Influencia de las variables relativas al sistema familiar en el ajuste y

Human genetic susceptibility towards diseases has been an important feature in discovering how alterations in the genome predispose an individual to certain adverse conditions. The study of epigenetics has been highlighted most notably as a potential mechanism for the regulation of the Nrf2-Keap1 pathway. It is defined as the study of heritable changes in gene expression that does not alter the DNA sequence [187]. Several epigenetic mechanisms exist that regulate gene expression including non-coding RNAs, chromatin remodelling, histone modification and DNA methylation [188].

The non-coding RNAs or miRs are of particular interest in Nrf2 regulation. They are a class of small non-coding ssRNAs that are approximately 21-23 nucleotides in length. They exert their control over cellular functions including cell proliferation, differentiation and apoptosis through post-transcriptional regulation of gene expression. RNA polymerase II transcribes miRs from genomic loci, which is then processed by Drosha and transported to the nucleus as short hairpin precursors. Dicer then cleaves the miR precursors to generate mature miRs. The mature miRs are then loaded onto Argonaut proteins to form the RNA-induced silencing complex (RISC).

This complex, together with base pairing of the miR onto the 3’- untranslated region (UTR) of target mRNAs, results in the inhibition of targeted gene expression. This occurs through either mRNA degradation or inhibition of protein translation (Figure 1.14) [24, 25, 189].

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Figure 1.14 microRNA Biogenesis. The transcription of miRs from genomic DNA by RNA polymerase II, its subsequent cropping by Drosha and exportation out of the nucleus via Exportin-5, is followed by cleavage of the precursor miR by DICER. This results in the formation of mature miRs that binds to Argonaut proteins to form the RISC complex which leads to the transcriptional inhibition of target genes (Prepared by author).

Several studies have identified miRs that regulate Nrf2 and Keap1 signalling. Sangokoya and colleagues demonstrated that the miR-144 inhibited the expression of Nrf2 mRNA within a myelogenous leukaemia cell line [190]. This was later verified by Yamamoto et al., who showed a negative association of miR-144 with Nrf2 and downstream enzymes in response to diesel exhaust exposure [24]. The miR-28 was found to post-transcriptionally regulate Nrf2 expression by binding directly to Nrf2’s mRNA 3’UTR which resulted in Nrf2 mRNA degradation. In addition, miR-28 also directly promoted Nrf2 protein degradation. This was not as a result of Keap1 protein expression or the Keap1-Nrf2 interaction but rather supressed mRNA expression through translational inhibition [25].

The controlled regulation and induction of Nrf2 is important to combat oxidative damage due to toxic insult from endogenous and exogenous sources to maintain homeostasis within the body.

This prevents and helps fight against oxidative injury which may lead to inflammatory diseases, cardiovascular disorders, adverse birth outcomes and carcinogenesis. Investigating this pathway in response to AAP, and understanding how AAP affects this pathway may identify specific targets for potential therapeutic interventions for diseases associated with OS-damage such as adverse birth outcomes.

35 1.7. Reactive nitrogen species

Nitric oxide plays a major role in a variety of physiological processes including: the regulation of vasodilation, modification of neurotransmission, memory formation and has anti-microbial activity [191–193]. However, its elevated production has also been implicated in several inflammatory diseases, neurotoxicity, ischaemia and adverse birth outcomes [34, 107]. The term

‘nitrosative stress’ was coined by Stamler and colleagues [194], to describe the excessive production and dysregulated formation of NO and NO-metabolites.

1.7.1. Chemistry of NO

NO is a simple heterodimeric molecule that is highly reactive and unstable. It has a half-life of about 6-10 seconds [47] and degrades rapidly to form nitrites and nitrates. NO is produced endogenously and has exogenous sources. Within the body, NO is synthesised by a family of enzymes known as nitric oxide synthases (NOS). There are three isoforms that produce NO:

neural (nNOS), endothelial (eNOS) and inducible (iNOS); each dependent on structural and functional properties including its tissue of origin [195, 196].

The constitutive NOS (cNOS) comprise eNOS and nNOS, which are found within vascular endothelial cells, neurons, smooth muscles and platelets. They have been shown to be positively modulated by IC levels of calcium and calmodulin binding [197]. NO produced via cNOS act as important signalling molecules within the cardiovascular and neural system.

The iNOS are found within immune cells such as macrophages, lymphocytes and neutrophils. It does not require activation by calcium and calmodulin, but is rather positively or negatively regulated by cell-cell contact in lymphocytes, inflammatory cytokines, bacterial and viral endotoxins. The production of NO via iNOS occurs over long periods of time and has been shown to be cytostatic or cytotoxic to tumour cells and microbial organisms [197].

Inflammatory cytokines that induce iNOS include interleukin (IL)- 1β, IL-6, IL-17, tumour necrosis factor (TNF)-α and interferon (IFN)- ƴ, whilst TNF-β, IL-4, 10, 11 and IL-13 suppress iNOS [198].

All NOS are haemoproteins that produce NO as a reaction by-product, during the catalytic conversion of L-arginine to L-citrulline. This reaction requires NADPH and O2 with flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), heme and tetrahydrobiopterin (BH4) acting as cofactors (Figure 1.15) [195, 199].

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Figure 1.15 Synthesis of nitric oxide as a reaction by-product from the conversion of arginine to L-citrulline [200].

NO is able to diffuse across the cell membrane and has high affinity for Hb. At low levels of NO, it is rapidly inactivated by binding to Hb to form methaemoglobin (methHb) followed by degradation to inorganic nitrites and nitrates [201]. However, at higher levels of NO, it rapidly reacts with superoxide and O2 to form peroxynitrite and dinitrogen trioxide, respectively. These NO intermediates and NO are highly reactive and result in macromolecular damage. The modification of peptides and proteins occurs as a result of S-nitrosylation, where NO directly modifies cysteine AA of target proteins to form S-nitrothiol adducts, as well as nitration of tyrosine in proteins. NO has been shown to directly oxidise to nitrite, which induces DNA damage, along with highly reactive peroxynitrite [107, 195, 196, 199].

Peroxynitrite is the most reactive intermediate of NO oxidation. It exists in two forms:

nucleophilic or protonated peroxynitrite, it is highly reactive with a half-life <1second.

Depending on its cellular environment and availability of reactive targets, it undergoes a variety of chemical reactions that not only cause nitration of tyrosine but also triggers LP, inactivate aconitases, inhibit the ETC and oxidise biological thiol-containing compounds. Peroxynitrite decays to form nitrates and undergoes homolytic decomposition to form highly reactive and toxic hydroxyland nitrite radicals [196, 199].