Evolución de la normativa vigente en cuanto a emisiones de olor

Intense physical activities, such as resistance exercise, with a strong eccentric component, cause micro-injury to skeletal muscle, and inflammation appears to play an important role in the repair and regeneration of skeletal muscle after damage (Dennis et al., 2004; Mann et al., 2011). There is evidence that susceptibility to inflammation is influenced by genetic variation in cytokine genes, and accumulated data have indicated that both pro- and anti-inflammatory responses can play a role in athletic performance. They may influence muscle repair, hypertrophy and strength (Cauci et al., 2010), particularly those SNPs located in the promoter regions of the cytokine genes that may alter their expression. SNPs in the cytokine genes and alterations in associated gene expression may also influence risk for upper respiratory symptoms (URS) in some athletes (Cox et al., 2010).

7.5.1. Tumor Necrosis Factor Alpha (TNF-)

With micro-injury to the muscle, the first genes activated by the quiescent resident macrophages are the pro-inflammatory cytokines TNF-α and IL-1β (Dennis et al., 2004). It is of note that accumulation and activation of muscle resident macrophages is a rich source of growth factors postulated to stimulate myogenesis. Thus, inflammation may serve as a mechanism promoting hypertrophy. However, pre- and post-exercise levels of inflammatory factors display considerable variation among people, and this is likely influenced, at least partially, by genetic variation (Cauci et al., 2010).

TNF- is a multifunctional proinflammatory cytokine that has effects on lipid metabolism, coagulation, insulin resistance and endothelial function. It is secreted predominantly by monocytes/macrophages, although significant amounts are also secreted by several other cell types (Skoog et al., 1999). TNF-α is also a potent catabolic factor to skeletal muscle (Liu et al., 2008), besides stimulating the production of interleukin (IL-6) and thereby inducing the hepatic production of C-reactive protein (CRP), a sensitive biomarker of the

3 STRs or microsatellites are hypervariable short sequences of DNA, normally of length 2-5 base pairs, that are interspersed throughout the human genome and repeated numerous times in tandem (in a head-to-tail fashion at a specific chromosomal locus). Ex.: the 16 bp sequence of "gatagatagatagata" would represent 4 head-tail copies of the tetramer "gata". The polymorphisms in STRs are due to the dfferent number of copies of the repeat element that can occur in a population of individuals.

inflammatory status of the individual and exercise-induced oxidative stress (Djarova et al., 2011).

TNF- is synthesized as a 26-kD membrane-bound protein (pro-TNF) that is cleaved by TNF-processing enzymes to release a soluble 17-kD TNF-α molecule. The mature TNF-α protein can then bind to its main receptors TNFR1 and TNFR2, which are expressed in most nucleated cells. After interacting with its receptors, a variety of responses are elicited which affect the regulation of a large number of genes (Skoog et al., 1999).

TNF-α gene is located on the short arm of chromosome 6 (6p21.3), which is within the highly polygenic and polymorphic major histocompatibility complex (MHC) region of the human genome (Liu et al., 2008). TNF- expression is indicated to be partly genetically determined, and polymorphic sites closely linked to the TNF-α locus (inside the MHC region) are associated with differences in cellular TNF-α secretion. While there is evidence for transcriptional regulation of TNF-α gene expression, polymorphisms in the promoter region of the TNF-α gene may be important for TNF-α gene expression and protein production (Skoog et al., 1999).

Many SNPs and microsatellites have been identified in the TNF locus, and the ones in the promoter region are thought to influence TNF transcription rate and to affect the circulating CRP levels (Lakka et al., 2006; Liu et al., 2008). It is also believed that the interaction between nuclear proteins and these TNF SNPs is an important pathway for the allele-specific modulation of TNF expression (Liu et al., 2008). Supporting this hypothesis, five SNPs in the promoter region have been shown to influence gene expression (Liu et al., 2008), being linked to various infectious and autoimmune diseases, obesity and obesity-associated insulin resistance, age-related diseases, including sarcopenia (age-related loss of muscle mass and strength), as well as longevity (Lakka et al., 2006; Liu et al., 2008). Among them, a guanine (G) to adenine (A) substitution located at position −308 of the transcription start site in the promoter region (308G/A; rs1800629) has been reported to affect the transcription rate of the TNF-α gene, with association between the AA genotype with higher plasma CRP levels and less favorable CRP response to regular exercise (Lakka et al., 2006). Because CRP can amplify the proinflammatory response through complement activation, tissue damage, and activation of endothelial cells (Libby et al. 2002), this polymorphism may contribute to a worse performance and also, over time, to loss of muscle mass and strength and CVD risk in athletes, particularly middle-aged athletes who exercise extensively.

