Subclinical Hypothyroidism in Families Due to Chronic Consumption of Nitrate-Contaminated Water in Rural Areas with Intensive Livestock and Agricultural Practices in Durango, Mexico

Abstract

Nitrate is a widely disseminated water pollutant and has been linked to health disorders, including hypothyroidism. Here, we evaluated the relationship between thyroid function and chronic exposure to nitrates in rural zone families, in addition to the genetic and autoimmune factors. Exposure and effect biomarkers, thyroid hormones, and autoantibodies of tiroperoxidase were measured, as well the presence of two FOXE1 polymorphisms (rs965513, rs1867277). Pearson’s correlation, principal component analysis, Kruskal–Wallis, and chi-squared tests were used for statistical analysis. A total of 102 individuals were analyzed; 45% presented subclinical hypothyroidism, a negative correlation was observed between methemoglobin and the total T3 (r = −0.43, p = 0.001) and free T3 levels (r = −0.34, p = 0.001), as well as between TSH and the free T4 (r = −0.41, p = 0.0001) and total T4 (r = −0.36, p = 0.0001). A total of 15.7% had positive antithyroid ab-TPO, while the polymorphic genotype (AA) represented only 3% (rs965513) and 4% (rs1867277) among subjects with subclinical hypothyroidism. The high frequency of subclinical hypothyroidism in the population under study could be related, mainly, to chronic exposure through the consumption of nitrate-contaminated water.

1. Introduction

Groundwater is the main source of drinking water, as well as a stable and reliable resource that meets the demand for human consumption [1]. Access to water is important to sustain the basic activities of all living beings and is closely related to the development of urban and rural areas. [2]. However, in recent years, the contamination of these has been conditioned by the increase in the levels of nitrogen compounds, specifically nitrate, as a result of leaching, mainly due to anthropogenic activities such as the use of soils for intensive agriculture [3], intensive livestock farming [4], the use of nitrogenated chemical fertilizers [5], the use of animal manure as an organic fertilizer, the production and disposal of urban wastewater [6], the disposal of septic waste and effluents from septic tanks or leaks that also contribute to pollution in settlement environments [7,8]. The latter, in conjunction with geological features [9], rainfall fluctuations [10], interactions with surface water [11], changes in water tables [12], and the presence of biological processes such as denitrification [13], can promote the variation of nitrate concentrations, thus exceeding the permissible limit in water for human consumption established by the WHO, which is 50 mg/L nitrate ion—equivalent to 11.3 mg/L NO₃-N (nitrate-nitrogen) [14].

Nitrate is a nitrogenous compound, widely found and disseminated in nature [15] through plants, soil, air, and water [16]. Some of the characteristics of nitrate salts, mainly sodium nitrate and calcium nitrate, indicate that these compounds are colorless, odorless, tasteless, and highly soluble, making them difficult to detect in drinking water [17]. By the action of nitrifying algae and bacteria, nitrite and ammonia are easily converted via oxidation into nitrate, which is the most abundant stable nitrogenated form [18]. Nitrogen is a component of many biomolecules, which is why it can also be obtained via an endogenous pathway that metabolizes nitric oxide via L-arginine-nitric oxide synthase. In the exogenous pathway, nitrogen is obtained via the diet from the consumption of vegetables, green leafy vegetables, processed foods containing nitrogen as an additive, and water contaminated with high nitrate concentrations. Both endogenous and exogenous pathways are found in humans [19]. Once ingested, nitrogen is absorbed via the gastrointestinal mucosa and the proximal duodenum, reaching a bioavailability of over 90% [20]. It is then disseminated around the entire body via systemic circulation, with approximately 25% absorbed via salivary gland uptake, and then transported to the stomach by means of enterosalivary circulation. Finally, 65% of the nitrate ingested is eliminated in urine in the form of nitrate, ammonium, or urea [21]. The exogenous nitrate–nitrite–NO pathway plays an important role due to its precursor action in the formation of nitrite and, in turn, of the genotoxic N-nitroso compounds present in endogenous nitrosation [22]. Moreover, the ingestion of high nitrate concentrations in water used for human consumption has been found to be related to the following: the appearance of methemoglobinemia, [23] alterations in biochemical parameters [24] and metabolic functions [25], the development of cancers, such as bladder [26], colorectal [27], gastric [28], and thyroid [29], and the development of thyroid function disorders [30], such as goiter [31], thyroid hypertrophy [32,33], and subclinical hypothyroidism (SCH) [34].

