PCB153 reduces apoptosis in primary cultures of murine pituitary cells through the activation of NF-κB mediated by PI3K/Akt

https://doi.org/10.1016/j.mce.2020.111090Get rights and content

Highlights

  • PCB153 is a non-dioxin-like PCB frequently found in biological samples.

  • PCB153 has been associated with proliferative and anti-apoptotic effects.

  • PCB153 reduced pituitary apoptosis acting by the extrinsic and intrinsic pathways.

  • Akt, Erk, p38, and p21/p53 contribute to the pituitary effects of PCB153.

  • PCB153 induced an Akt-mediated activation of NF-κB in the pituitary.

Abstract

Polychlorinated biphenyls (PCBs) are persistent pollutants involved in human tumorigenesis. PCB153 is a ubiquitous non-dioxin-like PCB with proliferative and anti-apoptotic effects. To explore the impact of PCB153 in the survival of pituitary cells, we exposed murine pituitary primary cells to PCB153 10 μM for 24 h. Apoptosis was assessed by RT-qPCR, Western-blot, immunoprecipitation, caspase activity, and immunofluorescence. We found that PCB153 decreased pituitary apoptosis through both the extrinsic and intrinsic pathways. PCB153 reduced the level of the pro-apoptotic protein p38-MAPK. Otherwise, PCB153 activated PI3K/Akt and Erk1/2 pathways and enhanced the expression and nuclear translocation of NF-κB. Cotreatments with specific inhibitors revealed that only PI3K/Akt changed the caspase−3 expression and NF-κB activation induced by PCB153. Also, PCB153 decreased the expression of the pro-apoptotic and pro-senescent cyclins p53 and p21. In summary, exposure to PCB153 leads to a downregulation of apoptosis in the pituitary driven by a PI3K/Akt-mediated activation of NF-κB.

Introduction

Polychlorinated biphenyls (PCBs) are a class of persistent organic pollutants extensively used in the 20th century as dielectric fluids and coolants in electrical apparatuses, and as additives to hydraulic lubricants, carbonless copy paper, and paints (International Agency for Research on Cancer, 2015; Quinete et al., 2014).

Recognition of the detrimental effects of PCBs on health led to the progressive limitation of these substances and ultimately their worldwide ban in 2001. Nevertheless, PCBs remain a threat for living beings due to their chemical stability and the improper disposal of residual sources. This explains the persistence of measurable levels of PCBs in soil, water, and air samples from all over the world (Arinaitwe et al., 2018; Chakraborty et al., 2018; Hens and Hens, 2017; Kolarik et al., 2017; Nøst et al., 2019).

Humans can be exposed to PCBs not only in the workplace or the environment but also, indirectly, by the ingestion of contaminated foods. In fact, PCBs frequently contaminate biological tissues due to their great lipophilicity and tendency to bio-accumulate in the food chain through animal fats (Hens and Hens, 2017; International Agency for Research on Cancer, 2015; Quinete et al., 2014). It is therefore not surprising that traces of PCBs are still even now detected in human tissues and fluids (including milk) (Chen et al., 2017; Lauritzen et al., 2018; Müller et al., 2017; Quinete et al., 2014; van den Berg et al., 2017; Wielsøe et al., 2017).

The biological effects of PCBs are complex and vary according to the structure of the individual congener (i.e., dioxin- and non-dioxin-like PCBs), the dose, and duration of exposure. The response to PCBs also depends on the biological characteristics of the exposed organism (e.g. species, sex, age) or tissue. In humans, PCBs influence the immune, reproductive, nervous, and endocrine systems (International Agency for Research on Cancer, 2015; Quinete et al., 2014). They are also involved in the induction and progression of some cancers and are thus classified as humans carcinogens by the International Agency for Research on Cancer (IARC) (International Agency for Research on Cancer, 2015; Quinete et al., 2014; Zani et al., 2017).

The pituitary is a classic target for PCB toxicity (Brandt, 1975). Most studies have focused on the disruption of the endocrine function, showing that specific PCBs can affect the synthesis and release of pituitary hormones by the interaction with aryl hydrocarbon, estrogen, and thyroid hormone receptor signalling (Bansal and Zoeller, 2008; Bestervelt et al., 1998; Cocchi et al., 2009; Desaulniers et al., 1999; Gauger et al., 2007; Khan and Hansen, 2003; Kitamura et al., 2005; Otake et al., 2007). Together with the modulation of endocrine functions, PCBs have also been associated with effects on the death of pituitary cells, with discrepant results in relation to the specific congener and the type of treated cells (Fortunati et al., 2017; Ghisari and Bonefeld-Jorgensen, 2005; Johansson et al., 2006). In recent years, we have tested the impact of various PCBs on the cell fate of primary cell cultures from the murine pituitary. Our results suggest that the dioxin-like PBCs 77 and 126 do not affect apoptosis. On the other hand, apoptosis was increased or reduced by the non-dioxin-like PCBs 180 and 153 in the pituitary, respectively (Raggi et al., 2016).

