Raw Water and ALS: A Unifying Hypothesis for the Environmental Agents Involved in ALS

Abstract

Different studies identified the presence of several altered genes in familial and sporadic amyotrophic lateral sclerosis (ALS) forms. The experimental data, together with the epidemiological data, would seem to suggest the existence of molecular mechanisms (e.g., axonal transport) related to these genes, together with a susceptibility of the same genes to certain environmental factors that would therefore suggest an impact of the environment on the etiopathogenesis of ALS. In our review, we considered the most relevant environmental clusters around the world, collecting different hypotheses and underlining common environmental factors among the different clusters. Moreover, further epidemiological data identified a higher risk of ALS in professional athletes and, in particular, in soccer and football players. Despite this increased risk of ALS highlighted by the epidemiological evidence in aforementioned sports, the mechanisms remain unclear. At last, the use of raw water has been associated with ALS risk. The aim of the present review is to characterize a possible relationship between these clusters, to be explored in the context of the interaction between genetic and environmental factors on the etiopathogenesis of ALS.

Introduction

Amyotrophic lateral sclerosis (ALS) is a rare degenerative disease affecting the 1st and 2nd type motor neurons, with secondary muscle degeneration and subsequent irreversible paralysis; however, despite a growing number of scientific evidence, its etiopathogenesis remains unknown. A recent review, which investigates ALS as a multistep process, focused on the definition of the gene-time-environment hypothesis, suggesting an interaction between the genetic predisposition and the environmental exposures over time and leading to the development of the disease.^{1, 2} A new challenge to identify the causes of ALS and the modifiable risk factors associated with the disease, and which would allow preventive approaches, is the definition of the exposome in the context of a multistep model, which is the measure of all exposures during an individual’s lifetime and related to the health status. As for the environmental factors, it is important to underline that these are also necessary steps in individuals with penetrant mutations for the disease. ² A recent meta-analysis showed an association between ALS and environmental factors as a result of the exposure to lead, heavy metals, pesticides, agricultural chemicals, and solvents. ³ In particular, ALS has been associated with rural environments, ⁴ with farmers, agricultural workers, ⁵ and people most exposed to agricultural chemicals, pesticides, and/or other environmental toxins. ⁶ A recent Italian study reported the association between an occupational history in the agricultural sector and the risk of ALS when the agricultural occupational history was longer than 10 years. ⁷ The presence of more susceptible individuals could be explained by a strong genetic susceptibility, together with the interaction with unknown environmental factors.

However, despite the evidence in animal models on the interaction between genetic and environmental factors in the onset of ALS, ⁸ there are a few studies with relevance to humans. One highlights the association between a paraoxonase 1 (PON1) promoter polymorphism and exposure to organophosphates, ⁹ another hypothesizes the role of the C9orf72 gene in modulating the inflammatory response in relation to the gut microbiota. ¹⁰ The challenge for the scientific community is to design rigorous epidemiological studies to evaluate complex hypotheses in the etiopathogenesis of ALS. ¹¹

Indeed, epidemiological findings suggest the presence of some disease-associated clusters in different parts of the world. ¹²

This review reports a synthesis of the evidence on three specific areas of the environmental theory on ALS, and in particular, the description of environmental clusters, the higher risk in sports, and the use of raw water. Then, the aim of the present review is to characterize a possible relationship between these three specific areas, suggesting, in the last paragraph, a unifying hypothesis and contributing to the debate on the etiopathogenesis of ALS, focusing on an element of its complexity.

Discussion

The Clusters of ALS and the Environmental Hypothesis

The largest known clusters of ALS in the world occurred immediately after World War II in the following three foci: the American island of Guam, ¹³ the Japanese peninsula of Kii, ¹⁴ and the island of New Guinea. ¹⁵ In these clusters, populations often showed the presence of ALS in a complex including also Parkinson’s disease and dementia amyotrophic lateral sclerosis and parkinsonism-dementia complex (ALS-PDC). As for the cluster on Guam Island, which perhaps represents the clearest example, the observed incidence at its peak in 1950s was a hundred times higher if compared to the rest of the world: 200 cases per 100,000 per year. ¹⁶ This incidence decreased in the following 40 years is probably not due to genetic factors. Instead, it was most likely the result of radical socioeconomic, ethnographic, and ecological changes caused by the rapid westernization of Guam. ¹²

