foods Perspective Could Global Intensification of Nitrogen Fertilisation Increase Immunogenic Proteins and Favour the Spread of Coeliac Pathology? Josep Penuelas 1,2,3, * , Albert Gargallo-Garriga 1,2,3 , Ivan A. Janssens 4 , Philippe Ciais 5 , Michael Obersteiner 6 , Karel Klem 3 , Otmar Urban 3 , Yong-Guan Zhu 7,8 and Jordi Sardans 1,2,3 1 CSIC, Global Ecology Unit CREAF-CSIC-UAB, Bellaterra, 08193 Catalonia, Spain; a.gargallo@creaf.uab.cat (A.G.-G.); j.sardans@creaf.uab.cat (J.S.) 2 CREAF, Cerdanyola del Valles, 08193 Catalonia, Spain 3 Global Change Research Institute, Czech Academy of Sciences, CZ-60300 Brno, Czech Republic; klem.k@czechglobe.cz (K.K.); otmar@brno.cas.cz (O.U.) 4 Research Group Plants and Ecosystems (PLECO), Department of Biology, University of Antwerp, B-2610 Wilrijk, Belgium; ivan.janssens@uantwerpen.be 5 Laboratory of Climate and Environmental Sciences, Institute Pierre Simon Laplace (PSL), 91191 Gif-sur-Yvette, France; philippe.ciais@lsce.ipsl.fr 6 Ecosystems Services and Management, International Institute for Applied Systems Analysis (IIASA), A-2361 Laxenburg, Austria; michael.obersteiner@gmail.com 7 Key Laboratory of Urban Environment and Health, Chinese Academy of Sciences, Xiamen 361021, China; ygzhu@iue.ac.cn 8 State Key Laboratory of Urban and Regional Ecology, Research Centre for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China * Correspondence: josep.penuelas@uab.cat  Received: 9 October 2020; Accepted: 2 November 2020; Published: 4 November 2020  Abstract: Fertilisation of cereal crops with nitrogen (N) has increased in the last five decades. In particular, the fertilisation of wheat crops increased by nearly one order of magnitude from 1961 to 2010, from 9.84 to 93.8 kg N ha−1 y−1 . We hypothesized that this intensification of N fertilisation would increase the content of allergenic proteins in wheat which could likely be associated with the increased pathology of coeliac disease in human populations. An increase in the per capita intake of gliadin proteins, the group of gluten proteins principally responsible for the development of coeliac disease, would be the responsible factor. We conducted a global meta-analysis of available reports that supported our hypothesis: wheat plants growing in soils receiving higher doses of N fertilizer have higher total gluten, total gliadin, α/β-gliadin, γ-gliadin and ω-gliadin contents and higher gliadin transcription in their grain. We thereafter calculated the per capita annual average intake of gliadins from wheat and derived foods and found that it increased from 1961 to 2010 from approximately 2.4 to 3.8 kg y−1 per capita (+1.4 ± 0.18 kg y−1 per capita, mean ± SE), i.e., increased by 58 ± 7.5%. Finally, we found that this increase was positively correlated with the increase in the rates of coeliac disease in all the available studies with temporal series of coeliac disease. The impacts and damage of over-fertilisation have been observed at an environmental scale (e.g., eutrophication and acid rain), but a potential direct effect of over-fertilisation is thus also possible on human health (coeliac disease). Foods 2020, 9, 1602; doi:10.3390/foods9111602 www.mdpi.com/journal/foods Foods 2020, 9, 1602 2 of 21 Keywords: global intensification of N fertilisation; wheat; allergenic proteins; gluten proteins; coeliac pathology 1. Introduction The demand for, and application of, nitrogen (N) fertilizer in cropland at a global scale has been continuously increasing. The global use of N fertilizers has increased substantially from 11.3 Tg N y−1 (0.9 g N m−2 y−1 ) in 1961 to 107.6 Tg N y−1 (7.4 g N m−2 y−1 ) in 2013 [1]. The data provided in the last International Nitrogen Initiative Conference indicated that the global consumption of N fertilizers increased 33% from 2000 to 2013 [2]. FAOstat data [3] indicated that the recent (2014–2018) intensification of N fertilisation at global and regional scales has affected most of the world, but with regional differences, with increases, from highest to lowest, of 29.1, 24.5, 17.6, 9.0, 5.4, 4.8, 4.1, 2.5 and 1.3% in eastern Asia, southern Asia, Latin America and the Caribbean, eastern Europe and central Asia, Sub-Saharan Africa, North America, western Asia, northern Africa and Oceania, respectively, and a decrease of 1.5% in western Europe [4]. Although continuous N fertilisation reduces nitrogen use efficiency on wheat yield production [5], this large increase in N fertilisation of wheat crops is the effective driver of the increased wheat yield [6,7]. This effect is especially important in wheat because it comes associated with a positive relationship between N fertilisation and wheat protein concentration [7,8]. Wheat (Triticum sp.) is currently the most widely planted crop and continues to be the most important food grain for humans [9]. Furthermore, despite a decrease of direct flour food products intake has occurred in some countries such as the United States of America, there is still a net increase in per capita annual wheat flour intake due to an additional flour intake from the extra flour used as food additive that has increased the net gluten annual intake per person from 4.1 kg in 1970 to 5.4 kg in 2000 [10]. Wheat crops currently cover an area of 217 × 106 ha globally [11]. Global wheat yield in recent decades (1961–2015) has continuously increased despite representing a similar area of land (Figure 1) [3,12,13]. The