poor fertility. Further studies will be needed to evaluate the in vivo fertility of sperm treated with EVs and understand how the content of EVs is involve in the sperm-vesicle interaction and in the improved sperm performance.

Endometrial and oviduct extra-cellular vescicles for in vitro equine sperm hyperactivation and oocyte fertilization.

Unlike humans and many other mammalian species, conventional in vitro fertilization (IVF) in equine species is not successful. To mimic in vitro equine spermatozoon-oviduct interaction as close as possible to that which occurs in vivo, extracellular vesicles (EVs) secreted by the female genital tract were used. Three female genital tracts were collected at slaughterhouse from mares in late estrus. Ipsilateral proximal and apical horn endometrial explants were digested with collagenase and trypsin and cells obtained were cultured on insert system to allow their polarization. Ipsilateral oviducts were squeezed out to obtain spheroids. To produce EVs, proximal and apical horn endometrial cells and oviductal spheroids were cultured for three days in serum free medium. To trace interaction between spermatozoa and EVs by fluorescence microscopy, EVs were differently labeled. Pooled samples of ejaculated spermatozoa from three stallions were incubated in capacitating medium (CM) for 6 h and to induce hyperactivation for other 6 h in CM supplemented with different kind of EVs alone or in combination. A control was performed in absence of EVs. Sperm were assessed for motility by CASA system, EV incorporation by confocal microscopy and acrosomal reaction (AR) by staining with FITC-PNA/PI. In vitro fertilization was performed, and presumed zygotes were subjected to chromatin configuration. The results show that incorporation of EVs of the proximal horn does not take place, while apical horn EVs are incorporated in the head of the spermatozoon in 4 h. The EVs of oviductal spheroids are incorporated in the middle tract in 1 h. The rate of AR with EVs of the apical horn and oviductal spheroids were respectively 50.25% and 57.14%. When these EVs were added in combination, the rate of AR was 71.42%. In the control, the rate of AR was of 15%. After in vitro fertilization, 44% of oocytes showed male and female pronuclei, whereas no fertilization is obtained in the control. In conclusion, EVs from apical horn and oviduct could be involved in cell trafficking during equine semen hyperactivation, and their possible use in vitro could facilitate the development of equine reproductive biotechnologies.

Effects of endocrine active contaminating pesticides on RACK1 expression and immunological consequences in THP-1 cells.

We have previously demonstrated that RACK1, which expression is under steroid hormone control, plays an important role in the activation of immune cells and its expression can be useful to evaluate the immunotoxic profile of endocrine disrupting chemicals (EDCs). Hence, we investigated the effects of three contaminating and persistent pesticides: the fungicide vinclozolin (VIN), the herbicide atrazine (ATR) and the insecticide cypermethrin (CYP) on RACK1 expression and on innate immune response. VIN resulted in modest alteration of RACK1 while ATR and CYP reduced in a dose dependent manner RACK1 expression, ultimately leading to the decrease in lipopolysaccharide-induced IL-8 and TNF-α release and CD86 and CD54 surface marker expression. Moreover, our data indicate that, after exposure to EDCs, alterations of RACK1 expression can also occur with mechanisms not directly mediated by an interaction with a nuclear or membrane steroid receptors. Therefore, RACK1 could represent a useful EDCs screening tool to evaluate their immunotoxic potential and to dissect their mechanisms of action.

Effects of endocrine disrupting chemicals on the expression of RACK1 and LPS-induced THP-1 cell activation.

The existence of a complex hormonal balance among glucocorticoids, androgens and estrogens involved in the regulation of Receptor for Activated C Kinase 1 (RACK1) expression and its related immune cells activation, highlights the possibility to employ this protein as screening tool for the evaluation of the immunotoxic profile of endocrine disrupting chemicals (EDCs), hormone-active substances capable of interfering with the physiologic hormonal signaling. Hence, the aim of this work was to investigate the effect of the exposure of EDCS 17α-ethynylestradiol (EE), diethyl phthalate (DEP) and perfluorooctanesulfonic acid (PFOS) on RACK1 expression and on lipopolysaccharide (LPS)-induced activation of the human monocytic cell line THP-1, a validated model for this investigation. In line with our previous results with estrogen-active compounds, EE treatment significantly induced RACK1 promoter transcriptional activity, mRNA expression, and protein levels, which paralleled an increase in LPS-induced IL-8, TNF-α production and CD86 expression, previously demonstrated to be dependent on RACK1/PKCß activation. EE mediates its effect on RACK1 expression through G-protein-coupled estrogen receptor (GPER) and androgen receptor (AR) ligand-independent cascade, as also suggested by in silico molecular docking simulation. Conversely, DEP and PFOS induced a dose-dependent downregulation of RACK1 promoter transcriptional activity, mRNA expression, and protein levels, which was mirrored by a reduction of IL-8, TNF-α production and CD86 expression. Mifepristone pre-treatments abolish DEP and PFOS effects, confirming their GR agonist profile, also corroborated by molecular docking. Altogether, our data confirm that RACK1 represents an interesting target of steroid active compounds, which expression offers the opportunity to screen the immunotoxic potential of different hormone-active substances of concerns due to their human exposure and environmental persistence.

Direct Effects of Glyphosate on In Vitro T Helper Cell Differentiation and Cytokine Production.

