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New perspectives on cytokine pathways modulation by pesticide exposure.
Journal Pre-proof New perspectives on cytokine pathways modulation by pesticide exposure. Chiara Costa, Giusi Briguglio, Rosaria Catanoso, Federica Giambò, Irene Polito, Michele Teodoro, Concettina Fenga PII:
S2468-2020(20)30004-8
DOI:
https://doi.org/10.1016/j.cotox.2020.01.002
Reference:
COTOX 235
To appear in:
Current Opinion in Toxicology
Received Date: 23 July 2019 Revised Date:
23 December 2019
Accepted Date: 6 January 2020
Please cite this article as: C. Costa, G. Briguglio, R. Catanoso, F. Giambò, I. Polito, M. Teodoro, C. Fenga, New perspectives on cytokine pathways modulation by pesticide exposure., Current Opinion in Toxicology, https://doi.org/10.1016/j.cotox.2020.01.002. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier B.V.
CRediT author statement
MS title: New perspectives on cytokine pathways modulation by pesticide exposure.
Authors Chiara Costa Giusi Briguglio Rosaria Catanoso Federica Giambò Irene Polito Michele Teodoro Concettina Fenga
Role Methodology; Roles/Writing – original draft; Writing – review & editing Writing – original draft; Visualization; Formal analysis; Visualization; Formal analysis; Investigation; Writing – review & editing Conceptualization; Roles/Writing – original draft; Writing – review & editing
New perspectives on cytokine pathways modulation by pesticide exposure. Chiara Costa1, Giusi Briguglio2, Rosaria Catanoso2, Federica Giambò2, Irene Polito2, Michele Teodoro2, Concettina Fenga2
1
Department of Clinical and Experimental Medicine - University of Messina, Italy
2
Department of Biomedical and Dental Sciences and Morpho-functional Imaging - Occupational Medicine Section - University of Messina, Italy
*Corresponding Author: Michele Teodoro Department of Biomedical and Dental Sciences and Morpho-functional Imaging Occupational Medicine Section University of Messina, Italy Policlinico Universitario “G. Martino” – pad. H Via Consolare Valeria 1 98125, Messina, Italy Tel: +39 090 2212052 Fax: +39 090 2212051 e-mail: [email protected] KEYWORDS: pesticides, immune system; occupational exposure, cytokine pathways.
Word count: [2376]
Abstract Immune cells are able to release a variety of inflammation mediators, activating pro- and antiinflammatory processes and regulating intracellular pathways. Consequences of chronic or earlylife exposure to pesticides may be extended beyond innate immune dysfunction to the increased risk of late-life chronic inflammatory-based diseases. This study aims to summarize some of the most recent advancements in occupational toxicology, focusing on biological mechanisms linking environmental exposure to pesticides, inflammation and cytokine modulation, as well as genetic polymorphisms or epigenetic modifications which can represent factors of vulnerability for exposed workers. Choosing appropriate toxicity biomarkers is also one of the main concerns in the field of immunotoxicology; for this purpose new technologies have been introduced for the monitoring of pesticides blood levels along with molecular alterations. These approaches will allow the assessment of the actual body burden of environmental pollutants associating it with a screening for the early diagnosis of pathologies.
Highlights • • • • •
Pesticides exposure and dysregulated formation of ROS may conduct immune stem cells into premature senescence Pesticide immunotoxicity involves signaling pathways by reciprocal triggering with the inflammatory cascade through cytokine modulation NF-κB, TLR and IFN-γ -mediated signaling pathways are involved in pesticide immunotoxicity New methods to toxicity testing like in silico studies and computational modeling can add new perspectives to pesticide risk assessment Study protocols have been proposed for hazard acknowledgement of some selected mixtures to better assess the actual risk
Graphical abstract
Introduction Organochlorine pesticides (DDT, pentachlorophenol, hexachlorobenzene, dieldrin, etc.) have been used to control the spread of pests that may compromise the healthiness and characteristics of foodstuffs. Owing to their ability to resist chemical degradation and to bioaccumulate they are classified as persistent organic pollutants (POPs). Although the production and use of some compounds has been banned or limited, they continue to persist in several ecosystems, representing a serious risk for humans and exposed living species [1,2]. Pesticides can interact with normal human biochemical processes such as immune system homeostasis [3]. Recent studies reported that exposure to pesticides can impair immunity reducing antimicrobial activity, and disturb the endocrine system altering the production of inflammatory mediators such as cytokines and chemokines [4–8]. Cytokines act with both synergistic and antagonistic mechanisms in order to maintain homeostasis, in particular between oxidant and antioxidant signals [9,10]. This equilibrium is very sensitive to the action of substances such as pesticides, which are responsible for structural and functional alterations of the network [11,12]. Although the identification of xenobiotics associated with immunotoxicity and the mechanisms by which they perturb the immune system has grown in importance in recent years, there are significant gaps in their understanding. This review aims to focus on the most recent advances in biological mechanisms linking exposure to pesticides, inflammation and cytokine modulation.
