Philosophiae Doctor - PhD / Pesticides are designed to control or eliminate pests such as insects, rodents, weeds, bacteria, and fungi. They are used at a global scale for agricultural produce. Although pesticides playa significant role in increasing food production and eliminating diseases, exposure to pesticides may be harmful to non-target organisms. As a result concern over safety and resistance to pesticides has increased and there is pressure to reduce use and search for more environmentally and toxicologically safe and efficacious pesticides. Most pesticides currently in use are synthetic; therefore an alternative to synthetic pesticides is the use of naturally occurring products! botanicals with pesticidal properties. Two plants indigenous to South African with pesticidal properties were chosen for this study. Dicerothamnus rhinocerotis (D. rhinocerotis) and Galenia africana (G. africana) have potential antifungal properties thus, may have potential use on agricultural produce as fungicides. Galenia africana and D. rhinocerotis extracts inhibit growth of B. cinerea (a fungal pathogen) at concentrations greater than 31.25 mg/ml and 125mg/ml respectively. A major consideration in approving pesticides for use is whether they pose an unreasonable risk to humans and to the environment. Toxicity studies are required to determine the safety of the plant extracts. The purpose of this study was to evaluate potential toxicity of ethanol extracts of D. rhinocerotis and G. africana, which is important when designing practices to reduce or eliminate excess exposure to them. Natural plant products with pesticidal properties could provide an alternative to synthetic pesticides and may thus effectively reduce resistance levels. This first objective of this study was to assess the cytotoxicity of D. rhinocerotis and G. africana on human cell cultures. Human whole blood and the human breast adenocarcinoma cell line (MCF-7) were treated with varying concentrations of the plant extracts and cytotoxicity determined. Cytotoxicity was measured using several biomarkers. Inhibiotory concentration for a 50% effect (ICso) and no observable effect level (NOEL) values were obtained for D. rhinocerotis and G. africana. The results showed that extracts of D. rhinocerotis and G. africana had cytotoxic effects on the cell cultures. The second objective of this study was to determine the ecotoxicity of D. rhinocerotis and G. africana. A series of acute toxicity tests, with effective concentration for a 50% effect (ECso) and lethal concentration for a 50% effect (LCso) as biomarkers, were conducted to estimate the potential environmental effect of the two plants. The tests were carried out using Vibrio fischeri, Selenastrum capricornutum, Daphnia pulex, and Poecilia reticulata as bioindicators. Results obtained showed that G. africana had higher toxicity units than D. rhinocerotis, thus showing that G. africana is more toxic to the aquatic species used as compared to D. rhinocerotis. The third objective of this study was to investigate the immunomodulatory effects of the two plant extracts. This was achieved by using mouse spleen cell cultures. Exposure of pesticides to the immune system may result in alteration of the normal immune functions. The cytokines IFN-y and IL-4 were used as biomarkers to determine the T-cell activity of the immune system when exposed to the two botanical products. The results obtained showed that both D. rhinocerotis and G. africana decrease of the cytokines interferon gamma (IFN-y) and interleukin-4 (IL-4), thus may have immunotoxic effects. The fourth objective was to investigate the hepatotoxicity of the two plant extracts. Injury to the liver was investigated using a range of clinical biochemical tests that monitor liver enzyme activity and metabolic activity. Primary liver cell cultures were exposed to the plant products in question, after which the biochemical tests were carried out. The enzymes that were monitored were alanine aminotrasferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and lactate dehydrogenase (LDH). The results obtained showed that both of D. rhinocerotis and G. africana may have effects on the liver, as shown by the increased levels of enzymes released from cells upon exposure to plant extracts. The final objective of this study was to investigate the effect of the two plants on the male reproductive system. Injury to the male reproductive system was investigated using testicular cell cultures. Primary cell cultures were stimulated with luteinizing-hormone (LH) and exposed to the plant extracts. LH results in the production of testosterone, thus testosterone was used as a biomarker for assessing reproductive toxicity. The results obtained showed that both of D. rhinocerotis and G. africana have effects on the male reproductive system, as shown by the decreased testosterone secretion. Botanicals provide a simple, inexpensive and environmentally friendly (non-pollution and lesser toxicological concerns) alternative for pest control. However, motivation for the commercial use of botanicals as pesticides requires validating the efficacy of the plant as a pesticide, and also assessing its effects on human health and the environment. An important component of this evaluation involves toxicity studies, which enables cautions of dangerous practices and toxic effects of the plants to be issued.