Glyphosate (G) is the active ingredient of the most used herbicides worldwide. Its use is currently very debated, as several studies indicating its hazard and toxicity are emerging. Among them, there is evidence of adverse effects on the immune system. The aim of this work was to investigate if G could directly affect immune cells. Peripheral blood mononuclear cells (PBMC) obtained from healthy donors were used as experimental model. PBMC were expose to G and stimulated with PMA/ionomycin, T helper (Th) cell differentiation and cytokine production were assessed by flow cytometry and enzyme-linked immunosorbent assay, respectively. A reduction of Th1/Th2 ratio, mainly due to a decrease in Th1 cells, was observed following G exposure. Results show an enhancement of IL-4 and IL-17A production, and a reduction of IFN-γ. Based on literature evidence that suggest G being an endocrine disruptor, we investigated the role of nuclear estrogen receptors (ER). ERα/ERß inhibition by ICI 182,780 abolished the effects of G on IFN-γ and IL-4 release, suggesting a role of ER in the observed effects. To further characterize the mechanism of action of G, miRNAs, both in exosome and intracellular, were investigated. A statistically significant increase in miR-500a-5p was observed following G treatment. The blockage of miR-500a-5p, using a specific antagomir, prevented G-induced reduction of IFN-γ production. Finally a relationship between miR-500a-5p up-regulation and ER was observed. Overall, these results suggest that G can directly act on T cells, altering T cell differentiation and cytokines production.

The Modified THP-1 Activation Assay for the In Vitro Identification of Drug-Inducing Systemic Hypersensitivity.

The development of new low molecular weight drugs has many chances of failure and is an expensive process. Currently, there are no screening methods and/or models to assess the hazard of hypersensitivity reactions to drugs (DHRs) in the preclinical phase. DHRs represent 6-15% of adverse drug reactions. Although rare, DHRs represent a serious health problem for predisposed individuals, resulting, in some cases, in life-threatening pathologies. To date, there are no in vitro or in vivo sensitive models able to predict the sensitizing potential of drugs in the preclinical tests, and these reactions are highlighted only after the drug has been placed on the market, affecting both population and public health. This article describes a novel approach methodology for the study of the sensitizing potential of drugs based on the use of the human promyelocytic cell line THP-1 as a surrogate for dendritic cells. The method is based on the upregulation of specific surface markers (CD86 and CD54) and on the production of IL-8. In our experience, the THP-1 activation assay allowed the correct identification of drugs known to induce systemic hypersensitivity in humans, including the one associated with specific HLAs. This method may help to discover possible systemic hypersensitivity reactions early in the preclinical phase of drug development.

Effects of Bisphenols on RACK1 Expression and Their Immunological Implications in THP-1 Cells.

Receptor for activated C kinase 1 (RACK1) has an important role in immune activation, and is regulated through a balance between glucocorticoid and androgen levels. We have previously demonstrated that RACK1 expression can serve as a marker for evaluation of immunotoxic profiles of hormone-active substances, such as endocrine-disrupting chemicals (EDCs). In this study, we investigated the effects of three bisphenols (BPA, BPAF, BPS) on RACK1 expression and on the innate immune responses in the THP-1 human promyelocytic cell line, a validated model for this investigation. BPA and BPAF reduced RACK1 promoter transcriptional activity, mRNA expression, and protein levels. However, BPS had the opposite effect. As expected, these results on RACK1 were paralleled by lipopolysaccharide (LPS)-induced interleukin-8 (IL-8) and tumor necrosis factor-α (TNFα) production. Since BPA and BPAF induced RACK1 expression in the presence of glucocorticoid receptor (GR) antagonist mifepristone, a role of G-protein-coupled estrogen receptor (GPER) has been considered due to their known estrogenic profile. Therefore, additional molecular effects of BPA and BPAF were unmasked after treatment with different inhibitors of well-known pivotal players of GPER-mediated signaling. BPA exerted its effects on RACK1 via NF-κB, as shown using the NF-κB inhibitor BAY11-7085 and NF-κB-specific luciferase reporter assay. Conversely, BPAF induced RACK1 up-regulation via androgen receptor (AR) activation, as confirmed by treatment with AR antagonist flutamide. Indeed, a biased agonism profile for BPA and BPAF for GPER was suggested based on their different binding modes revealed by our molecular docking. Altogether, our data suggest that RACK1 could represent an important target of EDCs and serves as a screening tool for their immunotoxic potential. Furthermore, RACK1 can be exploited to unmask multiple molecular interactions of hormone-active substances to better dissect out their mechanisms of action.

Glyphosate-based herbicides: Evidence of immune-endocrine alteration.

Glyphosate (G) is the active ingredient of the most widely used herbicide products. It targets the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), which lacks in humans, suggesting to confer a low mammalian toxicity to G-based herbicides (GBHs). Despite this, the use of G is currently under intense debate. Many studies indicating its hazard and toxicity on non-target organisms are emerging, and associations between GBHs and immune-endocrine disturbances have been described. This review aims to investigate, based on recent epidemiological studies and studies performed in vitro and in vivo in animals, the possible association between GBHs and immune-endocrine alterations. Published data suggest that GBHs have endocrine disrupting potentiality targeting sex and thyroid hormones, although its relevance for humans will require further investigations. Evidence of immunotoxicity are limited compared to those on endocrine effects, but overall highlight possible noxious effects, including lung inflammation and rhinitis. An attractive hypothesis could be the one that connects microbiota dysbiosis with possible immune-endocrine outcomes. Indeed, several intestinal microorganisms express the enzyme EPSPS and, studies are emerging that highlight a possible G-induced dysbiosis. Considering the wide use of GBHs in agriculture, further studies investigating their noxious effects at levels relevant for human exposure should be performed. A critical analysis of emerging evidence of G toxicity is required to better characterize its safety profile. In addition, attention should be paid to the differences between G alone and its formulations, which, containing substances able to increase G absorption, may present a different toxicity profile.

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.