Implicaciones prácticas

The imbalance of ROS, as a result of endogenous or exogenous toxicants, leads to the disruption of cellular calcium homeostasis [105].

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The ER acts as a major reservoir of IC calcium and therefore plays a critical role in calcium homeostasis [17, 18]. This homeostasis is necessary for the proper functioning of the protein folding machinery; disruption results in the activation of the UPR pathway that endeavours to restore balance within the ER (Figure 1.17). This is achieved through co-ordinated steps that reduce the misfolded/unfolded protein (UP) concentration through the suppression of protein synthesis, facilitation of protein degradation and increasing the protein folding capacity of the ER (reviewed elsewhere [18, 19, 228]). Failure to mitigate ER stress can lead to cellular death [105].

Figure 1.17 The unfolded protein response pathway. In stress free conditions, luminal protein chaperone binding immunoglobulin protein (BiP) also known as 78 kDa glucose-regulated protein (GRP78) binds to the intraluminal domain of the three UPR sensors: PERK, inositol-requiring enzyme 1α (IRE1α) and ATF6, rendering them inactive. Upon ER stress and the accumulation of UP and increased protein cargo within the ER, BiP dissociates from the UPR sensors and sequesters UP within the ER lumen, due to its higher affinity for UP. The dissociation of BiP from IRE1α and PERK causes the oligomerisation, auto-phosphorylation and activation of these sensors and downstream signalling pathways. The activation of the PERK arm leads to the phosphorylation of eukaryotic initiation factor 2 (eIF2) α which subsequently induces ATF4 mRNA translation and the inhibition of global protein translation. Pro-apoptotic genes

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such as CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP) are induced by ATF4 resulting in cellular death. Global translational inhibition results in decreased translation of antioxidant proteins leading to increased ROS and apoptosis. Whilst the dissociation of BiP from ATF6, allows the translocation of ATF6 to the Golgi apparatus where it is processed by serine protease 1 (S1P) and metalloprotease site-2 protease (S2P) to yield an active transcription factor which enters the nucleus. The UP sequestered on BiP are translocated to the cytosol for proteasomal degradation by ER-associated degradation (ERAD) machinery. These three arms of UPR pathway endeavour to mitigate ER stress by facilitating protein degradation, increasing the ER’s protein folding capacity by inducing protein chaperone production and by suppressing protein synthesis [18, 19, 228].

A reciprocal interplay between ROS production and increased ER stress due to calcium release has been suggested. Calcium release channels on the ER membrane, including the ryanodine receptor and the inositol-1,4,5-triphosphate receptor (IP3R), are activated by ROS. This induces calcium ions (Ca²⁺) to migrate into the cytosol from the ER lumen. Increased IC Ca²⁺ results in the loss of ER chaperone proteins which impairs ER function. This leads to increased UP levels that can generate ROS in its attempt to repair UP. Increased IC Ca²⁺ also adversely affects mitochondrial activity which further increases ROS production [105]. This will be discussed in detail in sections: 1.8.1. and 1.8.2. below.

1.8.1. Mitochondrial ROS generation as a result of increased intracellular calcium occurs via the following mechanisms:

The mitochondria experiences increased calcium loading; which generates increased ROS production. Calcium loading inhibits the ETC at complex III through the opening of the permeability transition pore (PTP). This releases cytochrome c from the inner mitochondrial membrane which blocks complex III. Calcium inhibition of complex III results in the increase of ubisemiquinone radical intermediate (QH·) (the quantity of QH· reflects the amount of mitochondrial ROS produced) which increases ROS production. There is also an increase in QH· generation observed when the ETC turnover occurs more rapidly [229, 230].

Additional increases in ROS occur via the following mechanisms:

 Increased cytosolic Ca²⁺ stimulates the tricarboxylic acid cycle leading to increased O2

consumption and ROS production.

 The Ca²⁺ induced opening of the PTP may result in GSH leakage into the cytosol from the mitochondrial matrix. This indirectly causes increased ROS generation as a result of reduced antioxidant capability.

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The amplified levels of mitochondrial ROS generation; further activates Ca²⁺ release from the ER due to their close proximity. Increased Ca²⁺ close to the mitochondria causes increased ROS production, as a result of Ca²⁺ loading and the opening of the PTP. Generation of ROS then acts on Ca²⁺ release channels further increasing Ca²⁺. A feedback loop is then established where increased IC Ca²⁺ induces ROS production which increases Ca²⁺ release into the cytosol, which loops back around. Therefore, suggesting a reciprocal interaction between ROS and ER stress that threatens cell survival [105, 229, 231].

1.8.2. Unfolded protein repair induces ROS generation by the following proposed mechanism:

The ER is a vast membranous organelle [18] that is the site of synthesis, folding, maturation and modification and trafficking of secreted and transmembrane proteins [19, 232]. The addition of a disulphide bonds are required for the stability, function and maturation of secretory proteins.

Misfolded proteins, formed as a result of incorrectly paired cysteine residues, may have an inappropriate disulfide bond that requires removal [229]. The formation of disulfide bonds generates about 25% of ROS in the cell, during ER oxidation protein folding [233]. Disulfide bond formation is driven by protein disulfide isomerase (PDI), where PDI becomes oxidized and subsequently reduced by ER oxidoreductin 1 that transfers electrons onto O2. This leads to the generation of ROS. Reduced GSH may assist this process in UP which results in the generation of oxidized GSSG. The depletion of GSH induces ROS formation [229, 234].

Reactive oxygen species can be generated independent of disulfide bond formation. The accumulation of UP, within the ER lumen, causes Ca²⁺ to leak into the cytosol which generates ROS production. Alternatively, due to the high energy dependent processes of folding and refolding proteins within the ER, ATP depletion could result from UP accumulation. This would induce the mitochondrial oxidative phosphorylation pathway to increase ATP turnover thereby increasing ROS production [229].

1.8.3. Ambient air pollution, Adverse birth outcomes and Endoplasmic reticulum stress

There are a few studies that have investigated the effect of AAP on ER stress. Andersson et al.

demonstrated that exposure to low levels of 1-nitropyrene (diesel exhaust) increased DNA damage which resulted in increased ROS and GRP78, a marker of ER stress [64]. Watterson et al. associated PM levels with the induction of ER stress [235]. Laing et al. demonstrated that

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PM2.5 induced PERK-dependent CHOP expression in mouse lung, liver and macrophage models [236]. This induction was dependent on ROS production for PM2.5 induced apoptosis to occur.

This gives evidence for AAP induced ER stress as a result of ROS production; demonstrating a reciprocal interaction between OS and ER stress.

The effect of ER stress on LBW has only been briefly investigated. Kawakami et al. has demonstrated that ER stress markers are increased in LBW neonates [237]. This area of research is important due to the reciprocal interaction shown between OS and ER stress, as well as the strong relationship observed between OS and LBW. It could be a potential mechanism linking OS and adverse birth outcome pathology to AAP toxicity.

1.9. Future considerations for ambient air pollution induction of adverse birth outcomes