annual amounts of N fertilizer applied to wheat crops have increased globally in the same period from approximately 10 to 100 kg N ha−1 y−1 . This increase in N fertilisation is associated with an increase in the production of wheat grains and flour per hectare. The fertilisation (kg ha−1 ) to yield (t ha−1 ) ratio, however, increased from 0.9 to 3.1 kg N t grains−1 during 1961–2010, i.e., the yield-to-fertilisation ratio is now only 3.5-fold what it was 50 years ago. Foods 2020, 9, 1602 3 of 21 Foods 2020, 9, x FOR PEER REVIEW 3 of 21 Figure 1. Global N-fertilisation rates (kg N ha−1 y−1 ) in wheat crops. Wheat grain yield (t ha−1 y−1 ) Figure and global1. Global annualN-fertilisation area of wheat rates (kg (10 crops N ha −1 y−1) in wheat crops. Wheat grain yield (t ha−1 y−1) and 7 ha) during 1961–2016 (1961–2010 for N-fertilisation rates) global annual area of wheat crops (10 −1 7 ha) during 1961–2016 (1961–2010 for N-fertilisation rates) (A). −1 (A). Efficiency of N fertilisation (kg N ha per tonne of wheat grains (t wheat-1 )) (B). Sources: [3,13–15] Efficiency of N fertilisation (kg−1 N ha−1 per tonne of wheat grains (t wheat-1)) (B). Sources: [3,13–15] The protein composition of wheat grains varies depending on genotype and environmental conditions, but wheat proteins are generally deficient in some fundamental amino acids, such as lysine and threonine [16]. Structural proteins of wheat grains are mostly albumins, globulins and Foods 2020, 9, 1602 4 of 21 amphiphilic proteins [16], whereas storage proteins are gliadins (monomeric proteins) and glutenins (polymeric proteins) [17]. N fertilisation generally influences the quantity and quality type of storage proteins (gliadins and glutenins) [18,19], but has little effect on structural proteins [20]. Martre et al. (2006) [21] modelled N-gliadin contents in wheat grains as a function of N partitioning among plant-protein groups and validated the model using 18 experimental studies. They observed a positive relationship between N-fertilisation rates and the amount of N allocated to gliadins after sowing. Ingestion of wheat gluten can trigger several intolerances and allergic diseases, among which coeliac disease (CD) is the most widespread in humans [22]. The mean prevalence of CD in the general population in Europe and the United States of America (USA) is approximately 1% [23,24], with some regional differences, e.g., the prevalence of CD is as high as 2–3% in Finland and Sweden but is only 0.2% in Germany. The overall prevalence of CD is now clearly increasing everywhere. The prevalence of CD in the USA was only 0.2% in 1975 but increased 5-fold during the next 25 years [25]. The causes remain elusive but are likely linked to the environmental components of CD (e.g., changes in the quantity and quality of ingested gluten, patterns of infant feeding, the spectrum of intestinal infections and colonization by gut microbiota) [25]. Among the components of gluten, glutenins have been also associated with coeliac disease [26], but the group of gliadin proteins appears to be the primary cause of coeliac disease by gluten intake [22,27] in genetically susceptible individuals [28]. All three gliadin families, α/β, γ and ω, have been associated with allergic reactions to gluten and with the development of CD in humans [29–37]. The autoimmune response is due to the deamidation of glutamine residues in gliadins by human transglutaminase 2 (tTG2) produced in the gut mucosa [22,28,38,39]. These deamidated peptides can bind to histocompatibility leukocyte antigen (HLA) class II in some humans, which stimulates lymphocyte T cells and triggers an inflammatory response in the gut [22,23]. Most studies have reported that gliadins are most responsible for CD [29–37], but some studies have reported that the high-molecular-weight glutenins can also induce these autoimmune responses in some people [26,40,41]. Glutenins, however, are easily degraded by digestive enzymes, providing mostly di- and tripeptides, whereas gliadins are more resistant to enzymatic degradation, producing mostly oligopeptides that are the main cause of inflammatory responses [27]. We hypothesize that the increase in N fertilisation could be related to a potential increase of gluten in wheat grains and flours and thus to the spread of coeliac disease. More detailed information is now available from experimental data in field conditions including studies with a great variety of wheat genotypes growing in distinct areas of world. By gathering all this information, we aimed to analyse the gluten and gliadin concentrations in the grains and flour of wheat as a function of N-fertilisation levels and their potential association with higher coeliac disease prevalence at global scale. 