The scientific community focused on other possible environmental factors such as mineral excess or deficiency^17–19 and possible infectious agents, such as viruses ²⁰ and prions. ²¹ Spencer et al.^{22, 23} suggested another interesting hypothesis with the identification of an amino acid present in the seeds and leaves of cycas circinalis: β-methylamino-L-alanine (BMAA). This amino acid has neurotoxic properties, and it is characterized by a molecular structure similar to glutamate and β-N-oxalyl-L-diaminopropionic acid (BOAA), which has been identified as a possible cause of underlying lathyrism.^{24, 25}

The Chamorro people of Guam prepared tortillas with cycas circinalis leaves for food purposes. Furthermore, Obendorf and Spencer ²⁶ examined the occurrence of cycad use for medicinal purposes and reported widespread utilization of crushed cycad as a poultice for open sores or abrasions. Although Spencer’s studies demonstrated the remarkable neurotoxicity of BMAA in vivo, ²⁷ the concentrations in washed cycas leaves of this neurotoxic amino acid were found to be too low for its neurotoxic action in humans. ²⁸ Cox and Sacks ²⁹ suggested the hypothesis of a bioconcentration process with a subsequent neurotoxic effect in humans. Indeed, the Chamorro people of Guam used to sed to feed on the flying foxes in which a bioaccumulation process of BMAA occurred, due to the diet of cycas seeds with the subsequent toxic effect on humans, as hypothesized by Spencer. To confirm this theory, some studies^{30, 31} identified a reduction in the incidence of ALS-PDC consistent with the reduction in the flying fox population.

BMAA exists in tissues in the following two forms: free and bound to the proteins. ²⁸ BMAA is not produced directly by cycas circinalis, but it is produced by symbiotic bacteria in the roots of cycas: cyanobacteria.^29–37 As for cyanobacteria, one of the most interesting findings is that these bacteria are ubiquitous and occur in terrestrial and aquatic environments worldwide, able to survive in extreme climatic conditions. ³⁸ These cyanobacteria were found in a wide variety of habitats including fresh, brackish, and marine waters, soil, hot springs, and limestone caves.^{24, 39} Cyanobacteria can form huge populations, blooms and foam, floating on the water surface used for recreation activities and in drinking water supplies. ⁴⁰ Codd et al. ⁴¹ described the contamination of the plant food, lettuce (Lactuca sativa), focusing on the terrestrial origin of cyanobacterial toxins, with microcystins after spray irrigation with water containing cyanobacteria (Mycrocistis aeruginosa). In addition, this review also described the possible exposure pathways through which these toxins could affect human health, highlighting the importance of a targeted approach to understanding them. The authors also emphasize the importance to identify risk mitigation measures arising from the effects of cyanobacterial toxins on water quality, through an approach that requires multidisciplinary skills. The presence of detectable levels of BMAA in some species of fish and seafood sold in Swedish supermarkets was identified by Jiang et al. ⁴² Another study on wheat (Triticum aestivum) demonstrated that seedlings could absorb BMAA from the water used to irrigate the fields. ⁴³ Cox et al. ⁴⁴ examined microbial mats produced by cyanobacteria in the deserts of Qatar detecting the presence of BMAAs, although no quantitative data were provided.

This finding led to consideration of a possible link between inhaled BMAA and an increased incidence of ALS among soldiers who were stationed in Qatar, ⁴⁵ where Richer et al. ⁴⁶ identified two isomers of BMAA in soil: 2,4-diamino butyric acid (DAB) and N- (2-aminoethyl) glycine (AEG). Other possible links between BMAA exposure and ALS due to consumption of blue crabs ⁴⁷ and exposure to aerosolized cyanobacteria from cooling towers ⁴⁸ have been suggested. Furthermore, BMAAs have been researched in the brain tissues of patients with degenerative neurological conditions outside Guam. Pablo et al. ⁴⁹ examined the brains of 13 people who died from ALS, 12 from AD, 8 from Huntington’s disease, and 12 from other causes. In all the brains of the 13 patients who died from ALS and the 12 patients who died from AD, it was possible to identify BMAA with levels ranging from 141 ± 14 µg/g. However, this data has not been confirmed by other authors. In fact, it was not possible to identify BMAA in any of the tissues examined of patients who died from ALS, ALS-PDC, as well as in Chamorro.^50–53 All these authors used different methods of identifying BMAA: fluorescence detection (FD), high-performance liquid chromatography (HPLC), mass spectrophotometry (MS), gas chromatography (GC), liquid chromatography (LC), and ultrahigh-performance liquid chromatography (UHPLC). However, the use of all these different methods could explain the heterogeneous results found.^{24, 54}