Further to its well-established carcinogenic action [35], nitrate has been suggested as a thyroid disruptor [36], as it is one of the major inhibitors of the sodium–iodide symporter channel. Acting competitively in the uptake of iodine by the thyroid glands, nitrate alters the hypothalamic–pituitary–thyroid axis, thus conditioning the appearance of hypothyroidism [37]. The presence of nitrates in the thyroid follicle has been associated with changes in the gene expression of sodium–iodide symporter, thyroid peroxidase, thyroglobulin, and the FOXE1 transcription factor, which are key elements in the biosynthesis of thyroid hormones [38].

SCH is characterized by thyroid-stimulating hormone (TSH) levels that exceed the normal parameters for thyroid hormone levels in both total and free T4 tests [39]. On a global level, the main cause of SCH is a low level of iodine ingestion [40], while auto-immune disorders are the second most common cause [41], followed by genetic factors [42] and endocrine disruptors [43]. The global prevalence of SCH is 10% [44], while in Mexico, it is 8% [45].

Some rural areas of the state of Durango are characterized by intensive agricultural practices, including one of the main producing regions of dairy and beef cattle and one of the largest milk basins in Mexico. These practices cause serious deterioration and contamination of the region’s groundwater reserves due to the high nitrate levels, which are mainly attributed to agriculture and livestock farming. Across different rural locations in the municipality of Lerdo, Durango, studies have been conducted to ascertain the extent of the levels of nitrate in water used for human consumption. A high nitrate level in potable water is >11 mg/L NO₃-N, as established in the 2019 amendment to the corresponding Official Mexican Standard, NOM-127-SSA-1994 [46]. Moreover, impacts on the health of the region’s inhabitants have been reported, such as high levels of methemoglobin (MetHb), effects on sperm quality, and the presence of Heinz bodies. In our research group, a study was carried out in women living in areas with high levels of nitrates in drinking water, and we found alterations in metabolic parameters and thyroid hormone levels, as well as genotoxic damage and a higher incidence of SCH. In addition, the study participants reported that more than one member of their family suffers from thyroid disorders that were detected in their primary health care centers. Therefore, the objective of this study was to analyze the families of women previously identified with SCH in order to analyze the possible environmental, genetic, and immunological factors that could explain the high incidence of SCH in rural populations of Lerdo, Durango, Mexico.

2. Materials and Methods

2.1. Study Population

A familial, observational, descriptive, and cross-sectional study was conducted on individuals previously identified with thyroid dysfunction and residing in rural areas of the municipality of Lerdo, Durango, Mexico. Lerdo is located in central northern Mexico in the Chihuahuan Desert Region. The average annual temperature is 21 °C, with 200–300 mm of annual rainfall. Mainly, the territory of this municipality is used for agricultural and livestock activities. Due to the low rainfall in the area, people extract water from wells at high depth both for consumption and for the irrigation of crops, so they are exposed to pollutants, among which the presence of nitrates has been determined. The study population comprised families residing in properties of five localities within the municipality— 21 de Marzo, Juan E. García, La Loma, Nazareno, and San Jacinto, which are located between 1200 and 1300 m asl (Figure 1).