2. Methods We collected these data searching PubMed, ISI Web of Science and Google Scholar using the following terms spanning 1960–2019: coeliac, coeliac disease, nitrogen, fertilisation, gliadins, gluten, glutenin grain, wheat, and flour. We selected only studies providing information of the concentrations of these compounds in grain and/or flour and data on N-fertilizer doses that could be expressed in Kg ha−1 y−1 . To obtain the data of the prevalence (percentage of coeliac cases in the total population) or incidence (new cases per 1000 inhabitants and year) of coeliac disease from studies with large and representative sets of population data, adjusted for age and sex, from 1961 to 2019, we have also searched PubMed, ISI Web of Science and Google Scholar using the following terms spanning 1960–2019: celiac, coeliac, celiac disease, coeliac disease, gluten, health, time, incidence and prevalence. We also used the FAO data: FAOSTAT http://www.fao.org/faostat/en/#data. (2019a) and the other sources cited in each figure caption. In the first bibliographic exploration, we gathered 172 articles. After data quality selection, we finally used the information provided by 47 articles (Table 1). We examined the effects of the intensification of N fertilisation by a meta-analysis of the studies reporting the differences of total contents of gluten, total gliadins, α/β-gliadins, γ-gliadins, Foods 2020, 9, 1602 5 of 21 ω-gliadins and gliadin transcripts (operational taxonomic units) in wheat grains and/or flour under different levels of N fertilisation by calculating the response ratios from each study (Table 1), as described by Hedges et al. [42]. The natural-log response ratio (lnRR) was calculated as ln (Xi /Xn ) = lnXi − lnXn , where Xi and Xn are the values of each observation in treated and control plants, respectively. The sampling variance for each lnRR was calculated as ln[(1/ni ) × (Si /Xi )2 + (1/nn ) × (Sn /Xn )2 ] using the R package metafor 1.9–2 [43], where ni and nn , Si and Sn and Xi and Xn are the sample sizes, standard deviations and mean response values of the treatments and controls, respectively. The natural-log response ratios were determined by specifying studies as random factors using the rma model in metafor. The differences of contents of total gluten, gliadins, α/β-gliadins, γ-gliadins, ω-gliadins and gliadin transcripts in wheat grains and/or flour under different levels of N-fertilisation were considered significant if the 95% confidence interval of lnRR did not overlap zero. All statistical analyses were performed in R 3.6.0 (2019) (Copenhagen Business School, Copenhagen, Denmarck). We analyzed only the variables with >30 observations available at the global scale. We then examined the sensitivities of contents of total gluten, total gliadins, α/β-gliadins, γ-gliadins, ω-gliadins and gliadin transcripts in wheat grains and/or flour under different levels of N-fertilisation using REML estimation in the rma.unl model for metafor. We also analysed the relationship between the prevalence of coeliac disease and the per capita wheat intake at country level using a regression type II analysis conducted with the package lmodel2 [44] (R-Forge, Vienna, Austria). Foods 2020, 9, 1602 6 of 21 Table 1. Responses of the concentrations of total gluten, total gliadins, α/β-gliadins, γ-gliadins and ω-gliadins and gliadin transcripts in wheat grains after an experimental increase in N fertilisation rates. Bibliographic Source Experimental Traits Result Tested Changes in Concentrations in Grain and/or Type of Reference Site N-Fertilisation Rates (Kg N ha−1 Genotypes Flour in Response to Increasing Experiment y−1 if not Specified) N-Fertilisation Experimental Farm Shiraz University [45] Field 0, 120, 240, 360 Shiraz variety of winter wheat Increases in gluten concentrations in grain (Iran) Increases in total protein and gluten [46] Bezek experimental station (Poland) Field 0, 50, 80 Triticum aesticum ssp. spelta concentrations in grain Chili, Biskri, Mahmoudi, [47] Two field areas of Tunis Field 0, 67 INRAT69, Karim, Razzak, Increases in gluten concentrations in grain Omrabiaa and Khiar varieties Experimental Station J. Hirschhorn [48] Field 0, 70, 140 Increases in gluten concentrations in grain (Argentina) Upland crop experimental farm of Increases in gluten concentrations in flour. [49] National Institute of crop Science Field 25, 50, 75 Five Korean wheat varieties Increases in α + β-gliadin and decreases in ω (Korea) and γ-gliadin concentrations Five breadmaking wheat varieties [50] Replicates in five field sites in U.K. Field 100, 200, 350 (Cordial, Hereward, Malacca, Increases in total gliadin concentrations in grain Marksman and Xi19) [50] Canada Field 0, 100 Neepawa variety Increases in total gliadin concentrations in grain 0, 50, 100, 150, 200, 250, 300, 350, Increases in total protein and total gliadin [51] Canada Field Neepawa variety 400 concentrations in grain Increases of total epipodes expression of [30] Eight different site sources Field 0, 105, 165, 225 Triticum aesticum ssp. spelta a-gliadin in grain Plant Breeding Station of Increases in total protein and gluten [52] Field 120, 140 Winter wheat Sladkovicovo-Novy (Slovakia) concentrations in grain Experimental Station J. Hirschhorn [53] Field 0, 70, 140 Increases in total gluten concentrations in flour (Argentina) [54] Alava (Spain) Field 0, 100, 140, 180 Soissons variety Increases in total gliadin concentrations in grain 37, 48 mg ammonium or nitrate per Increases in total protein and gliadins [55] Spain Pot experiment Cezanne variety pot concentrations in flour Experimental field station Teramo Increases in total protein, gluten and gliadins [56] Field 50, 100, 150, 250 Triticum turgidum L. subsp. durum University (Italy) concentrations in flour Increases in total, α, ω and γ-gliadin and total [57] Spain Greenhouse 0, 22.2, 66.7, 200 Bobwhite variety protein concentrations in flour National Center of Irrigation [58] Field 0, 120 Winter wheat Increases in total gliadins concentrations in flour Technology station (Spain) Foods 2020, 9, 1602 7 of 21 Table 