ALS and Sport: A Summary of the Hypotheses

In the last decade, a growing amount of scientific evidence has highlighted a strong interest in the issue of neurodegenerative diseases (NDs) within the context of professional sports. Supporting evidence has shown that sport and, in general, physical activity are factors that play a crucial role in the prevention of chronic diseases, with an overall mortality risk reduction (e.g., cardiovascular disease, ischemic heart disease, lung cancer)^{55, 56} with lifelong health benefits, ⁵⁷ even when considering sport at a professional level.^{58, 59} Nevertheless, several epidemiological studies reported an association between sport and NDs, such as Alzheimer’s disease (AD), ALS, and chronic traumatic encephalopathy (CTE), investigating the participation in contact sports as a potential risk factor, particularly among soccer^60–64 and American football athletes.^{65, 66} In particular, two recent large cohort studies were conducted in order to compare mortality from NDs between former soccer players and controls from the general population.^{59, 67} Both studies observed a higher risk of NDs among the former soccer players, almost 4 times higher if compared to the general population, with a hazard ratio (HR) of 3.66 (P < .001) and 4.10 (P < .001), respectively, and an increased risk among former professional soccer players with longer careers and in those playing in specific roles.

Focusing on the link between ALS/motor neuron disease (MND) and professional sport, several studies have explored the phenomenon by delving into epidemiological and neuropathological aspects.

A study, part of an investigation to assess the possible long-term effects of doping, using several sources, identified a set of soccer players and former soccer players (about 24,000 subjects) who played in the three top major leagues in the period 1960–1996. A total number of 375 deceased players before 1996 were identified with a substantial adherence of the observed and expected mortality. Only one exception was identified in mortality due to NDs, with ALS having an excess standardized mortality ratio of 11.6. ⁶⁸ Another study, investigating the increased risk of ALS in soccer, identified 5 cases of ALS among 7,325 professional players, active between 1970 and 2001 in the first and second divisions with an overall standardized morbidity ratio of 6.5. ⁶⁹ The same study also observed that the duration of professional activity and the midfielder position was associated with an increased risk of developing ALS. Moreover, the affected players were younger than ALS patients from the general population, and this evidence was confirmed by another recent study which also showed a shorter survival and an earlier onset of the disease (age at onset < 45 years). ⁷⁰ A recent systematic review ⁷¹ highlighted a significantly higher risk of ALS/MND among former professional athletes and, in particular, former professional soccer players. A significantly higher risk of ALS/MND has been also identified in former soccer players, if considering the same cohort investigated in the aforementioned studies^{54, 62} with an HR of 3.52 (P < .001) and 4.33 (P < .001), respectively. This increased risk of ALS/MND could be related to a complex interaction between genetic and environmental risk factors.

In a recent study of 19,423 male former and current National Football League players a significantly higher incidence of ALS diagnosis (standardized incidence ratio, 3.59; 95% CI, 2.58–4.93) and mortality (standardized mortality ratio, 3.94; 95% CI 2.62–5.69) were observed. ⁷²