The sample comprised 102 subjects who are members of 26 families residing in the abovementioned communities. The inclusion criteria were the following: voluntary participation; signed informed consent; any sex; any age; more than a year of residence in the locations of interest; consumption of water from the municipal network; not receiving medication with either anti-thyroid effects or compounds containing nitrate salts. The following were specified as exclusion criteria: persons with an established diagnosis of congenital, secondary, or tertiary hypothyroidism; persons under treatment with radiation or chemotherapy in the neck region; persons who had any type of resection of the thyroid gland, thyroid cancer, or any diagnosed pathophysiology of the thyroid gland; syndromes or conditions that had any effect on the hypothalamic–pituitary–thyroid axis. All those fulfilling the inclusion criteria were tested for exposure biomarkers (plasma and urinary nitrite), an effect biomarker (percentage of MetHb), and thyroid function (thyroid profile). The genotypes corresponding to the single nucleotide polymorphisms (SNPs) rs965513 and rs1867277 of the FOXE1 gene were also determined. The presence of anti-thyroid peroxidase antibody (TPO-Ab) was determined in the subjects who were found to have SCH. Other variables, such as age, sex, and body mass index, were considered for a better description of the population under study.

2.2. Biological Sample Collection

Study participants were asked to fast for eight hours prior to the collection of a 10 mL peripheral blood sample and a 10 mL sample of the first urine void in the morning, with both samples collected in sterile containers. The blood sample collection was conducted using BD Vacutainer^® tubes containing 6 mL of coagulation activator for the recovery of the serum to be used for establishing both the thyroid profile and TPO-Ab levels. Tubes containing 4 mL of ethylenediamine tetraacetic acid (EDTA) were also used to measure plasma nitrite and MetHb levels and for the purification of genomic DNA. Once obtained, the samples were kept at −70 °C until processing.

2.3. Nitrate Exposure via Drinking Water

Although there is a distinction in the literature between low, medium, and high exposure, there is no consensus about the ranges and doses in each area. One strategy is to stratify according to the concentrations found in the specific study to observe the exposure gradient. In the study area, the nitrate levels in the water for human consumption were established by Gandarilla et al. [48], where the study areas were then stratified according to their exposure level. A low level was classified as 4.7 ± 3.3 mg/L (21 de Marzo), a medium level was classified as 32.1 ± 3.7 mg/L (San Jacinto and Juan E. García), and a high level as 56.9 ± 14.7 mg/L (La Loma and Nazareno). The number of subjects/families studied in each nitrate category area was as follows: low, 25/5; medium, 46/12; high, 31/9, respectively.

2.4. Measurement of Methemoglobin Percentage

The percentage of MetHb was established with the spectrophotometry method reported by Sakata et al. [49], using 100 μL of the blood collected in the EDTA tubes and following the manufacturer’s instructions (FAR Diagnostics, Verona, Italy) for measurements conducted via four readings at 630 nm (HACH-DR5000). The first reading (D1) was a hemolyzed preparation, and the second reading (D2) was conducted via the addition of sodium azide. The third (D3) corresponded to a hemolyzed preparation to which potassium ferricyanide was added. Finally, sodium azide was added for the fourth reading (D4). The percentage of MetHb was calculated with the following equation:

$$\mathrm{MetHb} \mathrm{percentage}=\left[\left(\mathrm{D}1-\mathrm{D}2\right)÷\left(\mathrm{D}3-\mathrm{D}4\right)\right]\times 100$$

Values > 1.5% of the. total MetHb were considered to be higher than the reference values. The reagents used complied with high purity standards (Merck KGaA, Darmstadt, Germany and/or its affiliates).

2.5. Nitrite Measurement

The nitrite concentration was determined, as a stable metabolite, for the plasma and urine samples, which were then deproteinized using absolute ethanol at a 1:7 ratio and centrifuged at 3500 rpm for 15 min. The nitrite levels in the plasma and urine were measured by subjecting 100 μL of supernatant to the Griess colorimetric method, as was reported by Miranda et al. [50], using spectrophotometric quantification (HACH-DR5000) at 540 nm. As a reference, a curve with sodium nitrite and a detection limit between 0.5–50 µmol/mL was performed in triplicate, and each sample was measured by duplicate. All reagents used were high purity (Sigma-Aldrich, Darmstadt, Germany).