1. Cont. Sport, Dacke, Dragon and Thasos Increases in total proteins and gliadins [59] Field (Sweden) Field 0, 70, 140 varieties concentrations in flour Increases in total proteins and gliadins [60] UK Field 0, 40, 80, 120, 160, 200, 240 Option and Riband varieties concentrations in grain [61] Malice (Poland) Field 0, 40, 80, 120 Tybalt variety Increases in gluten concentrations in grain Agricultural experimental Staion of [62] University of Technology and Life Field 80, 120 Spring wheat Increases in gluten concentrations in grain Sciences of Minikowo (Poland) Peterlauki research and Study Farm [6] Field 0, 60, 90, 120, 150, 180, 210, 240 Skagen variety Increases in gluten concentrations in grain (Latvia) Henan Agricultural University [63] Field 0, 90, 180, 270, 360, 450 Yumai and Lanko Aizao varieties Increases in total gliadins concentrations in flour Experimental Satation (China) Durabon, Durabonus, Swadzim Experimental Station [64] Field 0, 50, 100, 150 Duraprimus and Rusticano Increases in gluten concentrations in flour (Poland) varieties Lincoln Research Farm (New Batten, Kotare, Oroua, Rongotea, [65] Field 0, 50, 100 Increases in total gliadins concentrations in flour Zealand) Ruapuna and Tui varieties [66] Mira (Italy) Field 70, 120, 130, 160, 180, 200, 240 Biensur variety Increases in gluten concentrations in flour Three varieties: Capo, Renan and Increases in total, α, ω and γ gliadin [67] Two different sites (Austria) Field 0, 180 Lindos concentrations in flour Experimental Farm of Helsinki Scandinavian, Kadett, Ruso and Increases in total proteins concentrations but not [68] Field 0, 110 University (Finland) Reno wheat varieties changes in gliadin concentrations in flour [69] Hungary Field 30–300 Winter wheat Increases in gluten concentrations in flour [70] Chile Field 0, 220, 250 Increases in gluten concentrations in flour [71] France Field 40, 60 Seedling from INRA Increases in total gliadins concentrations in flour [72] Minokowo (Poland) Field 0, 60, 90, 120 Zebra variety Increases in gluten concentrations in flour Field experimental Station of Increases in total gluten concentrations in grain [73] Mediterranean Agronomic Institute Field 30, 40, 50, 70 Triticum turgidum subsp. durum and flour of Bari (Italy) [74] Brazil Field 0, 50, 100, 150 Quartzo variety Increases in gluten concentrations in grain Experimental farm of INRA, Grignon, [75] Field 40, 60, 120 Soissons variety Increases in total gliadin concentrations in grain France Research field sation of Faculty of [76] Field 0–194 Marija and Soissons varieties Increases in gluten concentrations in grain Agriculture (Croatia) Cordiale, Hereward, Istabraq, [77] Rothamsted Research station (UK) Field 100, 200, 350 Malacca, Marksman Increases in γ-gliadin gene expression and Xi 19 varieties Foods 2020, 9, 1602 8 of 21 Table 1. Cont. Cordiale, Hereward, Istabraq, [78] Rothamsted Research station (UK) Field 100, 200, 350 Malacca, Marksman Increases in ω-gliadin gene expression and Xi 19 varieties Dozent, Monopol, Rektor, Apollo, Ares, Astron, Basalt, Bussard, Increases of α/β -gliadin, ω- and γ-gliadins, [17] Germany Field 0, 40, 120, 180, 200 Herzog, Ignaz, Kanzler, Monopol, total gliadins and gluten concentrations in flour Obelisk, Sperber varieties Johann Heinrich von Thunen-Institute, Increases of α/β -gliadin, ω- and γ-gliadins, [79] Federal Research Institute for Rural Field 84, 168 Batis variety total gliadins and gluten concentrations in flour Areas, Forestry and Fisheries, in Braunschweig, Germany Research Station of Warmia and Increases of α/β -gliadin, no clear effects on ω- [80] Field 80, 120 Spring triticale cv. Andrus Mazury University (Poland) and γ-gliadins in grain Research Station of Warmia and Increases of total gliadins concentrations in in [81] Field 80, 120 Spring triticale cv. Andrus Mazury University (Poland) grain Uhrusk Experimental [82] Station belonging to the University of Field 90, 150 Opatka variety Increases in gluten concentrations in grain Life Sciences in Lublin (Poland) Fields research stations of Idaho and [83] Field 168, 224, 280 Spring wheat Increases in gluten concentrations in flour Monatana state Universities (USA) Futterkamp Increases in total gliadin and gluten [84] and Sonke-Nissen-Koog Field 220, 260 Tobak and Asano varieties concentrations in flour Northern Germany Grains Research Centre Kragujevac [85] Field 60, 90, 120 Increases in gluten concentrations in grain (Serbia) China Agricultural University [86] Research Center field station, Hebei Field 180, 240 Zhongmai variety Increases in gluten concentrations in grain province, China Chongzhou and Renshou Shumai 969, Shumai 482, Increases of total, α/β-gliadin and ω-gliadins [87] experimental stations of Sichuan Field 0, 75, 150, 225 Chuannong 16 and Mianmai 51 and gluten concentrations, no clear effects on Agricultural University, China varieties ω-gliadins in flour [88] Germany Pot experiment 0.25, 1.0 and 2.5 g N/pot Privileg variety Increases of total gliadin concentrations Foods 2020, Foods 9, 1602 2020, 9, x FOR PEER REVIEW 9 9ofof 21 21 3. Increasing Gluten and Gliadin Contents with N-Fertilisation 3. Increasing Gluten and Gliadin Contents with N-Fertilisation Our meta-analysis found that the increase in N-fertilisation rates was associated with increased Our meta-analysis found that the increase in N-fertilisation rates was associated with increased content of total gluten, total