A history of multiple traumatic events (e.g., head trauma) has been considered as a possible risk factor for ALS, with epidemiological studies and case reports suggesting that sports involving these repeated head traumas might be related to a long-term risk of developing NDs ⁷³ and, in particular, the risk of ALS.^74–76 Another environmental factor that contributes to the risk of developing ALS, identified in the literature and for which the biological plausibility has been investigated, is oxidative stress. It seems to be linked to the high amounts of oxygen consumed as well as the high tissue metabolism during intense physical activity, with the consequent production of reactive oxygen species (ROS). ⁷⁷ Although the evidence is still insufficient to support an association between the extensive use of medications and ALS, some studies suggested that nonsteroidal anti-inflammatory drugs may play an important role in increasing the risk of ALS.^78–81 Another hypothesis on susceptibility for ALS has been identified in the chronic use of food supplements containing substances like branched-chain amino acids (BCAAs). ⁸² Chronic use of BCAAs by professional athletes, in order to increase physical performance and muscle growth, could lead to an increased risk of ALS, ⁷⁸ based on genetic susceptibility, as suggested by animal model studies ⁸³ with their transformation into toxic substances leading to neurodegeneration. ⁸⁴ In particular, animal models also showed how dietary BCAAs could be responsible for alterations in specific oxidative stress pathways in the brain, ⁸⁵ inducing hyperexcitability at the neuronal level. ⁸⁶ In addition to the aforementioned risk factors, a further hypothesis has been suggested about the presence of a common phenotype among athletes, which would be responsible for a genetic predisposition leading to an increased risk of developing ALS.^{87, 88}

ALS and Raw Water: What Is the Epidemiological Relationship?

Raw water can be found in the environment in lakes, rivers, well water, and underground rainwater tanks, which has not been treated with treatment methods such as ozonation, chlorination, activated carbon adsorption, and flocculation to remove microbial agents and their toxins, ⁴⁰ including fungi. ⁸⁹ Several ALS clusters have been reported in relation to the use of raw water: In 1997, Poloni et al. ⁹⁰ described for the first time a particular cluster of ALS: a conjugal ALS of a couple married for 28 years who developed ALS within 30 months of each other. Both of them had been drinking water from the same artesian well for about 30 years. In the southern coastal region of Papua, Indonesia, cases of ALS and Parkinsonism were detected by Okumiya et al. ⁹¹ between 2001 and 2012 in villages located near the following three rivers: Ia, Dumut, and Edera. These authors reported that the Indigenous people of Papua drank water from wells near these rivers. In 2000–2009 on Oshima, a small island in the Kii peninsula of Japan, a high incidence of ALS has been reported after the drinking water source changed from regional water to the Kozagawa river in 1975. ⁹² Caller et al. ⁹³ noted the existence of a northeast US cluster of nine ALS cases in a town that was close to a lake that contained both Microcystis and Anabaena sp., two genera known to produce BMAAs, and suggested a possible causal link between BMAAs and ALS. Banack et al. ⁹⁴ revisited the same lake and found both microcystins and BMAAs in the fish brain (0.043 µg/g), liver, and muscle (1.28 µg/g). Masseret et al. ⁹⁵ and Réveillon ⁹⁶ both observed a significant ALS cluster surrounding the Thau lagoon in the Herault district in Southern France. These authors have quantified the BMAA with comparable results and they speculated the ingestion of mussels and oysters as a route of exposure. Gary et al.^{97, 98} observed a significant association between MND mortality and the percentage of population using private wells in the USA and Schwartz and Klug, ⁹⁷ with similar epidemiological results, assuming a possible association with the contamination of these wells with Legionella. Torbick et al. ⁹⁹ conducted a spatial analysis of water quality using Landsat data on chlorophyll-a, Sechi depth, and total nitrogen, and related these indicators of water quality to ALS hot spots. Data indicated an association of poorer water quality with ALS incidence. Andrew et al. ¹⁰⁰ in their case-control study demonstrated the presence of a water-body risk factor in ALS cases in New England. Most patients lived within 2 miles of a water body and they reported frequent participation of ALS cases in a water sport such as boating, sailing, kayaking and, more frequently, waterskiing. These authors hypothesize that the risk can be attributed to the presence of cyanobacteria and their toxins in the water. In a population-based case-control study, Filippini et al. ¹⁰¹ found a positive association between ALS risk and the use of private wells for drinking water in the Northern Italian population and a higher risk from the usage of private wells for irrigation in the Southern Italian population. In our Italian case-control study conducted in Terni, Umbria, ¹⁰² we have seen an association between ALS and in general raw water use.

Conclusion

A Unifying Hypothesis of the Three Previous Areas of the Environmental Theory of ALS

The presence of the clusters, neuroepidemiologically evident and described in this review, demonstrates a strong environmental component in the genesis of ALS in genetically susceptible patients in the framework of a multistep model of the etiopathogenesis of the disease.