PCB153 is currently considered as one of the most persistent PCBs due to its widespread use in the past and its great stability and propensity to bioaccumulate (Center for Disease Control and Prevention, 2018; Kraft et al., 2017; Pavuk et al., 2014). This has led to increasing focus on the biological effects of this congener, highlighting the anti-apoptotic and proliferative effects of PCB153 as indicated by in vivo (Liu et al., 2014; Tharappel et al., 2002) and in vitro studies (Abella et al., 2015; Ferrante et al., 2011; Ptak et al., 2011; Sánchez-Alonso et al., 2003).

The aim of the study was to examine the molecular mechanisms by which PCB153 exerts its anti-apoptotic effects in the murine pituitary. Our results indicated that PCB153 reduce pituitary apoptosis acting by both the death receptors and the mitochondrial pathways. We also found that the PCB153 anti-apoptotic action was induced by the activation of the PI3K/Akt pathway and by an Akt-mediated activation of NF-κB.

Section snippets

Animals

All the procedures were conducted in accordance with EC Directive 86/609/EEC for animal experimentation. The local Committee for Animal Experimentation approved the study protocol prior to its start (protocol n°: 11,033/2017, University of Pisa).

As donors of pituitary tissues, we used male C57BL/6J mice aged from 8 to 12 weeks. The animals were housed in standard cages with free access to water and standard pellet chows. The animal room had controlled conditions with a 12-h light/dark cycle,

PCB153 reduced apoptosis in the pituitary through the intrinsic and extrinsic pathways

Our previous results showed that the non-dioxin-like PCB153 reduced apoptosis in primary cultures of murine pituitary cells by both the intrinsic and extrinsic pathways (Raggi et al., 2016). To confirm our observations, we tested the expression and activity of the executioner caspase−3 and the initiator caspases −8 and −9 in pituitary primary cell cultures which had or had not been treated with PCB153 for 24 h. We observed that the treatment with PCB153 significantly decreased the expression of

Discussion

PCB153 is a persistent contaminant that is ubiquitous in the environment as well as in animal and human samples despite being banned globally in 2001.

Several studies have explored the consequences of PCB153 exposure on human health with a particular focus on reproduction, development and endocrine function. Among its adverse influences, in epidemiological studies PCB153 has also been associated with proliferative and anti-apoptotic effects in cellular models and with an increased risk of some

Conclusions

The study explored the molecular pathways by which PCB153 modulates apoptosis in an in vitro model of the murine pituitary. Our results confirm that low doses of PCB153 (10 μM) reduce apoptosis through both the extrinsic and intrinsic pathways. Exposure to PCB153 activates the PI3K/Akt and Erk1/2 pathways and lowers the level of p38-MAPK and p53/21. The selective inhibition of these pathways suggests that PI3K/Akt mediates the anti-apoptotic effects of PCB153. Lastly, we showed that PI3K/Akt

Fundings

This work was supported by University of Pisa, and MIUR (Italian Ministry of Instruction, University and Research) as a part of a Research Project of National Interest (PRIN) grant 2012 (2010TYCL9B_008) to Fausto Bogazzi. This work was also partly supported by MIUR PRIN Grant (2017YF9FBS) to Francesco Cardarelli. The sponsors have no role in the collection, analysis and interpretation of data, in the writing of the report, and in the decision to submit the article for publication.

Ethical committee approval

The local Committee for the Animal Experimentation approved the protocol of the study before its start (protocol n°: 11033/2017, University of Pisa).

CRediT authorship contribution statement

Claudio Urbani: Conceptualization, Methodology, Validation, Investigation, Data curation, Writing - original draft, Writing - review & editing, Visualization. Alessandro Mattiello: Conceptualization, Methodology, Software, Formal analysis, Investigation, Data curation, Writing - original draft, Writing - review & editing, Visualization. Gianmarco Ferri: Methodology, Formal analysis, Investigation, Writing - review & editing. Francesco Raggi: Methodology, Validation, Investigation, Resources,

Declaration of competing interest

None.

Acknowledgements

We would thank Prof. Enio Martino for the careful reading of the manuscript. We are also grateful to Dr Silvia Burchielli (Fondazione Toscana Gabriele Monasterio (FTGM), Pisa, Italy) for the precious support for animal handling.

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    Present address: Department of Surgical, Medical, Molecular and Critical Area Pathology, University of Pisa, Via Savi, 10–56,126 Pisa, Italy.

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