Plan nacional de evaluación de la calidad de las universidades

The WHO Regional Office for Europe indoor air quality guideline (2010) noted that acute exposures (hours) in the range of 0.04–1.0 ppm were rarely observed to cause effects in animals. The California Environmental Protection Agency Air Resources Board (CARB,

2007) noted acute effects (hours) in rats and mice at 0.2–0.8 ppm NO2 (increased mast cells, quoted from Hayashi & Kohno (1985) in CARB, 2007)*11 and increased synthesis of the carcinogen dimethylnitrosamine (Iqbal, Dahl & Epstein, 1981)* at 0.2 ppm. Also, there were: effects on liver detoxification enzymes at 0.25 ppm (Miller et al., 1980)*; effects on macrophages at 0.3–0.5 ppm (e.g. Robison et al., 1993)*; and increased bronchiolar proliferation at 0.8 ppm (Barth et al., 1994)*. The EPA (2008b) also highlighted the study by Barth et al. (1994). The effects at 0.2–0.25 ppm are harder to interpret (the mast cells may not have been degranulating, dimethylnitrosamine synthesis relied on an additional administered precursor and the effect on liver enzymes may not be adverse), but it is clear that the effects are adverse above 0.3 ppm. Tables in these reports quote other studies that report acute no effect levels from 0.4 ppm to 0.8 ppm.

A literature search, and literature abstracts already held from 2008 onwards (the literature cut-off for WHO Regional Office for Europe (2010)),12 did not indicate any animal studies with short-term exposure to NO2 alone that would change the CARB (2007) or EPA (2008b) view. Urea selective catalytic reduction-treated diesel engine emissions (0.78 ppm NO2 and dilutions) were generally less toxic to rat lungs than conventional diesel engine emissions (0.31 ppm NO2 and dilutions), for various endpoints over durations of 1, 3 or 7 days. However, there were differences in the nature of the oxidative stress produced with either increases in 8-hydroxy-2-deoxyguanosine with conventional diesel engine emissions, or increases in haemoxygenase-1 mRNA expression with urea selective catalytic reduction- treated diesel engine emissions (Tsukue et al., 2010). The latter suggests oxidative stress related to NO2, but is not conclusive, given the mixture of constituents present.

Using exposures lasting from hours to days, recent mechanistic animal studies show: protein S-glutathionylation in the lung at 25 ppm NO2 (Aesif et al., 2009); a decrease in aggregating activity of surfactant-protein D at 10 ppm or 20 ppm (Matalon et al., 2009); and increases in markers of oxidative stress, endothelial dysfunction, inflammation and apoptosis in the hearts of rats from exposures of 2.7 ppm to 10.6 ppm (Li H et al., 2011). Zhu et al. (2012) found that 2.6 ppm NO2 delayed recovery from stroke (slowed reduction in infarct volume) in a rat stroke model and increased behavioural deficits. Dose-related endothelial and inflammatory responses were also found in the range 2.6–10.6 ppm. Channell et al. (2012) found that plasma from healthy adults exposed to 0.5 ppm NO2 for 2 hours was able to activate cultured primary human coronary endothelial cells. This suggested that a circulating factor was mediating, at least in part, the endothelial response that may underly the cardiovascular epidemiological findings. One study in mice (Alberg et al., 2011) found no increased sensitization to intranasal ovalbumin at 5 ppm or 25 ppm NO2 when diesel exhaust particles did show an increase, whereas another study in mice (Hodgkins et al., 2010) at 10 ppm NO2 showed sensitization to inhaled ovalbumin due to increased antigen uptake by antigen- presenting cells. A study in mice suggested that effects in the lung due to 20 ppm NO2 for 10 days are worse with a small increase in vitamin C dose than with a large increase (Zhang et al., 2010), perhaps due to vitamin C increasing NO2 absorption into the epithelial lining fluid (Enami, Hoffmann & Colussi, 2009). All these concentrations (apart from Channell et al., 2012) are far in excess of the ambient concentrations linked to health effects in population studies, and the studies are not designed to show whether the mechanisms extend down to lower concentrations – that is, the mechanisms may or may not be relevant.