Obesity is a disease that increases risk of developing metabolic diseases including insulin resistance (IR), metabolic syndrome (MS), and type 2 diabetes (T2D). Adipose tissue expansion during obesity leads to immune cell infiltration, causing local inflammation and disruption of lipid homeostasis. There is an association between exposure to environmental chemicals, like p,p’-DDE, a metabolite of p,p’-DDT, and diagnosis of obesity, dyslipidemia, IR, and prevalence of MS and T2D. DDE accumulates in fatty tissues and has been shown to have immunomodulatory properties, affecting macrophage and T cell populations. Potential mechanisms were studied by which DDE could modulate adipocyte and immune cell function and facilitate an increased risk of obesity and immune dysregulation, potentially through cyclooxygenase-2 (COX-2). 3T3-L1 preadipocytes and J774A.1 macrophages were studied for the effects of DDE on adipogenesis and macrophage reactivity, respectively. 3T3-L1 cells were induced to differentiate to adipocytes using a sub-optimal differentiation cocktail with increasing concentrations of DDE (0.5uM-100uM). It was determined that DDE enhanced adipogenesis in a concentration dependent manner and the expression of adipogenic and lipogenic genes, indicating that DDE enhances adipogenesis. In J774A.1 cells, the ability of DDE or 10uM NS-398, a specific COX-2 inhibitor, to inhibit the production of the prostaglandins PGE2, PGD2, PGF2a, was assessed in vitro and in a cellree system. DDE or NS-398 followed by immune challenge reduced cellular PG secretion and reduced PG production in a cell free system, indicating that DDE may interfere with lipid mediator signaling. Additionally, DDE or NS-398 exposure altered gene expression in J774A.1 cells following M1 or M2 polarization stimulus. Lastly, male C57Bl mice were exposed to 2mg/kg DDE for 5 days and the macrophage population of the adipose stromal vascular fraction was analyzed by flow cytometry. Adipose from DDE treated animals contained approximately 40% F4/80+CD11b+ macrophages. These results indicate that DDE may alter the homeostasis of adipose tissue by both enhancing adipogenesis and altering the reactivity of the resident macrophage population in a manner that may contribute to adipose dysfunction. These data suggest a possible mechanism by which DDE exposure may contribute to adiposity and adipose tissue dysfunction commonly seen in metabolic disease.

The use of the plasticizer bis(2-ethylhexyl)phthalate (DEHP) and other plasticizers in the manufacture of plastic products has been restricted due to adverse health outcomes such as obesity, metabolic syndrome, and asthma, for which inflammation has been described to be a driving factor. The emerging alternative plasticizer 1,2-cyclohexanedioic acid diisononyl ester (DINCH) still lacks information regarding its potential effects on the immune system. Here, we investigated the effects of DINCH and its naturally occurring metabolite monoisononylcyclohexane-1,2-dicarboxylic acid ester (MINCH) on the innate immune response. Human THP-1 macrophages were exposed to 10 nM–10 μM DINCH or MINCH for 4 h, 16 h, and 24 h. To decipher the underlying mechanism of action, we applied an untargeted proteomic approach that revealed xenobiotic-induced activation of immune-related pathways such as the nuclear factor κB (NF-κB) signaling pathway. Key drivers were associated with oxidative stress, mitochondrial dysfunction, DNA damage repair, apoptosis, and autophagy. We verified increased reactive oxygen species (ROS) leading to cellular damage, NF-κB activation, and subsequent TNF and IL-1β release, even at low nM concentrations. Taken together, DINCH and MINCH induced cellular stress and pro-inflammatory effects in macrophages, which may lead to adverse health effects.