gliadins, α/β-gliadins, γ-gliadins, ω-gliadins and gliadin transcripts in content of total gluten, total gliadins, α/β-gliadins, γ-gliadins, ω-gliadins and gliadin transcripts in wheat grains (Figure 2). Our analyses also identified a significant relationship (R2 =2 0.30, p < 0.001) wheat grains (Figure 2). Our analyses also identified a significant relationship (R = 0.30, p < 0·001) between the increase in N fertilisation and the increase in total gliadin content in wheat grains (Figure 3). between the increase in N fertilisation and the increase in total gliadin content in wheat grains (Figure Although the analysis has been conducted with very different genotypes of wheat growing under 3). Although the analysis has been conducted with very different genotypes of wheat growing under different soil types and climates, and therefore under very diverse conditions, the level of N fertilisation different soil types and climates, and therefore under very diverse conditions, the level of N explained 30% of the change in gliadin content. These results are consistent with several studies fertilisation explained 30% of the change in gliadin content. These results are consistent with several observing a positive link of nutrient availability with the expression of gliadin genes [19,77,78,89] and all studies observing a positive link of nutrient availability with the expression of gliadin genes gluten proteins [17,90–92]. Furthermore, the results are also consistent with common farmer knowledge [19,77,78,89] and all gluten proteins [17,90–92]. Furthermore, the results are also consistent with on protein concentration in wheat grain being strongly affected by N availability, which leads farmers common farmer knowledge on protein concentration in wheat grain being strongly affected by N to adjust the level of nitrogen fertilisation to obtain the required protein concentration in grain for availability, which leads farmers to adjust the level of nitrogen fertilisation to obtain the required bread making [93]. protein concentration in grain for bread making [93]. (219) Total gluten *** (297) Total gliadins *** (62) α/β-gliadins *** (61) γ-gliadins *** (76) ω-gliadins *** (174) Gliadin transcripts *** −0.4 -0.4 −0.3 -0.3 −0.2 -0.2 −0.1 -0.1 0 0 0.1 0.1 0.2 0.2 0.3 0.3 0.4 0.4 Response ratio to N fertilization Figure 2. Response ratios (±95% CI) of contents of total gluten, total gliadins, α/β-gliadins, Figure 2.and γ-gliadins ratiosand ω-gliadins Response gliadin (±95% transcripts CI) of intotal contents of wheat grainstotal gluten, after an increase gliadins, in N fertilisation. α/β-gliadins, γ-gliadins See Table 1 and References therein for the sources. The number into parenthesis indicates and ω-gliadins and gliadin transcripts in wheat grains after an increase in N fertilisation. theSee number Table 1 of and studies. *** p < 0.0001. References therein for the sources. The number into parenthesis indicates the number of studies. *** p < 0.0001. Foods 2020, 9, 1602 10 of 21 Foods 2020, 9, x FOR PEER REVIEW 10 of 21 120 [Gliadin, mg g-1] = 42.93 + 0.16 kg N-fertilizer y−1 ha−1 R2 = 0.30, p < 0.00001 Gliadin concentration in grain (mg g−1) 100 80 60 40 20 0 0 50 100 150 200 250 N-fertilization (kg N y−1 ha−1) Figure 3. Increases Figure in total 3. Increases gliadin in total contents gliadin in wheat contents grains in wheat as a function grains of theof as a function increases in N-fertilisation the increases in N- (kg N ha−1rates ratesfertilisation y−1 ). See Table 1 for the sources. (kg N ha y ). See Table 1 for the sources. −1 −1 The The per per capita annual capita annualincrease increaseiningliadin gliadin intake fromwheat intake from wheat andand derived derived foods foods during during 1961–2010 1961–2010 −1 was estimated was estimatedto beto approximately be approximately −1 (+58% ±±7.5%) 1.41.4kgkgyy (+58% 7.5%)(Mean (Mean ± SE; ± SE; Figure Figure 4). This 4). This estimation estimation took took into into account account thethe annual annual intakeofofwheat intake wheat and derivatives derivativesatatthe global the scale, global [3] the scale, [3] increase in N in N the increase fertilisation fertilisation in wheat in wheat crops crops and and thethe relationshipsbetween relationships between NN fertilisation fertilisationand andgliadin gliadinincrease increase(Figure (Figure 3 3 and Table 1). The increase in N-fertilisation from approximate 10 to 100 kg and Table 1). The increase in N-fertilisation from approximate 10 to 100 kg N ha corresponded N ha −1 −1 corresponded to to an an increase in gliadin contents in grains/flour from 44 to 59 mg−1 g−1, respectively) (Figure 3 and Table increase in gliadin contents in grains/flour from 44 to 59 mg g , respectively) (Figure 3 and Table 1). 1). Foods 2020, 9, 1602 11 of 21 Foods 2020, 9, x FOR PEER REVIEW 11 of 21 4 120 Per capita intake of wheat and derivates (kg y−1) 3.8 110 Per capita gliadins intake (kg y−1) 3.6 100 3.4 90 3.2 80 3 70 2.8 2.6 60 2.4 50 1960 1970 1980 1990 2000 2010 2020 Year Figure 4. Per capita gliadin intake (g y−1 ) and per capita wheat and derivates intake (kg y−1 ). Figure 4. Per capita gliadin intake (g y−1) and per capita wheat and derivates intake (kg y−1). 