We do not know the specific environmental agents that can trigger or have a role in the complex mechanisms of the onset of ALS as well as the role of genetic susceptibility factors but some hypotheses can be formulated.

In the clusters present in soccer, exposure to some toxic substances and chemicals, heavy metals, and solvents ¹⁰³ has been considered as potentially responsible for NDs. These substances also include pesticides, commonly used for the treatment of football grounds. However, although pesticides may play an important role in the onset of ALS, the exposure in soccer players’ activity is not easy to measure. ⁷⁸ ALS has always been described more frequently in rural environments, but it is not so much the residing in a rural environment that creates this risk, rather it seems to be those who carry out active agricultural work who are at risk. ⁵

We have described ¹⁰² a statistically significant association between the use of untreated water and ALS. It is probable that agricultural work is the vehicle of unknown toxins. Additionally, the football world may be included in this hypothesis.

The study by Pupillo et al. ⁷⁰ showed that the teams from Como (5 observed cases vs. 0.41 expected cases) and Lecco (4 observed cases vs. 0.31 expected cases) were higher among teams (e-Tab3). These soccer teams play on soccer fields located respectively in the stadiums of Sinigaglia and Rigamonti-Ceppi, located close to the same lake: Lake Como. Similarly, in the same study, the football teams of Genoa (4 observed cases vs. 0.36 expected cases) and Sampdoria (5 observed cases vs. 0.29 expected cases) share the same football fields in the city of Genoa, whose main stadium, the Luigi Ferraris stadium, is located near the Bisagno river. In the same e-Tab3 of Pupillo et al., ⁷⁰ another football team studied was Fiorentina (4 observed cases vs. 0.29 expected cases) and Fiorentina’s football field, Artemio Franchi stadium, is located near the Arno River.

The standardized incidence ratios for these five specific football teams are higher among all teams and are statistically significant with a lower limit of 95% confidence intervals superior to one. ⁷⁰

In the case-control study of Andrew, ¹⁰⁰ the risk of getting sick with ALS was linked to the existence of water bodies within 2 miles and in relation to the performance of water sports. Football fields are frequently irrigated, even between the first and second half of a football match, to maintain the integrity of the turf.

It would be very important to know with what water the football fields have been or are still being irrigated. There may be a relationship between football, ground, and untreated water that should be further studied. In particular, the presence of toxic substances (heavy metals, solvents, pesticides, and fungicides) and/or microbiological agents such as cyanobacteria and their toxins in untreated water should be better investigated.

Finally, ALS-PDC in Guam and Papua New Guinea may be linked with ALS cases in the rest of the world by means of common environmental agents that may act with different mechanisms. In particular, a hypothesis on the imbalance of environmental minerals, resulting in long-term Ca and Mg deficiencies with consequent increase in transition metals, such as Al, Fe, and Cu in the cerebral cortex and spinal cord tissues has been also proposed. ¹⁰⁵ In Guam, the Chamorros used rainwater from tin roofs and ground surface water as drinking water, containing extremely low contents of Ca and Mg, and in Oshima, a small island opposite to the top of the mainland of the Kii Peninsula, the water source was changed from the Kozagawa river, in which the Ca and Mg contents were extremely low.^{88, 104}

At last, it is important to underline the recent evidence on the effectiveness of the UV/chlorine process for the treatment of BMAA-contained water. ¹⁰⁵

Overall, the use of raw water could be the vehicle by which the human organism is exposed to toxins, metals, microorganisms, or deficient concentrations of minerals that may be involved in the etiopathogenetic mechanism of ALS by interacting with genetic susceptibility factors.

Research on these genetic factors requires an extraordinary organizational effort to conduct quality studies on a rare disease focusing on the metabolic polymorphisms involved in detoxification processes ¹⁰⁶ using also the Mendelian randomization methodology. ¹⁰⁷

In conclusion, the involvement and the mechanisms through which environmental agents are potentially associated with the etiopathogenesis of ALS are still unclear. However, if we consider ALS as a multistep pathology, and thus as a possible consequence of all exposures during an individual’s lifetime, some environmental factors, such as raw water, should be further discussed and addressed, also if considering the growing number of evidence on this topic.