Recent literature data on mechanisms of immunotoxicity The immune system is a complex network distributed on all systems of the organism, with specialized cells able to guarantee a defense against pathogens. Immune cells are able to release a variety of inflammation mediators, including cytokines/chemokines, reactive oxygen or nitrogen species (ROS or NOS). By binding to specific receptors these mediators orchestrate this network, activating pro anti-inflammatory processes and regulating intracellular pathways; this balance can be altered by xenobiotics, such as pesticides. Thanks to the improvement of diagnostic tools and techniques, recent studies highlight various pathogenetic mechanisms modulating responsiveness to diseases related to exposure to pesticides. Recently, great interest is raising towards new methods to toxicity testing like in silico studies and computational modeling (as QSPR/QSAR); these novel approaches are likely to add new perspectives to pesticide risk assessment, allowing to provide also necessary answers to the concerns raised by the use of nano-pesticides [13,14]. Animal and in vitro models are traditionally used in experimental studies because it is not possible to test single toxic compounds in the human population for ethical reasons. In fact, numerous recent studies test the in vivo effects of pesticides in order to better understand some of the molecular mechanisms that could be reproducible in humans [15,16].
Alteration of immune cells Martyniuk et al. investigated the effect induced by two organochlorine pesticides, p,p'dichlorodiphenyldichlorethylene and methoxyclor, on Micropterus salmoides. Starting from the hypothesis that this exposure altered reproductive function, the authors failed to demonstrate this supposition. Rather the study led to another explanation, namely the target of these compounds is the immune system, and alterations in reproductive function would be secondary to the interaction between the immune system and the reproductive system. In particular, a decrease in platelet function, T cell suppression and interaction, adaptive immune responses (Th1 immune response), leukocyte function, and mast cell degranulation were observed [17]. Latorre et al. showed that exposure to glyphosate was able to lower the total white blood cell count in reptiles; as leukocytes are essential effectors for the correct functioning of the immune system exposure to glyphosate could have repercussions on the defensive capacity of the whole organism [18]. In agreement with these findings, a similar study evaluated exposure to pyrethroids (cypermethrin) and organophosphates (glyphosate and chlorpyrifos) and the results obtained are comparable [5]. A cross-sectional study conducted in Pakistan evaluated the toxic effects of pesticides belonging to different chemical classes including organophosphates (chlorpyrifos, profenofos, deltamethrin and imidacloprid) in sprayers, analyzing the values of some liver enzymes and blood parameters, including the total white blood cell count; a weakening of the immune system was observed and this condition could lead to the development of tuberculosis in exposed subjects [19]. Also deltamethrin has been shown to alter white blood cells count and to recall inflammatory cells and leukocytes in the lung [20]. Moreover pyrethroids are also been shown to reduce the activity of the immune system in both human and animals, reducing macrophage activity [21]. A recent study evaluated the immunotoxicity of mancozeb by analyzing rats splenocytes in vitro. Following exposure to mancozeb, a significant increase in splenocyte death was observed due to both apoptosis and necrosis processes [22]. Wang et al. observed that in rats the combined administration of cadmium and chlorpyrifos in vivo at environmentally-relevant low dose synergistically inhibits the proliferation of T and B lymphocytes. The same administration, in vitro, did not show the synergistic effect and chlorpyrifos showed the major inhibitory effects on cytokine production in isolated splenocytes [23]. A decrement in total G immunoglobulins (IgG) was observed in blood samples of workers employed in a pesticide production plant who were exposed to 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD), a compound used in the production of herbicides [24]. Leveque et al. demonstrated that a pesticide mixture commonly found in food, at noncytotoxic doses, can induce selective modifications on mesenchimal stem cells (MSC), suggesting a premature cellular aging profile [25].
Cytokine modulation Other studies investigated mechanisms behind immunotoxicity at a molecular level. The immunotoxicity of α-cypermethrin was assessed in greenhouse workers, using urinary 3phenoxybenzoic acid as an exposure biomarker; exposed workers showed no clinical sign of immunotoxicity, but proinflammatory cytokines involved in antitumor immunity and response to infection (IL-12p70, INF-γ, IL-2 and IL-8) were significantly reduced; findings support the hypothesis that pyrethroid exposure may reduce host defenses against infection and cancer, particularly in subjects with impaired immune capacity [26]. Other studies confirmed that
pyrethroids reduce the production of IL-2, IL-8, IL-12p70 and interferon γ (IFN-γ), and also increase the expression of the Toll-like receptor 4 (TLR4) and the necrotic factor-alpha (TNFα) [20,21]. In carbofuran-treated rats were found increased SMAD protein levels and a rise of TGF-β levels [27]. Still, Kumar et al. observed that organochlorine pesticides were associated with high levels of complement system markers, particularly C3 and C3a [28]. Pesticide mixture induced activation pathways common with aging process, such as IL-7 signaling, modifications in MSC secretome (IL-10 and IL-6), MCP-1 induction, reduced TGF- β signaling. IL-7 signaling plays a role in the body’s innate and adaptive immune responses and in regulating T cells. IL-6 is commonly described as a proinflammatory cytokine, while IL-10 suppresses macrophage and neutrophil functions and inhibits the Th1 immune response. MCP-1 is a key chemokine regulating the recruitment and migration of cells of the monocyte-macrophage system. TGF-β regulates multiple fundamental cellular functions and biological processes [25]. Overall these modifications play a role in inflammatory cascade.