No description available.

The present study examined adverse effects of sunlight exposure (mimicked by intradermal cis-urocanic acid, cUCA) on local and systemic immune responses, with or without co-exposure to the immunotoxic insecticide permethrin. A single exposure to cUCA caused diminished splenic macrophage phagocytosis that was persistent up to 30 days post-exposure. Five-day exposure to cUCA subtly increased splenocyte proliferation in response to the T cell mitogen Concanavalin A. Four-week exposure to cUCA caused increased splenic lymphocyte cellularity, thymic hypocellularity, and enhanced hydrogen peroxide production by splenic leukocytes. Single exposure to topical permethrin resulted in decreased thymic and splenic weight and cellularity, and inhibited antibody production by splenic B cells. cUCA worsened the negative effect of permethrin on both thymic weight and cellularity, and depressed splenocyte blastogenesis, hydrogen peroxide production, and antibody production. Five-day exposure to either cUCA or permethrin also caused persistent decreased contact hypersensitivity responses, an effect that became more than additive when the chemicals were administered concurrently. Defects in antigen processing and presentation by cutaneous Langerhans cells were evaluated as possible contributing mechanisms to the cutaneous immunosuppression, using mice with deleted genes. Vehicle-exposed IFNg knockout mice displayed approximately a 22.1% depression in the ear swelling response as compared to control C57BL/6N mice, suggesting that this cytokine may be required for mounting a control-level hypersensitivity response. Ear swelling in cUCA-exposed IFNg knockout mice displayed a 21.4% depressed response as compared to cUCA-exposed wild-type C57BL/6N mice, again suggesting that IFNg is an important cytokine in the contact hypersensitivity (CH) response. TNFaR knockout mice exposed to cUCA displayed 33.9% greater ear swelling than cUCA-exposed wild-type C57BL/6N mice, suggesting that increased TNFa may be involved in inhibited CH by cUCA. TNFaR knockout mice exposed to permethrin displayed 33.9% greater ear swelling than permethrin-exposed C57BL/6N mice, suggesting that increased TNFa may also be involved in inhibited CH by permethrin. C57BL/6N mice exposed to cUCA + permethrin displayed severe reduction of the CH response to 8.7% of the control level. IFNg knockout mice exposed to permethrin + cUCA showed essentially identical depression of the CH response as IFNg knockout mice exposed to either permethrin or cUCA alone. These results suggest that IFNg is required for the greater than additive immunotoxic effect that occurred when these two agents were co-administered. TNFaR knockout mice exposed to cUCA + permethrin displayed 8.7 fold greater ear swelling than similarly exposed C57BL/6N mice, again suggesting that increased TNFa is involved in inhibited CH by both cUCA and permethrin. / Ph. D.

Les nanoparticules (NP) ont été introduites en médecine pour développer des médicaments intelligents ou des agents d'imagerie. En raison de leur petite taille (<200 nm), les NP permettent la mise en place de thérapies ciblées, augmentent la diffusion et l’efficacité des drogues, tout en facilitant les modes d’administration et en diminuant les couts de santé publique. Malgré les promesses que présentent les NP pour la médecine, les risques potentiels pour la santé humaine associés à une exposition aux NP restent mal documentés ; en particulier en ce qui concerne leurs effets sur le système immunitaire. Les cellules présentatrices d’antigène (CPA) (comprenant les macrophages et les cellules dendritiques) sont recrutées au site d’inflammation induite par des pathogènes et constituent une ligne de défense majeure pour notre organisme. Les CPA sont dotées d’une activité de phagocytose et endocytose conduisant à une forte internalisation des NP. Ainsi, ces cellules seront parmi les plus affectées par une exposition aux NP et sont un modèle expérimental pertinent pour l’étude du devenir cellulaire des NP et de leurs effets sur l’hôte.Dans cette étude, nous avons étudié si les fonctions de ces cellules pourraient être modifiées par une exposition aux NP. Comme modèles de NP, nous avons choisi l'or (AuNP) et le gadolinium-polysiloxane (GdSi) utilisés comme agent de contraste ou de théranostique, le poly lactic-co-glycolic acid (PLGA) et une nano émulsion lipidique (LNP) développés comme plateforme de délivrance d’antigènes ou de médicaments. Tout d'abord, en utilisant des microsphères fluorescentes comme sonde, nous avons montré que toutes les NP testées n'altèrent pas la capacité de phagocytose de la lignée cellulaire de macrophages J774. Ensuite, l’activation des cellules a été analysée par cytométrie de flux, basée sur l'expression des marqueurs de surface CD-86 et MHC-II. Nous avons établi