Santoro, Anna (2014) LEVELS OF POLYCHLORINATED BIPHENYLS (PCBs) AND ORGANOCHLORINE PESTICIDES (OCPs) IN BIOINDICATOR SPECIES AND EVALUATION OF TOXIC EFFECTS OF NON-DIOXIN LIKE PCBs 101, 153 AND 180 ON IMMUNE AND ENDOCRINE SYSTEMS. [Tesi di dottorato]

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Tipologia del documento:	Tesi di dottorato
Lingua:	English
Titolo:	LEVELS OF POLYCHLORINATED BIPHENYLS (PCBs) AND ORGANOCHLORINE PESTICIDES (OCPs) IN BIOINDICATOR SPECIES AND EVALUATION OF TOXIC EFFECTS OF NON-DIOXIN LIKE PCBs 101, 153 AND 180 ON IMMUNE AND ENDOCRINE SYSTEMS
Autori:	Autore Email Santoro, Anna anna.santoro@unina.it
Data:	30 Marzo 2014
Numero di pagine:	175
Istituzione:	Università degli Studi di Napoli Federico II
Dipartimento:	Farmacia
Scuola di dottorato:	Scienze farmaceutiche
Dottorato:	Scienza del farmaco
Ciclo di dottorato:	26
Coordinatore del Corso di dottorato:	nome email D'Auria, Maria Valeria madauria@unina.it
Tutor:	nome email Meli, Rosaria [non definito]
Data:	30 Marzo 2014
Numero di pagine:	175
Parole chiave:	bioaccumulation, pollutants, toxicity
Settori scientifico-disciplinari del MIUR:	Area 05 - Scienze biologiche > BIO/14 - Farmacologia
Depositato il:	15 Apr 2014 09:24
Ultima modifica:	15 Lug 2015 01:01
URI:	http://www.fedoa.unina.it/id/eprint/9831

Abstract

During my PhD course, the residue levels of Polychlorinated biphenyls (PCBs) and Organochlorine pesticides (OCPs) in the edible tissues of two bivalve species (Mytilus galloprovincialis and Ensis siliqua) from Tyrrhenian Sea (Mediterranean Sea) were measured. Factors explaining differences in bioaccumulation levels were also considered and an evaluation of the health risk for human consumer was provided. A gas chromatography–electron capture detection (GC-ECD) was used for the analysis; the concentration levels of five OCPs—among which the 1,1,1-trichloro-2,2-bis-(4-chlorophenyl)-ethane (p,p’-DDT)—and twenty PCBs, including the seven indicator-PCBs, were determined. Differences in residue levels were revealed between the two examined species; in some cases they were statistically significant. PCBs were the most abundant pollutants (mean values of 422.19 ng g-1 and 399.33 ng g-1, respectively, for mussels and clams on fat weight), followed by DDTs, Dieldrin and hexachlorobenzene (HCB). In particular, the PCBs nos. 101, 118, 138 and 153 were the dominant congeners, accounting for 66% and 56% of the total residue levels of PCBs in mussels and clams, respectively. From the human health point of view, OCPs residue levels were below the national limits established for fish and aquatic products. Conversely, the mean concentrations of PCBs exceeded the limits set by the EU for terrestrial foods in both species. Non-dioxin-like (NDL)-PCBs are stable and lipophilic chemicals that persist in the environment and tend to bioaccumulate in the food chain. Epidemiological studies show that PCB exposure is associated with modifications of innate and adaptive immunity, including effects on immune cells and signalling molecules involved in the response against foreign antigens. It is noteworthy that no in vitro studies are available regarding the impairment of immune innate response due to NDL-PCB exposure. Therefore, here we investigated the effects of NDL-PCBs 101, 153, and 180, alone or differently associated, on lipopolysaccharide (LPS)-activated J774A.1 murine macrophages. Interestingly, concentrations of the aforesaid NDL-PCBs inactive by themselves induced immunesoppression when NDL-PCBs were differently combined. In particular, the exposure to NDL-PCB mixture caused a significative suppression of LPS-induced cytokine synthesis, as well as nitrite (NO-2) production and proinflammatory enzyme expression. The involvement and role of nuclear factor-kB (NF-kB) in the effects of these pollutants was also demonstrated. Western blot analysis of NF-kB showed that LPS-induced NF-kB activation was significantly decreased by the exposure of macrophages to NDL-PCB associations. These results demonstrated the impaired capability of macrophages to respond properly to noxious stimuli, such as LPS, mimicking the environmental co-exposure to these compounds. In conclusion, our findings suggest that, although less toxic than dioxin like (DL) congeners, the NDL-PCBs tested are equally dangerous as well as immunotoxic pollutants, also considering their presence as mixtures and at higher levels than DL-PCBs in biotic and abiotic matrices. Interestingly, NDL PCBs tend to accumulate in adipose tissue. Therefore, we evaluated the alteration of the mature 3T3-L1 adipocyte metabolism induced by PCB 101, 153 and 180 alone or associated two by two or all together. We observed an increase in lipid content and leptin gene expression and a concomitant reduction of hormone receptor expression and activity. These modifications support the induction of leptin-resistance, a typical metabolic complication of obesity. Consequently, we investigated how these PCBs affect the expression of important proteins involved in the signalling of leptin receptor. In particular, the phosphatase PTP1B and the suppressor of cytokine signalling (SOCS) 3, two negative regulators of leptin signalling, were induced by the association of the PCB 153 with the 180 or of all PCBs. Conversely, the same associations caused a significant decrease in the phosphorylation of STAT3, a downstream activator of the transcription of leptin gene targets. This effect has been also associated to the inactivation of AMPK/ACC pathway through the reduction of the phosphorylation of these enzymes, and hence the increase in lipid content. Furthermore, it was highlighted the ability of these pollutants to increase the transcription of inflammatory cytokines, such as IL-6 and TNF alpha. Interestingly, it is important to highlight that PCB concentrations used in this study are comparable to levels detectable in human adipose tissue. Our data strongly support the hypothesis that these substances may interfere with the lipid metabolism contributing to the development of obesity and related diseases. Obesity is a clear risk factor for Osteoarthitis (OA). Adipokines are factors, dysregulated in obesity and that play an increasing pathogenic role in OA. Apoptosis is involved in extracellular matrix (ECM) degradation, thus the identification of inductors of this process is important for the understanding pathogenesis/progression of OA. We also evaluated the adverse effect of PCBs 101, 153 and 180 on human and murine chondrocytes by assessing apoptosis pathways. Murine chondrogenic ATDC5 cell line and human T/C-28a2 immortalized chondrocytes were exposed to NDL-PCBs 101, 153 and 180. Cell viability was examined using MTT assay. Necrosis was evaluated by LDH assay and Annexin V flow cytometric assay. Expression of apoptotic related proteins, such as caspase 3, Bcl-2 and Bax, was assessed by Western blot analysis. Oxidative stress was evaluated by malondialdehyde (MDA) assay and the Oxidative Stress Index (OSI). Exposure to examined PCBs caused the loss of cell viability and accelerated apoptosis in a concentration-dependent manner both in murine and human chondrocytes. Data from Annexin V and LDH assays showed necrosis induction. Caspase 3 activation, as well as, altered Bcl-2/Bax ratio and p38 phosphorylation suggested apoptosis induction. Finally, MDA levels and OSI revealed that PCBs drive chondrocytes towards oxidative stress. Our results indicate that the activity of PCBs on cell viability is likely to be mediated by alterations in the mechanisms of regulation of apoptosis and necrosis. Overall, these data highlight a novel role of environmental pollutants in the pathophysiology of chondrocytes.

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