Signaling pathways Alterated levels of cytokines induce the activation or inhibition of some biological pathways mainly related with inflammatory processes. Christen and Fent showed that chlorpyrifos, malathion, cypermethrin and chlorantraniliprole are able to induce significant alterations in genes related to the immune system in honeybees, in particular the three pathways involved are TLR, Imd and Jak/STAT. Among the four pesticides, cypermethrin showed the greatest downregulating effects on all three pathways. The downregulation of abaecin and apidaecin, and the up-regulation of defensin-1 was determined by chlorpyrifos, the up-regulation of abaecin, apidaecin and defensin-1 transcripts by malathion, the down-regulation of relish, hopscotch and domeless by cypermethrin, and the upregulation of apidaecin and relish by chlorantraniliprole [29]. Previously the same Authors had shown that also neonicotinoids were able to modify the gene expression of the immune system [30]. Furthermore, neonicotinoids have also been shown to reduce antimicrobial defense in bees [31]; in particular clothianidin has negative effects on the immune system of bees as it negatively modulates the activation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway, suppressing antiviral activity [32] and known to be involved also in neoplastic transformation [33]. Midic et al. analyzed in vitro the immunosuppressive effects of atrazine on rhesus monkey trophoblast stem cells. A suppression of IFN-γ mediated signaling pathways, such as TNF, IL-1β, STAT1, STAT2 and STAT3, was observed, as well an increase in mRNA expression encoding various immunomodulation genes; overall, suppression of immune and antiviral function shows that prolonged low-dose exposure to atrazine can alter the integrity and functionality of the embryo implantation structures, compromising gestation [34].
Role of oxidative stress Experimental studies support the hypothesis that organochlorine pesticides induce both an oxidative stress response in tissues and disrupt the immune system in vertebrates, from humans to fish. OCPs alter the immune system with effects mainly on the antibody response; a study conducted on rats, which were administered dichlorodiphenyltrichloroethane (DDT) and lindane orally, showed that in addition to the antibody deficiency there is immunosuppression following production of free
radicals [35]. Moreover, background exposure to OCPs has been positively related to the percentage of CD8+ T lymphocytes exhibiting cell surface characteristics associated with senescence in the general population, and this relationship was stronger than those for known risk factors of immunosenescence, such as age [36]. The self-renewal ability of stem cells is known to decline with advancing age. Increasing evidence suggests that also dysregulated formation of ROS may conduct stem cells into premature senescence [37]. A recent review by Asghari et al. has described the protective role that melatonin can play in relation to pesticide exposure. In fact, since oxidative stress is one of the main mechanisms involved in pesticide-induced damage, melatonin does exert its protective effect by reversing the state of oxidative stress induced by exposure, reducing the amount of reactive species and regulating the antioxidant enzymes such as glutathione and catalase [38]. Melatonin has also shown protective effects against chloranil-induced neuroinflammation through the TLR4 pathway [39]. A recent study evaluated the immunotoxicity of mancozeb by analyzing rats splenocytes in vitro. Following exposure to mancozeb, a significant increase in splenocyte death was observed due to both apoptosis and necrosis processes, conversely this toxic effect was mitigated by vitamin E, suggesting an oxidative mechanism [22]. A study conducted on zebrafish evaluated the effects induced by dieldrin. Mitochondrial dysfunction has been observed in the central nervous system; furthermore, the authors point out that dieldrin interferes with T-cell signaling cascades in the hypothalamus, acting globally as an immunosuppressor. Dieldrin exposure also resulted in the production of ROS in primary human neutrophils, increased mortality for infection in mice, suppression of primary IgM response in sheep blood; therefore, there can be a range of effects on both immune systems, innate and adaptive [40]. Even palmitoleic acid, used as an algicide, seems to have effects on the immune system of bivalves; it resulted capable of modulating a series of immune responses including the expression of related genes (FREP, PGRP, HSP90, MnSOD and Cu/ZnSOD) in these organisms [41]. Although it is well known that excessive ROS production can cause toxicity through oxidative damage to key cellular components [10], interference with normal signaling pathways also has detrimental consequences [42,43]. In this complex scenario, signaling pathways involving oxidative stress, such as NF-κB, TLR, Imd and Jak/STAT, as well as other IFN-γ mediated signaling pathways, such as TNF and IL-1β, are important for various biological processes including regulation of cell proliferation, differentiation, apoptosis and network function of the immune system.