2.6. Measurement of Thyroid Function

Thyroid function (TSH), total (tT3) and free T3 (fT3), and total (tT4) and free T4 (fT4) were measured by chemiluminescent immunoassay (IMMULITE^®1000 Siemmens™, Gwynedd, UK). The TSH assay had a sensitivity of 0.004 μIU/mL and an upper limit of 75 μIU/mL. The reference ranges for thyroid hormones were TSH, 0.4–4.0 μIU/mL, tT3, 82–179 ng/dL, fT3, 1–40 pg/mL, tT4, 4.5–12.5 μg/dL, and fT4, 0.3–6.0 ng/dL.

2.7. Measurement of TPO-Ab Concentration

For those individuals with TSH levels above the normal range, 100 μL of blood serum was used to conduct a sandwich-type enzyme-linked immunosorbent assay (ELISA) using a commercial TPO-Ab detection kit in accordance with the manufacturer’s instructions (Merck KGaA, Darmstadt, Germany and/or its affiliates). The levels of TPO-Ab+ were determined once values of >35 UI/mL were observed.

2.8. DNA Extraction and Genotypification of FOXE1

Genomic DNA extraction was performed using whole blood following the protocol described by Lahiri and Nurnberger [51], while DNA concentration and purity was established using the NanoDrop 2000 (Thermo Fisher Scientific Inc., Germering, Germany). The SNPs rs965513 and rs1867277 of FOXE1 were genotyped in the StepOne Real-Time PCR (polymerase chain reaction) (Applied Biosystems, Foster City, CA, USA), using hybridization probes (Integrated DNA Technologies rhAmp^®, ID Hs.GT.rs965513. G.1 and Hs.GT.rs1867277. G.1) in accordance with the manufacturer’s instructions.

2.9. Statistical Analysis

The data for 16 variables were compiled for 102 individuals, with the database including numerical and categorical variables. Measures of central tendency were analyzed for the description of the data, and Pearson’s correlation for quantitative variables with normal distribution. For non-normal distribution, the variables were standardized by subtracting the average corresponding to the variable from the value of each data point and then dividing by the standard deviation. The multivariate analysis comprised a correlation analysis applied solely for the 11 numerical variables, given that principal component analysis (PCA) requires that the variables used present some level of correlation. For this reason, only the variables with a level of significance greater than or equal to 0.90 were considered. Once the correlations among the variables were identified, a Mahalanobis distance analysis was undertaken to discard the data considered atypical for this set of variables. The PCA was carried out to show, firstly, the distribution of the individuals based on the SCH condition and, secondly, on the nitrate exposure according to the levels detected in the communities in which they live. A heatmap analysis was carried out to group the individuals according to the 11 numerical variables and, at the same time, to show the levels of correlation of each of the variables for each individual. The cluster analysis was undertaken using the Euclidean distance index and the Ward algorithm to construct the groupings, while genotype, subclinical condition, sex, and the NO₃-N level to which they were exposed were mapped for the individuals in the heatmap. Moreover, in order to reassign those individuals who had been classified with a non-SCH condition prior to the analysis, a discriminant function analysis was conducted to establish the percentage of correct classifications, which was then statistically corroborated via the Wilkins test. Finally, Kruskal–Wallis and chi-squared tests were performed to determine the differences between the different exposure scenarios, where applicable. All statistical and multivariate analyses were performed with the R software version 4.1.0 [52]; a p-value of <0.05 was considered statistically significant.

2.10. Ethical Considerations

This study was approved by the Ethics Committee of the Faculty of Chemical Sciences at the Juarez University of the State of Durango, with a unique assigned registration number of R-2017-123301538X0201-026. The study followed the guidelines, as pertaining to health research, stipulated in articles 13, 14, 17, 21, and 22 of the General Health Law in Mexico.