que l'exposition aux NP n'active pas les cellules dendritiques dérivées de la moelle osseuse (BMDC). Dans le même sens, aucune de ces NP n’induit par elle-même de sécrétions de cytokines par les BMDC. En outre, l’activation de ces cellules par des activateurs connus, tels que le lipopolysaccharide bactérien (LPS) n'est pas modifiée par les NP. Cependant, l'exposition aux AuNP diminue l'expression des cytokines IL-6, IL-12 et IL-23 par les BMDC activées par LPS. Or, ces cytokines sont impliquées dans la polarisation des lymphocytes T CD4 + vers le phénotype T helper approprié (Th). Nous avons analysé si ces modifications de cytokines pourraient modifier la réponse Th. Dans un modèle in vitro de présentation d'antigène, les BMDC ont été incubés avec un antigène modèle (ovalbumine (OVA)) et co-cultivés avec des cellules T spécifiques de l'OVA. L'exposition aux AuNP a conduit à une augmentation des cytokines spécifiques des lymphocytes T: IL-13 (indiquant un déplacement de la balance Th1 / Th2 classique vers Th2) et IL-17 (permettant de diriger les cellules T vers Th17). L'exposition des BMDC aux autres NP de l'étude ne modifie que très faiblement leurs sécrétions de cytokines inflammatoires, et n'a donc pas d'impact sur le destin des lymphocytes T après la présentation de l'antigène.L’ensemble de ces résultats démontrent que GdSi, PLGA et LNP ne modifient pas la phagocytose, l'activation des DC et la présentation antigénique. Cependant, l'exposition aux AuNP modifie les réponses inflammatoires des DC et le devenir des cellules T vers les phénotypes Th2 et Th17. Ces modifications pourraient entraver la physiologie du système immunitaire et contribuer aux maladies chroniques ou à l'auto-immunité. / Nanoparticles (NP) have been introduced in medicine to develop intelligent drugs or imaging agents. Due to their small size (<200 nm), NPs allow the development of targeted therapies, increase drug diffusion and effectiveness while facilitating modes of administration and decreasing public health costs. Nevertheless, the potential risks for human health associated to NP exposure remain poorly documented; especially about their effects on the immune system. Antigen-presenting cells (APC) (including macrophages and dendritic cells) are recruited at the site of pathogen-induced inflammation and constitute to the maintenance of body integrity, engulfing pathogens and delivering signals to other components of the immune system. Due to their internalization abilities, APC accumulate NP in their cytoplasm. Thus, these cells will be among the most affected by exposure to NP and constitute a relevant experimental model for the study of the cellular fate of NP and their effects on the host.In this study, we investigated whether the functions of these cells could be modified by an exposure to NP. As models of NP, we selected gold (AuNP) and gadolinium-polysiloxane (GdSi) used as contrast agent for therapeutic and diagnostic applications, and poly lactic-co-glycolic acid (PLGA) and lipid nano emulsion (LNP) developed as a platform for the delivery of antigens or drugs.First, using fluorescent microspheres as probe, we have shown that all of the tested NP did not alter the phagocytosis capacity of the J774 macrophage cell line. Then, cell activation was analyzed by flow cytometry, based on the expression of the surface markers CD-86 and MHC-II. We have established that NP exposure did not activate bone marrow derived dendritic cells (BMDC). In this way, none of these NP induced cytokine secretions by the BMDC. Furthermore, activation of these cells by known activators, such as bacterial lipopolysaccharide (LPS) was not modified by NP.However, in this case, the cytokine response was altered by AuNP exposure, showing reduced inflammatory cytokine production such as IL-6, IL-12 and IL-23. Interestingly, these cytokines are involved in the polarization of CD4 + T lymphocytes to the appropriate T helper phenotype (Th). In a model of antigen presentation in vitro, this cytokine profile resulted into an altered development of specific immune responses. AuNP exposure increased T cell specific cytokines: IL-13 and IL-4 (indicating a shift of classical Th1/Th2 balance towards Th2) and IL-17 (standing for an alteration of T-cell fate towards Th17). The exposure of BMDC to the other NP of the study only very slightly altered their inflammatory cytokine secretions and therefore did not affect the fate of T lymphocytes after antigen presentation.All together, these results demonstrate that GdSi, PLGA and LNP do not modify phagocytosis, DC activation and antigen presentation. However, exposure to AuNP alters the DC induced inflammatory responses and polarizes the T cell fate towards Th2 and Th17 phenotypes. These changes could impair the physiology of the immune system and contribute to chronic diseases or autoimmunity.