Future directions Even though health concerns about pesticide exposure have mainly been focused on their direct mutagenic and neurotoxic potential, pesticides may exert many profound effects on human health through transient or permanent alteration of the immune system in case of both environmental or occupational exposures. Consequences of chronic or early-life exposure to pesticides may be extended beyond innate immune dysfunction to the increased risk of late-life chronic inflammatorybased diseases. Choosing appropriate toxicity biomarkers is one of the main concerns in the field of immunotoxicology where in combination with multivariate statistical analysis, it may provide a sensitive means for detecting disturbance to the immune system parameters. Moreover, since for
many pesticides, there are multiple targets and mechanisms of action, comprehension of direct and indirect pathways utilized by pesticides to affect immune system can lead to better understanding of the diagnostic potential of these biomarkers; consequently, it would lead to taking more effective, protective and therapeutic strategies. For this purpose, new technologies, e.g. droplet digital polymerase chain reaction (ddPCR), liquid biopsy and metabolomics, have been introduced for the monitoring of pesticides blood levels along with molecular alterations. These approaches will allow the assessment of the actual body burden of environmental pollutants associating it with a screening for the early diagnosis of pathologies [44]. Since laboratory experiments are not always typical of real-life situations, closing the gaps between real-life conditions and experimental settings is a crucial issue which should be considered more in immunotoxicity studies. For example, in most immunotoxicity studies, animals are exposed to high doses of a single pesticide, while in real-life conditions individuals are chronically contacted with low doses of a series of different chemicals and/or pesticides. Due to the probable synergies or antagonistic effects of chemicals when they are combined together, toxic effects and the deriving risk may be over or differ from their single effects. This is the main limit in standard toxicological trials performed by regulatory agencies to formulate safe exposure thresholds. In order to better encounter the real risk, study protocols have been proposed for hazard acknowledgement of some selected mixtures. A recent study proposed a draft protocol taking into consideration the realistic setting of long-term low-dose exposure to mixtures containing pesticides, either alone or in combination with common chemicals in commercial products, in order to establish a standardized method to evaluate the daily exposures of the general population. This could lead to a new cumulative risk assessment methodology and no longer to the current single risk approach [45,46]. Even if these trials simulate a setting closer to the daily reality, they cannot however replace the traditional experimental modelling that till today has seen the evaluation of the single pesticide at a known dose. Rather, these methods should go hand in hand to ensure a more proper risk assessment. Therefore, the awareness of the toxic effects of pesticides and the availability of molecular screening tests for the biological monitoring of exposure levels and early effects are fundamental to enhance the surveillance strategies, in order to hinder the growing incidence of chronicdegenerative diseases probably related to pesticide exposure.
Conflict of interest disclosure The authors declare no conflict of interest.
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Annotated References 1. A.M. Cowie, K.I. Sarty, A. Mercer, J. Koh, K.A. Kidd, C.J. Martyniuk, Molecular
2.
3.
4.
5.
networks related to the immune system and mitochondria are targets for the pesticide dieldrin in the zebrafish ( Danio rerio ) central nervous system, J. Proteomics. 157 (2017) 71–82. doi:10.1016/j.jprot.2017.02.003. a. The study provides new informations on the neurotoxic mechanisms of dieldrin. Through a quantitative proteomic analysis in zebrafish, alterations in proteins functionally associated with mitochondria have been identified. The resulting mitochondrial dysfunction and T-cell regulation may lie behind the association between this pesticide and the increased risk of neurodegeneration. V. Christen, F. Mittner, K. Fent, Molecular Effects of Neonicotinoids in Honey Bees (Apis mellifera), Environ. Sci. Technol. 50 (2016) 4071–4081. doi:10.1021/acs.est.6b00678. b. In bees it has been observed that neonicotinoids are able to alter immune response by interfering with 3 pathways (TLR, Imd and JAK / STAT); they also induce endoplasmic reticulum stress and have negative effects on cytochrome P450 enzymes. X. Leveque, M. Hochane, F. Geraldo, S. Dumont, C. Gratas, L. Oliver, C. Gaignier, V. Trichet, P. Layrolle, D. Heymann, O. Herault, F.M. Vallette, C. Olivier, Low-Dose Pesticide Mixture Induces Accelerated Mesenchymal Stem Cell Aging In Vitro, Stem Cells. (2019). doi:10.1002/stem.3014. c. Authors induced in vitro cellular senescence related to oxidative stress in human mesenchymal stem cells (MSCs) exposed to low doses of common pesticides, resulting in a non-reversible modification of the phenotype. Changes in metabolic markers and in cytokine production associated with cellular aging have also been observed. In conclusion, they hypothesize that pesticides can induce MSC premature cellular aging. A.P. Mestre, P.S. Amavet, A.I. Vanzetti, M.S. Moleón, M.V. Parachú Marcó, G.L. Poletta, P.A. Siroski, Effects of cypermethrin (pyrethroid), glyphosate and chlorpyrifos (organophosphorus) on the endocrine and immune system of Salvator merianae (Argentine tegu), Ecotoxicol. Environ. Saf. (2019). doi:10.1016/j.ecoenv.2018.10.057. d. Authors focused on the immune system and endocrine alterations that have been observed in pesticide-exposed reptiles. In particular, a mixture of cypermethrin, glyphosate and chlorpyrifos caused a reduction in the total white blood cell count and antibody titers, suggesting a reduction in global immune function. P. Wang, J. Wang, Y.-J. Sun, L. Yang, Y.-J. Wu, Cadmium and chlorpyrifos inhibit cellular immune response in spleen of rats, Environ. Toxicol. 32 (2017) 1927–1936. doi:10.1002/tox.22415. e. A mixture of Cadmium and Chlorpyrifos at low doses is able to induce an inhibition of interferon-γ production and on the production of interleukin 2 in the splenocytes of the exposed rats, establishing a synergistic effect. Furthermore, proliferation of the immune cells was reduced, suggesting that exposure to both pesticides simultaneously may be more dangerous than exposure to the single compound.