7.5.2. Interleukins (IL)

The interleukin-1 (IL-1) family of cytokines and IL-6 are other cytokines involved in the inflammatory and repair reactions of skeletal muscle during and after exercise (Cauci et al., 2010; Eynon et al., 2011). IL-6 also plays a role in the regulation of metabolism during physical exercise, improving skeletal muscle energy supply and assisting in the maintenance of stable blood glucose levels by stimulating lipolysis in the adipose tissue and augmenting glycogenolysis in the liver (Pedersen et al., 2004; Huuskonen et al., 2009).

In general, IL-1 acts synergistically with TNF-, activating proinflammatory responses in a wide range of cells and promoting the acute phase response. IL-1 is able to induce the secretion of several inflammatory factors, including 6 and TNF- (Cauci et al., 2010). IL-6 plays an important role in the homeostasis of the neuroendocrine and immune systems, in the balance of pro- and anti-inflammatory pathways and in response to oxidative stress,

besides regulating hematopoiesis and bone resorption (Chung et al., 2003; Pereira et al., 2011). Additionally, it may modify the regulation of energy balance, by actings as an energy sensor, being dependent on the glycogen content in the muscle. IL-6 is released from contracting muscles in high amounts and exerts its effect on adipose tissue, inducing lipolysis and gene transcription in abdominal subcutaneous fat (Pedersen et al., 2004).

During exercise, IL-6 is produced by muscle fibers in contraction, even without any muscle damage, via a TNF-independent pathway increasing plasma IL-6 levels dramatically (100-fold) (Pedersen et al., 2004). IL-6 stimulates the appearance in the circulation of the anti-inflammatory cytokine IL-10 and the cytokine inhibitors such as IL-1 receptor antagonist (IL-1ra or IL-1RN) and TNF- receptor, inhibiting the production of the proinflammatory cytokine TNF- (Pedersen et al., 2004; Petersen and Pedersen, 2005). Consequently, IL-6 induces an anti-inflammatory environment, may inhibit TNF--induced insulin resistance and have an important role in mediating the beneficial health effects of exercise in inactivity and obesity-related disorders such as diabetes and CVD (Pedersen et al., 2004; Petersen and Pedersen, 2005; Petersen and Pedersen, 2006). Polymorphisms in the promoter region of the IL-6 gene (locus 7p21) that affect the IL-6 expression level may consequently influence performance, immunodepression and risk of upper respiratory symptoms (URS), besides contributing to insulin resistance and CVD risk.

The IL-1 and IL-1 genes are located on the long arm of chromosome 2 (2q14) and are tightly linked (D'Eustachio et al., 1987; Nicklin et al., 1994). They are synthesized as a large precursor of 30.6 and 30.7 kD, respectively, which is processed to a smaller form (March et al., 1985). Another gene map close to IL-1  and  genes is the IL-1ra gene (locus 2q14.2) (Nicklin et al., 1994). IL-1ra is a protein that binds to IL-1 receptors (IL-1RI) and inhibits the binding of IL-1 and IL-1, neutralizing the biologic activity of these 2 cytokines in physiologic and pathophysiologic immune and inflammatory responses (Arend, 1991).

Because IL-1ra acts as an antagonist of IL-1RI and prevents IL-1-dependent signaling, deficiency of IL-1ra in humans, which may be caused by certain polymorphisms, can lead to IL-1-mediated systemic and local inflammation (Cauci et al., 2010).

Several studies showed that polymorphisms in the IL-1 and IL-1ra genes correlate with altered protein expression (Cauci et al., 2010). Two SNPs in IL-1 representing C-to-T base transitions have been studied for disease predisposition, one at position 511 in the promoter region and another at position +3954 in exon 5 (TaqI restriction site polymorphism). In addition, a polymorphism in the intron 2 region of the IL-1ra gene consisting of a variable number of tandem repeats (VNTR)⁴ of 86 base pairs (bp) has been extensively investigated in relation to a variety of pathological conditions, including inflammatory myopathies (Cauci et al., 2010).