4. Increased Prevalence of Coeliac Disease 4. Increased Part of Prevalence the increase of Coeliac Disease in CD prevalence in populations in recent decades has frequently been attributed to improved diagnosis [94], with Part of the increase in CD prevalence in populations some studies suggesting that thehas in recent decades increase in diagnoses frequently been was due to increased awareness [95]. Some studies of populations over attributed to improved diagnosis [94], with some studies suggesting that the increase in diagnosestime, however, have reported wasandueactual increase awareness to increased in CD development [95]. Some in recentofdecades studies beyond populations overthe time,improvement however, have of reported diagnostic efficiency [96]. The increase in diagnosed cases of new coeliac an actual increase in CD development in recent decades beyond the improvement of diagnosticpatients may thus be due to more efficient diagnosis and higher awareness, but also to changes in environmental efficiency [96]. The increase in diagnosed cases of new coeliac patients may thus be due to more variables associated with this efficient increaseand diagnosis in the percentage higher awareness,of a population but also toaffected changesbyinCD. Our study provides environmental variablesgood evidence associated with this increase in the percentage of a population affected by CD. Our study provides good in of a strong potential increase in the average human intake of gliadins by associating the changes global per evidence of acapita strongintakes of wheat potential and derivatives increase in the average withhuman the empirical intakeeffects of a global of gliadins 10-fold increase by associating the in intensification of N fertilisation during 1961–2010. The contents of digested changes in global per capita intakes of wheat and derivatives with the empirical effects of a global peptides derived from gliadin in the gut have been demonstrated to be a determinant for the appearance 10-fold increase in intensification of N fertilisation during 1961–2010. The contents of digested of autoimmunological responses peptides and CD derived frommanifestation gliadin in [97,98], the gutand thebeen have amount of gluten/gliadin demonstrated to benecessary to trigger a determinant for CD the in susceptible people can vary [99]. appearance of autoimmunological responses and CD manifestation [97,98], and the amount of A comparison gluten/gliadin necessaryof the changesCD to trigger in in global per capita susceptible intake people canofvary gliadins [99]. from 1961 to 2010, with data forACD prevalence comparison of(percentage the changes of in coeliac cases global per in the capita totalof intake population) gliadins from or incidence (new 1961 to 2010, cases with dataper 10,000 inhabitants per year) provided by studies of large and representative for CD prevalence (percentage of coeliac cases in the total population) or incidence (new cases per 10 sets of population data, 000adjusted for age inhabitants perand year) sex is shownbyinstudies provided Figure of 5. large The increases in prevalence/incidence and representative sets of populationduring this data, adjusted for age and sex is shown in Figure 5. The increases in prevalence/incidence during thisthe period coincided with the per capita increase in gliadin intake associated to the increase in application period coincided of Nwithfertilizer the per percapita tonneincrease of wheat in grains gliadinproduced and withtothethe intake associated per capita increase increase in the application of N fertilizer per tonne of wheat grains produced and with the per capita increase in gliadin intake (Figure 5). The higher per capita ingestion of gluten/gliadin globally in recent decades could thus account for, at least partially, the spread of coeliac pathology in the global human Foods 2020, 9, 1602 12 of 21 Foods 2020, 9, x FOR PEER REVIEW 12 of 21 in gliadin intake (Figure 5). The higher per capita ingestion of gluten/gliadin globally in recent decades could thus account for, at least partially, the spread of coeliac pathology in the global human population. New research is though warranted to test this possibility and to figure out why instead population. New research is though warranted to test this possibility and to figure out why instead the prevalence of CD is comparable between countries in which the intake of gluten is much higher, the prevalence of CD is comparable between countries in which the intake of gluten is much higher, e.g., Italy with its high consumption of pasta, than in other EU countries where the intake is much e.g., Italy with its high consumption of pasta, than in other EU countries where the intake is much lower [100]. Long-term evolutionary adaptation may play a role there. However, several studies have lower [100]. Long-term evolutionary adaptation may play a role there. However, several studies demonstrated the link between probability of coeliac disease development and gluten intake. For have demonstrated the link between probability of coeliac disease development and gluten intake. example, Makharia et al. [101] observed that northern Indian populations had much higher rates of For example, Makharia et al. [101] observed that northern Indian populations had much higher rates CD than southern Indian populations despite having similar predisposing HLA susceptibility genes of CD than southern Indian populations despite having similar predisposing HLA susceptibility genes across the country. This was primarily attributed to the mainly wheat-based diet in the North and across the country. This was primarily attributed to the mainly wheat-based diet in the North and the rice-based diet in the South. We have checked the available data at country level for the period the rice-based 2001–2017 diet in and found the South. a positive We have checked relationship betweenthe availableof prevalence data at country coeliac diseaselevel and for perthe period capita 2001–2017 and found a positive relationship wheat intake across 38 countries (Figure 6). between prevalence of coeliac disease and per capita wheat intake across 38 countries (Figure 6). 