S2468-2020(20)30004-8
DOI:
https://doi.org/10.1016/j.cotox.2020.01.002
Reference:
COTOX 235
To appear in:
Current Opinion in Toxicology
Received Date: 23 July 2019 Revised Date:
23 December 2019
Accepted Date: 6 January 2020
Please cite this article as: C. Costa, G. Briguglio, R. Catanoso, F. Giambò, I. Polito, M. Teodoro, C. Fenga, New perspectives on cytokine pathways modulation by pesticide exposure., Current Opinion in Toxicology, https://doi.org/10.1016/j.cotox.2020.01.002. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier B.V.
CRediT author statement
MS title: New perspectives on cytokine pathways modulation by pesticide exposure.
Authors Chiara Costa Giusi Briguglio Rosaria Catanoso Federica Giambò Irene Polito Michele Teodoro Concettina Fenga
Role Methodology; Roles/Writing – original draft; Writing – review & editing Writing – original draft; Visualization; Formal analysis; Visualization; Formal analysis; Investigation; Writing – review & editing Conceptualization; Roles/Writing – original draft; Writing – review & editing
New perspectives on cytokine pathways modulation by pesticide exposure. Chiara Costa1, Giusi Briguglio2, Rosaria Catanoso2, Federica Giambò2, Irene Polito2, Michele Teodoro2, Concettina Fenga2
1
Department of Clinical and Experimental Medicine - University of Messina, Italy
2
Department of Biomedical and Dental Sciences and Morpho-functional Imaging - Occupational Medicine Section - University of Messina, Italy
*Corresponding Author: Michele Teodoro Department of Biomedical and Dental Sciences and Morpho-functional Imaging Occupational Medicine Section University of Messina, Italy Policlinico Universitario “G. Martino” – pad. H Via Consolare Valeria 1 98125, Messina, Italy Tel: +39 090 2212052 Fax: +39 090 2212051 e-mail: [email protected] KEYWORDS: pesticides, immune system; occupational exposure, cytokine pathways.
Word count: [2376]
Abstract Immune cells are able to release a variety of inflammation mediators, activating pro- and antiinflammatory processes and regulating intracellular pathways. Consequences of chronic or earlylife exposure to pesticides may be extended beyond innate immune dysfunction to the increased risk of late-life chronic inflammatory-based diseases. This study aims to summarize some of the most recent advancements in occupational toxicology, focusing on biological mechanisms linking environmental exposure to pesticides, inflammation and cytokine modulation, as well as genetic polymorphisms or epigenetic modifications which can represent factors of vulnerability for exposed workers. Choosing appropriate toxicity biomarkers is also one of the main concerns in the field of immunotoxicology; for this purpose new technologies have been introduced for the monitoring of pesticides blood levels along with molecular alterations. These approaches will allow the assessment of the actual body burden of environmental pollutants associating it with a screening for the early diagnosis of pathologies.
Highlights • • • • •
Pesticides exposure and dysregulated formation of ROS may conduct immune stem cells into premature senescence Pesticide immunotoxicity involves signaling pathways by reciprocal triggering with the inflammatory cascade through cytokine modulation NF-κB, TLR and IFN-γ -mediated signaling pathways are involved in pesticide immunotoxicity New methods to toxicity testing like in silico studies and computational modeling can add new perspectives to pesticide risk assessment Study protocols have been proposed for hazard acknowledgement of some selected mixtures to better assess the actual risk
Graphical abstract
Introduction Organochlorine pesticides (DDT, pentachlorophenol, hexachlorobenzene, dieldrin, etc.) have been used to control the spread of pests that may compromise the healthiness and characteristics of foodstuffs. Owing to their ability to resist chemical degradation and to bioaccumulate they are classified as persistent organic pollutants (POPs). Although the production and use of some compounds has been banned or limited, they continue to persist in several ecosystems, representing a serious risk for humans and exposed living species [1,2]. Pesticides can interact with normal human biochemical processes such as immune system homeostasis [3]. Recent studies reported that exposure to pesticides can impair immunity reducing antimicrobial activity, and disturb the endocrine system altering the production of inflammatory mediators such as cytokines and chemokines [4–8]. Cytokines act with both synergistic and antagonistic mechanisms in order to maintain homeostasis, in particular between oxidant and antioxidant signals [9,10]. This equilibrium is very sensitive to the action of substances such as pesticides, which are responsible for structural and functional alterations of the network [11,12]. Although the identification of xenobiotics associated with immunotoxicity and the mechanisms by which they perturb the immune system has grown in importance in recent years, there are significant gaps in their understanding. This review aims to focus on the most recent advances in biological mechanisms linking exposure to pesticides, inflammation and cytokine modulation.