Les hydrocarbures aromatiques polycycliques (HAPs), tels que le benzo(a)pyrène (B[a]P), sont des contaminants environnementaux ubiquistes générés lors de la combustion de matière organique. Ces composés ont été associés au développement d'effets toxiques sur la santé humaine, notamment des effets cancérigènes et immunotoxiques, principalement liés à l'activation du récepteur aux hydrocarbures aromatiques (RAh). Parmi les cellules du système immunitaire, les lymphocytes T apparaissent comme des cibles majeures des HAPs. Des résultats antérieurs, obtenus au laboratoire, ont montré que l'activation des lymphocytes T humains en culture primaire conduit à l’augmentation de l'expression et de la fonction du RAh, suggérant la capacité accrue de ces cellules à répondre à une exposition aux HAPs. Nos objectifs sont : (1) de déterminer les effets du B[a]P sur les profils d'expression génique dans les lymphocytes humains normaux en utilisant des approches à haut débit telle que l'analyse transcriptomique sur puce à ADN, (2) d’évaluer les effets génotoxiques et immunotoxiques du B[a]P en mesurant respectivement les dommages à l'ADN induits et leurs actions immunosuppressives et (3) d’analyser la modulation de ces effets en présence d'autres HAPs. Notre travail identifie les lymphocytes T humains normaux comme un bon modèle pour étudier les effets génotoxiques et immunotoxiques des HAPs, et pour prédire les problèmes de santé humaine liés à l’exposition à ces contaminants. Il permet également de mieux comprendre la régulation par les HAPs de la réponse immune et propose de nouveaux biomarqueurs potentiels de l'exposition à ces contaminants environnementaux. / Polycyclic aromatic hydrocarbons (PAHs), such as benzo(a)pyrene (B[a]P), are ubiquitous environmental contaminants generated during organic matter combustion. These compounds have been associated with the development of toxic effects on human health, including carcinogenic and immunotoxic effects, mainly related to Aryl hydrocarbon Receptor (AhR) activation. Among the immune system cells, T lymphocytes appear as major targets of PAHs. Previous results, obtained in the laboratory, have shown that activation of primary human T lymphocytes leads to a functional AhR expression increase, suggesting their ability to respond to PAH exposure. Our specific aims are: (1) to determine the effects of B[a]P on gene expression profiles in human normal lymphocytes by using large-scale approaches such as microarray-based transcriptome analysis, (2) to monitor the genotoxic and immunotoxic effects of B[a]P by measuring DNA damage and immunosuppressive actions, respectively and, (3) to analyze the modulation of these effects by the presence of other PAHs. Our work propose primary cultures of activated human T lymphocytes as a good model for studying both genotoxic and immunotoxic effects of environmental contaminants such as PAHs and predicting human health issues. It also gains a comprehensive insight into the immune response regulation after PAH exposure and provides potential new biomarkers of exposure to these environmental contaminants.