11

Studies followed by asterisks (*) are older studies not referenced in earlier guidelines.

12

Literature abstracting service was provided by the Institute of Environment and Health at Cranfield until 2009; it was funded by the United Kingdom Department of Health.

Recent studies indicate that the NO2 radical can be directly involved in nitration of tyrosines (Surmeli et al., 2010, for example) and also that it causes a nitration-dependent cis-trans- isomerization to trans-arachidonic acid (linked to microvascular injury), described by the authors as a characteristic process for NO2 (Balazy & Chemtob, 2008). This is not just relevant to the lung. While NO2 itself is not absorbed systemically, as it is likely to react first, its reaction products, nitrite and nitrate ions, are found in the blood after NO2 inhalation (Saul & Archer, 1983). Nitrite and nitrate anions in the blood are now regarded as carriers of nitric oxide (Lundberg, Weitzberg & Gladwin, 2008; Lundberg & Weitzberg, 2010; Weitzberg, Hezel & Lundberg, 2010).

The cycle whereby nitrate is excreted into the saliva and reduced to nitrite by oral bacteria and then converted to nitric oxide in the stomach or in the blood and tissues, after absorption of nitrite, may have a physiological role (to ensure nitric oxide production for vasodilation under hypoxic conditions when oxygen dependent nitric oxide synthases may fail), but may also have adverse consequences (Panesar, 2008). Nitrite can also lead to the NO2 radical via peroxidase catalysed oxidation with hydrogen peroxide, via formation of nitrous acid, which dissociates to nitric oxide and NO2, via formation of peroxynitrite from nitric oxide and superoxide and subsequent breakdown to the NO2 and hydroxyl radicals and, less commonly, direct oxidation of nitric oxide (d’Ischia et al., 2011; Signorelli et al., 2011). In other words, there is an indirect transfer of inhaled NO2 to the NO2 radical in tissues via reactive intermediates.

There is a great deal of literature on reactive nitrogen species (Abello et al., 2009; d’Ischia et al., 2011; Sugiura & Ichinose, 2011), including in literature on atherosclerosis (Upmacis, 2008). The review by Upmacis (2008) describes the occurrence of protein nitrotyrosine in atherosclerotic plaques and also in the bloodstream and describes an emerging view that this is a risk factor for cardiovascular disease. A major proportion of the nitrotyrosine is thought to come via nitric oxide from inducible nitric oxide synthase (induced under inflammatory conditions), but the source of the remainder is unknown. Nitrite and nitrate in the blood from NO2 inhalation provides a potential link with this literature. Human beings exposed to 0.1 ppm NO2 by inhalation have been inferred to form about 3.6 mg nitrite per day, more than the dietary intake of nitrite (Saul & Archer, 1983). More work is needed, however, to understand how significant any contribution of NO2 inhalation to systemic nitrative stress might be in quantitative terms.

Although this is a section on toxicology, some epidemiological studies are considered here as they concentrate on mechanisms. They provide a potential link between the mechanisms in animal studies discussed above and mechanisms operating in human beings, although they lose the advantage of confident specificity for NO2. These studies have mostly addressed cardiovascular end-points: no effects on blood coagulation and inflammatory markers (Zuurbier et al., 2011b; Steinvil et al., 2008) or a non-significant increase (significant for sP- selectin) (Delfino et al., 2008); increases in brachial artery diameter and flow mediated dilatation (perhaps actually due to nitric oxide) (Williams et al., 2012); adverse effects on heart rate variability in heart disease patients (Zanobetti et al., 2010) and in cyclists (Weichenthal et al., 2011) (independent of PM metrics); a non-significant increase in the QT interval in electrocardiograms (significant in diabetics) (Baja et al., 2010); and an increase in lipoprotein-associated phospholipase A2 (linked to inflammation in atherosclerotic plaques) (Brüske et al., 2011).