Recent literature data on mechanisms of immunotoxicity The immune system is a complex network distributed on all systems of the organism, with specialized cells able to guarantee a defense against pathogens. Immune cells are able to release a variety of inflammation mediators, including cytokines/chemokines, reactive oxygen or nitrogen species (ROS or NOS). By binding to specific receptors these mediators orchestrate this network, activating pro anti-inflammatory processes and regulating intracellular pathways; this balance can be altered by xenobiotics, such as pesticides. Thanks to the improvement of diagnostic tools and techniques, recent studies highlight various pathogenetic mechanisms modulating responsiveness to diseases related to exposure to pesticides. Recently, great interest is raising towards new methods to toxicity testing like in silico studies and computational modeling (as QSPR/QSAR); these novel approaches are likely to add new perspectives to pesticide risk assessment, allowing to provide also necessary answers to the concerns raised by the use of nano-pesticides [13,14]. Animal and in vitro models are traditionally used in experimental studies because it is not possible to test single toxic compounds in the human population for ethical reasons. In fact, numerous recent studies test the in vivo effects of pesticides in order to better understand some of the molecular mechanisms that could be reproducible in humans [15,16].
Alteration of immune cells Martyniuk et al. investigated the effect induced by two organochlorine pesticides, p,p'dichlorodiphenyldichlorethylene and methoxyclor, on Micropterus salmoides. Starting from the hypothesis that this exposure altered reproductive function, the authors failed to demonstrate this supposition. Rather the study led to another explanation, namely the target of these compounds is the immune system, and alterations in reproductive function would be secondary to the interaction between the immune system and the reproductive system. In particular, a decrease in platelet function, T cell suppression and interaction, adaptive immune responses (Th1 immune response), leukocyte function, and mast cell degranulation were observed [17]. Latorre et al. showed that exposure to glyphosate was able to lower the total white blood cell count in reptiles; as leukocytes are essential effectors for the correct functioning of the immune system exposure to glyphosate could have repercussions on the defensive capacity of the whole organism [18]. In agreement with these findings, a similar study evaluated exposure to pyrethroids (cypermethrin) and organophosphates (glyphosate and chlorpyrifos) and the results obtained are comparable [5]. A cross-sectional study conducted in Pakistan evaluated the toxic effects of pesticides belonging to different chemical classes including organophosphates (chlorpyrifos, profenofos, deltamethrin and imidacloprid) in sprayers, analyzing the values of some liver enzymes and blood parameters, including the total white blood cell count; a weakening of the immune system was observed and this condition could lead to the development of tuberculosis in exposed subjects [19]. Also deltamethrin has been shown to alter white blood cells count and to recall inflammatory cells and leukocytes in the lung [20]. Moreover pyrethroids are also been shown to reduce the activity of the immune system in both human and animals, reducing macrophage activity [21]. A recent study evaluated the immunotoxicity of mancozeb by analyzing rats splenocytes in vitro. Following exposure to mancozeb, a significant increase in splenocyte death was observed due to both apoptosis and necrosis processes [22]. Wang et al. observed that in rats the combined administration of cadmium and chlorpyrifos in vivo at environmentally-relevant low dose synergistically inhibits the proliferation of T and B lymphocytes. The same administration, in vitro, did not show the synergistic effect and chlorpyrifos showed the major inhibitory effects on cytokine production in isolated splenocytes [23]. A decrement in total G immunoglobulins (IgG) was observed in blood samples of workers employed in a pesticide production plant who were exposed to 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD), a compound used in the production of herbicides [24]. Leveque et al. demonstrated that a pesticide mixture commonly found in food, at noncytotoxic doses, can induce selective modifications on mesenchimal stem cells (MSC), suggesting a premature cellular aging profile [25].
Cytokine modulation Other studies investigated mechanisms behind immunotoxicity at a molecular level. The immunotoxicity of α-cypermethrin was assessed in greenhouse workers, using urinary 3phenoxybenzoic acid as an exposure biomarker; exposed workers showed no clinical sign of immunotoxicity, but proinflammatory cytokines involved in antitumor immunity and response to infection (IL-12p70, INF-γ, IL-2 and IL-8) were significantly reduced; findings support the hypothesis that pyrethroid exposure may reduce host defenses against infection and cancer, particularly in subjects with impaired immune capacity [26]. Other studies confirmed that
pyrethroids reduce the production of IL-2, IL-8, IL-12p70 and interferon γ (IFN-γ), and also increase the expression of the Toll-like receptor 4 (TLR4) and the necrotic factor-alpha (TNFα) [20,21]. In carbofuran-treated rats were found increased SMAD protein levels and a rise of TGF-β levels [27]. Still, Kumar et al. observed that organochlorine pesticides were associated with high levels of complement system markers, particularly C3 and C3a [28]. Pesticide mixture induced activation pathways common with aging process, such as IL-7 signaling, modifications in MSC secretome (IL-10 and IL-6), MCP-1 induction, reduced TGF- β signaling. IL-7 signaling plays a role in the body’s innate and adaptive immune responses and in regulating T cells. IL-6 is commonly described as a proinflammatory cytokine, while IL-10 suppresses macrophage and neutrophil functions and inhibits the Th1 immune response. MCP-1 is a key chemokine regulating the recruitment and migration of cells of the monocyte-macrophage system. TGF-β regulates multiple fundamental cellular functions and biological processes [25]. Overall these modifications play a role in inflammatory cascade.