Whether polymorphisms in the interleukin genes can affect the severity of the inflammatory response or the athletic status has been also investigated. A study performed on sedentary subjects selected on the basis of their haplotype pattern of specific combinations of five SNPs in the IL-1 gene cluster showed that the wild type genotype for IL-1 +3954 or the variant genotype for IL-1 −3737 in combination with the variant allele at IL-1ra +2018 were associated with inflammation of skeletal muscle, following acute resistance exercise. This

4 VNTRs or minisatellites are hypervariable regions of human DNA of length 10-100 base pairs that are repeated numerous times in tandem (in a head-tail manner, like STRs). VNTRs are similar to STRs, the difference being that in a VNTR, the repeated sequence is longer.

study indicated that IL-1 haplotype can influence the inflammatory response of skeletal muscle after exercise and that it is necessary to test whether the specific IL-1 haplotype is beneficial or detrimental for muscle repair and the adaptability to resistance training (Dennis et al., 2004). Another study carried out with professional and non-professional Italian athletes and non-athlete controls assessing the IL-1 511 and +3954 polymorphisms and the VNTR IL-1ra polymorphism showed that variants in the IL-1ra gene was associated with athletic status. The authors suggested that as VNTR IL-1ra polymorphism is implicated in several disease conditions, athlete status may constitute a confounding variable that will need to be accounted for when examining associations of this polymorphism with disease risk (Cauci et al., 2010).

For IL-6 gene, of the three main polymorphisms reported so far in the promoter region of the IL-6 gene, such as 174G/C, 572G/C and 597G/A (Wang et al., 2011), the −174G/C polymorphism has been reported as a candidate to explain individual variations in health and exercise related phenotypes, with GG genotype favoring sprint/power sports performance in European (Spanish) Caucasian males (Ruiz et al., 2010) but not in Israeli Caucasians (Eynon et al., 2011), and CG genotype favoring increased plasma IL-6 levels, greatest gains in VO2max and decreased BMI (Huuskonen et al., 2009). These apparently contradictory findings support the need to replicate association results between genetic polymorphisms and athletic status in populations of different ethnic backgrounds with the largest possible population, since they vary among different ethnicities.

The change of guanine bases to cytosine (G → C) at position 174 bp from the transcriptional start site seems to affect the transcription of the IL-6 gene and therefore the plasma levels of this cytokine in young, elderly and centenarian individuals (Pereira et al., 2011), with an increased inflammatory response associated with the G allele (Bennermo et al., 2004). It has been shown that, in German Caucasian surgical patients, the −174GG was not associated with the incidence of sepsis, although it increased their survival in sepsis (Schlüter et al., 2002), while in highly-trained athletes, this genotype has been associated with an increased likelihood of ≥ 3 URS episodes in a 12 month period (Cox et al., 2010). These contradictory results indicate the need to study cytokine haplotypes in association with studies involving elite and trained athletes, at least those involved in the systemic IL-6 response, such as IL-10, IL-1ra and TNF- receptor.

7.5.3. Methylenetetrahydrofolate Reductase (MTHFR) Gene

Polymorphisms in the MTHFR gene have been reported in association with an altered plasma homocysteine (Hcy) level (Morita et al. 1997; van Bockxmeer et al. 1997; Fujimura et al. 2000; Miller et al. 2005), which is in turn an independent factor risk for CVD (Graham et al. 1997; Morita et al. 1997; Eikelboom et al. 1999; Anderson et al. 2000). Because elevated Hcy levels are associated with coronary and peripheral vascular obstructive events (Misawa et al., 2008), the variant alleles of the MTHFR gene could contribute, in the course of time, to a lower oxygen supply to the heart in those endurance athletes who exercise strenuously, reducing cardiovascular fitness and thus performance, since they have been associated with increased Hcy. In fact, MTHFR C677T polymorphism has been associated with a lower hemoglobin level in a healthy exercise-trained population (Fortunato et al. 2007). Moreover, hemolysis can occur as a result of mechanical trauma in the capillaries of runners’ feet

(Carlson and Mawdsley, 1986), which would compromise performance still more in carriers of these variants.

Previous reports from our research group have demonstrated an association between both MTHFR polymorphisms (C677T and A1298C) with plasma CRP levels, and thus with a slightly higher inflammatory process (Miranda-Vilela et al., 2011c). Hence, these results indicate that MTHFR polymorphisms should be better investigated in the context of performance and future CVD risk in athletes, particularly middle-aged athletes who exercise extensively, because besides the facts discussed above, CRP is present in atherosclerotic plaques, where it might exert several potential proinflammatory and atherogenic actions that include the binding of oxidized LDL cholesterol, induction of adhesion molecule expression, activation of complement, and stimulation of tissue factor production by monocytes (Brull et al. 2003). Furthermore, the existence of a sport-related hyperhomocysteinemia has been reported, independent of the variables found in the general population such as decreased folate or vitamin B12 (Borrione et al. 2008), particularly if a race took place close to the anaerobic threshold speed (Benedini et al. 2010). This suggests that it would represent an adaptation to training but the possibility of secondary vascular damage cannot be excluded (Borrione et al. 2008).