4 4 Prevalence of coeliac dissease (% of population) 3.5 3.5 Global per capita gliadin intake (kg y−1) 3 3 2.5 2.5 2 2 Finland102 1.5 1.5 1 1 USA - 32 states of USA25 USA – Washington and 0.5 Maryland counties23 0.5 USA – Olmsted County, Minnesota103 0 0 1960 1970 1980 1990 2000 2010 2020 Year Figure 5. Changes in global per capita intake of gliadins, global per capita intake of gliadins per kg of N fertilisation, Figure 5. Changes amount in globalofper N fertilizer applied capita intake per tonne of gliadins, of harvested global wheat per capita grains intake and theper of gliadins prevalence kg of (percentageamount N fertilisation, of coeliac of cases in the applied N fertilizer total population) per tonne or of incidence harvested (new wheatcases perand grains 1000 theinhabitants prevalenceand year) of coeliac (percentage disease of coeliac casesfrom studies in the with large and total population) or representative incidence (newsets of population cases data, adjusted per 1000 inhabitants and for age year) ofand sex, disease coeliac from 1961 to 2010 from [23,25,102,103]. studies with large and representative sets of population data, adjusted for age and sex, from 1961 to 2010 [23,25,102,103]. Foods 2020, 9, 1602 13 of 21 Foods 2020, 9, x FOR PEER REVIEW 13 of 21 MEX 2.0 PRT TUR (% FIN disease (% coeliac dissease CZE URY DZA 1.6 HUN JOR ITA population) IND GBR ISR of coeliac NZL TUN population) 1.2 AUS SWE IRN EGY ARG RUS NDL USA GRC Prevalenceof SVNDNK IRL 0.8 AUTESP Prevalence BRA ISL CHE Regression type II EST Regression type II DEU POL F = 150 BFA F=150 0.4 t = 12.3 t=12.3 NOR R2 = 0.40 R2=0.40 p < 0.00001 P<0.00001 JPN 0.0 0 50 100 150 200 Per capita Per capitawheat wheatintake y−1)y-1) (kg(kg intake Figure 6. Relationship between the prevalence of coeliac disease and the per capita wheat intakes at Figurelevel. country DZA = Argelia. 6. Relationship betweenARGthe= prevalence Argentina. ofAUS = Australia. coeliac disease and AUT the=per capitaBFA Austria. = Burkina wheat intakes at Faso. BRA = Brazil. country level. DZACZE = Czech = Argelia. ARG = Argentina. Republic. AUS= DNK = Australia. Denmark. AUTEGY = Egypt. = Austria. EST = Faso. BFA = Burkina Estonia. BRA==Cuba. CUB FIN ==Czech Brazil. CZE Republic. Finland. DEUDNK = Germany. = Denmark.GRC EGY= Greece. = Egypt. EST HUN = Hungary. = Estonia. Cuba.=FIN CUB = IND India. ISL = Iceland. = Finland. DEU IRN = Iran. IRL = Germany. GRC==Ireland. ISR ==Israel. Greece. HUN Hungary. = Italy. ITAIND = India. JPN = =Japan. ISL Iceland. MEXIRN== Mexico. Iran. IRL = NLD = Ireland. ISR = Israel. Netherland. = New NZL ITA = Italy. JPN = Japan. Zealand. NORMEX = Norway. NLD = = Mexico. POL = Netherland. Poland. PRT NZL = =Portugal. New Zealand. NOR = Norway. POL = Poland. PRT = Portugal. RUS = Russia. SVN = RUS = Russia. SVN = Slovenia. ESP = Spain. SWE = Sweden. CHE = Switzerland. TUN = Tunisia.Slovenia. ESP = Spain. SWE==Turkey. TUR Sweden. CHE GRB ==Switzerland. TUN = Tunisia. United Kingdom. USA =TUR = Turkey. United StatesGRB = United Kingdom. of America. URY = USA = Uruguay. United States of America. Data from [3,15,104–108]. URY = Uruguay. Data from [3,15,104–108]. Themodern The modernprocedures proceduresofofmanagement managementand andprocessing processing (shortening (shortening thethe fermentation fermentation time, time, use use of of non-acid dough, add protein fortification and use of refined white flour) could have also non-acid dough, add protein fortification and use of refined white flour) could have also increased increased the the exposuretotoimmunoreactive exposure immunoreactive compounds compounds [109]. [109].The Theuse useofofdifferent differentgenotypes genotypesof of wheat wheatmay maybe be involved too since the gluten and gliadin composition are highly determined by environmental involved too since the gluten and gliadin composition are highly determined by environmental effects effects but also by genetic differences among wheat varieties. The genotypes and varieties of but also by genetic differences among wheat varieties. The genotypes and varieties of cultivated cultivated wheat have also changed in these last 60 years. Breeding practices in wheat genotypes have wheat have also changed in these last 60 years. Breeding practices in wheat genotypes have been been mainly addressed to achieve higher yield, rheological conditions and gluten quality and high mainly addressed to achieve higher yield, rheological conditions and gluten quality and high protein protein concentration for better baking and also for better livestock foods [7,110,111], providing concentration for better baking and also for better livestock foods [7,110,111], providing stronger stronger glutens rich in glutenins [89,112] but with scarce effects on gliadins and coeliac disease glutens rich in prevalence glutenins [89,112] but with scarce effects on gliadins and coeliac disease prevalence [10]. [10]. Foods 