Signaling pathways Alterated levels of cytokines induce the activation or inhibition of some biological pathways mainly related with inflammatory processes. Christen and Fent showed that chlorpyrifos, malathion, cypermethrin and chlorantraniliprole are able to induce significant alterations in genes related to the immune system in honeybees, in particular the three pathways involved are TLR, Imd and Jak/STAT. Among the four pesticides, cypermethrin showed the greatest downregulating effects on all three pathways. The downregulation of abaecin and apidaecin, and the up-regulation of defensin-1 was determined by chlorpyrifos, the up-regulation of abaecin, apidaecin and defensin-1 transcripts by malathion, the down-regulation of relish, hopscotch and domeless by cypermethrin, and the upregulation of apidaecin and relish by chlorantraniliprole [29]. Previously the same Authors had shown that also neonicotinoids were able to modify the gene expression of the immune system [30]. Furthermore, neonicotinoids have also been shown to reduce antimicrobial defense in bees [31]; in particular clothianidin has negative effects on the immune system of bees as it negatively modulates the activation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway, suppressing antiviral activity [32] and known to be involved also in neoplastic transformation [33]. Midic et al. analyzed in vitro the immunosuppressive effects of atrazine on rhesus monkey trophoblast stem cells. A suppression of IFN-γ mediated signaling pathways, such as TNF, IL-1β, STAT1, STAT2 and STAT3, was observed, as well an increase in mRNA expression encoding various immunomodulation genes; overall, suppression of immune and antiviral function shows that prolonged low-dose exposure to atrazine can alter the integrity and functionality of the embryo implantation structures, compromising gestation [34].
Role of oxidative stress Experimental studies support the hypothesis that organochlorine pesticides induce both an oxidative stress response in tissues and disrupt the immune system in vertebrates, from humans to fish. OCPs alter the immune system with effects mainly on the antibody response; a study conducted on rats, which were administered dichlorodiphenyltrichloroethane (DDT) and lindane orally, showed that in addition to the antibody deficiency there is immunosuppression following production of free
radicals [35]. Moreover, background exposure to OCPs has been positively related to the percentage of CD8+ T lymphocytes exhibiting cell surface characteristics associated with senescence in the general population, and this relationship was stronger than those for known risk factors of immunosenescence, such as age [36]. The self-renewal ability of stem cells is known to decline with advancing age. Increasing evidence suggests that also dysregulated formation of ROS may conduct stem cells into premature senescence [37]. A recent review by Asghari et al. has described the protective role that melatonin can play in relation to pesticide exposure. In fact, since oxidative stress is one of the main mechanisms involved in pesticide-induced damage, melatonin does exert its protective effect by reversing the state of oxidative stress induced by exposure, reducing the amount of reactive species and regulating the antioxidant enzymes such as glutathione and catalase [38]. Melatonin has also shown protective effects against chloranil-induced neuroinflammation through the TLR4 pathway [39]. A recent study evaluated the immunotoxicity of mancozeb by analyzing rats splenocytes in vitro. Following exposure to mancozeb, a significant increase in splenocyte death was observed due to both apoptosis and necrosis processes, conversely this toxic effect was mitigated by vitamin E, suggesting an oxidative mechanism [22]. A study conducted on zebrafish evaluated the effects induced by dieldrin. Mitochondrial dysfunction has been observed in the central nervous system; furthermore, the authors point out that dieldrin interferes with T-cell signaling cascades in the hypothalamus, acting globally as an immunosuppressor. Dieldrin exposure also resulted in the production of ROS in primary human neutrophils, increased mortality for infection in mice, suppression of primary IgM response in sheep blood; therefore, there can be a range of effects on both immune systems, innate and adaptive [40]. Even palmitoleic acid, used as an algicide, seems to have effects on the immune system of bivalves; it resulted capable of modulating a series of immune responses including the expression of related genes (FREP, PGRP, HSP90, MnSOD and Cu/ZnSOD) in these organisms [41]. Although it is well known that excessive ROS production can cause toxicity through oxidative damage to key cellular components [10], interference with normal signaling pathways also has detrimental consequences [42,43]. In this complex scenario, signaling pathways involving oxidative stress, such as NF-κB, TLR, Imd and Jak/STAT, as well as other IFN-γ mediated signaling pathways, such as TNF and IL-1β, are important for various biological processes including regulation of cell proliferation, differentiation, apoptosis and network function of the immune system.
Future directions Even though health concerns about pesticide exposure have mainly been focused on their direct mutagenic and neurotoxic potential, pesticides may exert many profound effects on human health through transient or permanent alteration of the immune system in case of both environmental or occupational exposures. Consequences of chronic or early-life exposure to pesticides may be extended beyond innate immune dysfunction to the increased risk of late-life chronic inflammatorybased diseases. Choosing appropriate toxicity biomarkers is one of the main concerns in the field of immunotoxicology where in combination with multivariate statistical analysis, it may provide a sensitive means for detecting disturbance to the immune system parameters. Moreover, since for
many pesticides, there are multiple targets and mechanisms of action, comprehension of direct and indirect pathways utilized by pesticides to affect immune system can lead to better understanding of the diagnostic potential of these biomarkers; consequently, it would lead to taking more effective, protective and therapeutic strategies. For this purpose, new technologies, e.g. droplet digital polymerase chain reaction (ddPCR), liquid biopsy and metabolomics, have been introduced for the monitoring of pesticides blood levels along with molecular alterations. These approaches will allow the assessment of the actual body burden of environmental pollutants associating it with a screening for the early diagnosis of pathologies [44]. Since laboratory experiments are not always typical of real-life situations, closing the gaps between real-life conditions and experimental settings is a crucial issue which should be considered more in immunotoxicity studies. For example, in most immunotoxicity studies, animals are exposed to high doses of a single pesticide, while in real-life conditions individuals are chronically contacted with low doses of a series of different chemicals and/or pesticides. Due to the probable synergies or antagonistic effects of chemicals when they are combined together, toxic effects and the deriving risk may be over or differ from their single effects. This is the main limit in standard toxicological trials performed by regulatory agencies to formulate safe exposure thresholds. In order to better encounter the real risk, study protocols have been proposed for hazard acknowledgement of some selected mixtures. A recent study proposed a draft protocol taking into consideration the realistic setting of long-term low-dose exposure to mixtures containing pesticides, either alone or in combination with common chemicals in commercial products, in order to establish a standardized method to evaluate the daily exposures of the general population. This could lead to a new cumulative risk assessment methodology and no longer to the current single risk approach [45,46]. Even if these trials simulate a setting closer to the daily reality, they cannot however replace the traditional experimental modelling that till today has seen the evaluation of the single pesticide at a known dose. Rather, these methods should go hand in hand to ensure a more proper risk assessment. Therefore, the awareness of the toxic effects of pesticides and the availability of molecular screening tests for the biological monitoring of exposure levels and early effects are fundamental to enhance the surveillance strategies, in order to hinder the growing incidence of chronicdegenerative diseases probably related to pesticide exposure.
Conflict of interest disclosure The authors declare no conflict of interest.
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Annotated References 1. A.M. Cowie, K.I. Sarty, A. Mercer, J. Koh, K.A. Kidd, C.J. Martyniuk, Molecular
2.
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networks related to the immune system and mitochondria are targets for the pesticide dieldrin in the zebrafish ( Danio rerio ) central nervous system, J. Proteomics. 157 (2017) 71–82. doi:10.1016/j.jprot.2017.02.003. a. The study provides new informations on the neurotoxic mechanisms of dieldrin. Through a quantitative proteomic analysis in zebrafish, alterations in proteins functionally associated with mitochondria have been identified. The resulting mitochondrial dysfunction and T-cell regulation may lie behind the association between this pesticide and the increased risk of neurodegeneration. V. Christen, F. Mittner, K. Fent, Molecular Effects of Neonicotinoids in Honey Bees (Apis mellifera), Environ. Sci. Technol. 50 (2016) 4071–4081. doi:10.1021/acs.est.6b00678. b. In bees it has been observed that neonicotinoids are able to alter immune response by interfering with 3 pathways (TLR, Imd and JAK / STAT); they also induce endoplasmic reticulum stress and have negative effects on cytochrome P450 enzymes. X. Leveque, M. Hochane, F. Geraldo, S. Dumont, C. Gratas, L. Oliver, C. Gaignier, V. Trichet, P. Layrolle, D. Heymann, O. Herault, F.M. Vallette, C. Olivier, Low-Dose Pesticide Mixture Induces Accelerated Mesenchymal Stem Cell Aging In Vitro, Stem Cells. (2019). doi:10.1002/stem.3014. c. Authors induced in vitro cellular senescence related to oxidative stress in human mesenchymal stem cells (MSCs) exposed to low doses of common pesticides, resulting in a non-reversible modification of the phenotype. Changes in metabolic markers and in cytokine production associated with cellular aging have also been observed. In conclusion, they hypothesize that pesticides can induce MSC premature cellular aging. A.P. Mestre, P.S. Amavet, A.I. Vanzetti, M.S. Moleón, M.V. Parachú Marcó, G.L. Poletta, P.A. Siroski, Effects of cypermethrin (pyrethroid), glyphosate and chlorpyrifos (organophosphorus) on the endocrine and immune system of Salvator merianae (Argentine tegu), Ecotoxicol. Environ. Saf. (2019). doi:10.1016/j.ecoenv.2018.10.057. d. Authors focused on the immune system and endocrine alterations that have been observed in pesticide-exposed reptiles. In particular, a mixture of cypermethrin, glyphosate and chlorpyrifos caused a reduction in the total white blood cell count and antibody titers, suggesting a reduction in global immune function. P. Wang, J. Wang, Y.-J. Sun, L. Yang, Y.-J. Wu, Cadmium and chlorpyrifos inhibit cellular immune response in spleen of rats, Environ. Toxicol. 32 (2017) 1927–1936. doi:10.1002/tox.22415. e. A mixture of Cadmium and Chlorpyrifos at low doses is able to induce an inhibition of interferon-γ production and on the production of interleukin 2 in the splenocytes of the exposed rats, establishing a synergistic effect. Furthermore, proliferation of the immune cells was reduced, suggesting that exposure to both pesticides simultaneously may be more dangerous than exposure to the single compound.