2020, 9, 1602 14 of 21 Some studies have reported that breeding activities in the last decades have contributed to increase the prevalence of coeliac disease [113], but there are more studies reporting no contribution of modern wheat breeding practices to coeliac disease prevalence during the last decades [109,112,114]. Most studies have reported that both modern and ancient wheat genotypes have similar concentrations of the pathogenic peptides responsible of inflammatory diseases [115–122], similar quantities of immunostimulatory epitopes [109,115,119,123], similar human T cell immunological responses [124–127] and similar immunogenic peptide sequences [128]. Some studies have even concluded that old varieties produced larger amounts of peptides containing immunogenic and toxic sequences than modern ones [129] and also that old varieties trigger more inflammatory processes in gut [130]. The current literature does not thus sustain the hypothesis that the shift in wheat genotypes could be a significant potential explanation for the rise in coeliac disease at global scale. Furthermore, breeding is now aiming to produce new wheat genotypes with less gliadin epitopes but without clear success yet [30]. The research to find wheat genotypes with less capacity to produce coeliac disease, i.e., less immune-reactive wheat genotypes, is very recent [116]. Independently of wheat subspecies, our results show that the increase in N fertilisation is related with increasing levels of gluten and gliadin concentrations in wheat grain and flour in all types of wheat genotypes and varieties studied, that the average per capita intake of wheat flour and grain food derivatives has been kept more or less constant in the last 60 years, and therefore the per capita ingest of gluten and gliadins has substantially increased. There are, moreover, other possible factors predisposing to the development of CD such as the many substances emitted by humans into the environment. For instance, some studies have reported significant relationships between the ingestion of glyphosate, an increasingly used herbicide, and the predisposition to the development of CD [39,131]. In fact, CD is a very complex pathology, whose development involves not only environmental factors (gluten) but also genetic factors. Recently, a gene Inc13 has been identified that encodes for a noncoding RNA that blocks and represses the expression of certain inflammatory genes under normal conditions. The Inc13 expression can be inhibited by stimulation and increased expression of inflammatory genes favoring CD [132]. Moreover, the inflammatory over-expression of T cells could be also favoured under the infestation of certain reovirus that would suppress peripheral regulatory T cell [133]. In any case, though, we now know that autoimmune responses are generally triggered by gliadin peptides [39], and we have shown here that these gliadin peptides increase their concentration in grains in response to the intensification of N fertilisation (Figures 2 and 3). 5. Conclusions The intensification of N fertilisation of wheat crops has been very high (ten-fold since 1961). Our meta-analysis of the literature has demonstrated that wheat growing under higher soil N availability produces not only higher yield but also grains and flours with higher gliadin concentrations in all type of wheat genotypes. Since gliadins are the main direct responsible triggering coeliac disease and the per capita intake of wheat products in the last decades has remained more or less constant, there has been a rise in per capita intake of gliadins at the global scale. We suggest that the rise in coeliac disease reported in several human populations around the world could be related, at least in part, to N fertilisation intensification of wheat crops. If this suggested link between N fertilisation intensification and coeliac disease expansion is demonstrated in future experimental studies, we will have an important tool to control and prevent the expansion of coeliac disease. Author Contributions: J.P. and J.S. conceived the idea and designed the research, J.S. and J.P. gathered and analysed the data. A.G.-G., I.A.J., P.C., M.O., K.K., Y.-G.Z. and O.U. assisted in the analyses. All authors contributed substantially to the writing and discussion of the paper. All authors have read and agreed to the published version of the manuscript. Foods 2020, 9, 1602 15 of 21 Funding: This research was funded by the Spanish Government grant PID2019-110521GB-I00, the Catalan Government grant SGR 2017-1005, the Czech SustEs project (CZ.02.1.01/0.0/0.0/16_019/0000797) and the European Research Council Synergy grant ERC-SyG-2013-610028 IMBALANCE-P. Conflicts of Interest: The authors declare no conflict of interest. References 1. Lu, C.; Tian, H. Global nitrogen and phosphorus fertilizer use for agriculture production in the past half century: Shifted hot spots and nutrient imbalance. Earth Syst. Sci. Data 2017, 9, 181–192. [CrossRef] 2. Heffer, P.; Prud’homme, M. Global Nitrogen Fertilizer Demand and Supply: Trend, Current Level and Outlook. In Proceedings of the International Fertilizer Association (IFA), 7th International Nitrogen Initiative Conference, Melbourne, Australia, 4–8 December 2016; pp. 1–11. 3. FAOSTAT. 2019. Available online: http://www.fao.org/faostat/en/#data (accessed on 11 July 2019). 4. 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