3. Results

The study population initially comprised 26 females who had been previously diagnosed with SCH and who described other cases in their families. The analysis was carried out on as many members of the subjects’ families as possible in order to explore the possible causes underlying the increased incidence of SCH in these rural populations that have been exposed to an endocrine-disrupting condition, such as nitrates through drinking water. Finally, the sample comprised 102 subjects with an average age of 22 years; 69% were women, 15% had no schooling, and 36% had been educated up to a lower secondary level, while 58% were overweight or presented some degree of obesity, and the majority worked in the home or were students. With regard to water consumption, 85.5% consumed water drawn from the public water system or from private wells, while only 14.5% occasionally consumed purified water from purification machines (see

Table 1. General characteristics of the total study population.

Variable		Subjects = 102 n (%)
Sex	Women	70 (69)
	Man	32 (31)
Age	Median (Min–Max) 22 (2–72)
Education	No schooling	15 (15)
	Primary education	37 (36)
	Middle school	36 (35)
	High school	10 (10)
	University	4 (4)
	Postgraduate	0 (0)
Body mass index (kg/m2)	Underweight	4 (4)
	Normal weight	39 (38)
	Overweight	28 (27)
	Class I obesity	20 (20)
	Class II obesity	5 (5)
	Class III obesity	6 (6)
Occupation	Housewife	56 (55)
	Seasonal farmer	8 (8)
	Student	32 (31)
	Other	6 (6)
Drinking water consumption	Public water system	63 (62)
	Well	24 (23.5)
	Purified	15 (14.5)
Consumption of water to prepare food	Public water system	70 (69)
	Well	26 (25)
	Purified	6 (6)
Boiled water for consumption	Yes	2 (2)
	No	100 (98)

).

The measurements of MetHb found 79% of subjects to fall outside the reference limit, while the average levels of nitrite in plasma and nitrite in urine were 24.62 µmol/mL and 3.46 µmol/mL, respectively. Regarding the parameters of thyroid function, 45% of subjects presented TSH levels over the reference limits (SCH), 49% had reduced tT3 levels, and 19.6% presented reduced tT4 levels. A total of 15.7% of the SCH cases were positive for the presence of TPO-Ab antibodies (see

Table 2. Determination of biomarkers of interest in the study population.

Parameters	n	Median (Min–Max)	Reference Rank	Out of Rank n (%)
MetHb (%)	102	2.44 (0.26–12.35)	>1.5	81 (79)
Nitrite in plasma μmol/mL	102	24.62(3.19–45.65)	>7	97 (95)
Nitrite in urine μmol/mL	102	3.46 (0.04–25.50)	>1	87 (85)
TSH μUI/mL	102	3.72 (0.49–95.49)	0.04–4.00	46 (45) †
tT3 ng/dL	102	89.70 (0.27–210.00)	82.00–179.00	51 (49) ‡
fT3 pg/mL	102	3.30 (1.00–10.20)	1.00–40.00	0 (0)
tT4 μg/dL	102	6.38 (1.30–23.10)	4.50–12.50	20 (20) ‡
fT4 ng/dL	102	1.17 (0.36–3.21)	0.30–6.00	0 (0)
TPO-Ab	38	1.00 (1.00–1268.70)	>35 UI/mL	6 (16) §

^§ The determination of TPO-Ab antibodies was only carried out in 38 of the 46 samples with a TSH level above the normal range. ^† Indicates increase, ^‡ indicates decrease.

).

Regarding the genotypic and allelic distribution of the SNPs, rs965513 and rs1867277 were determined for each exposure scenario to nitrates in water used for human consumption. The SNP rs1867277 was not found in the Hardy–Weinberg equilibrium (chi-square = 0.012), and its genotypic distribution across the exposure scenarios showed a statistically significant difference (chi-square = 0.019) (see

Table 3. Genotypic and allelic frequencies of polymorphisms in the FOXE1 gene observed in the study population, according to the exposure scenarios.

All Subjects n = 102		Low n = 25	Medium n = 46	High n = 31	p-Value
FOXE1 rs965513
Genotype
GG	47 (46%)	12 (48%)	25 (54%)	10 (32%)
GA	49 (48%)	12 (48%)	20 (43%)	17 (55%)	0.177
AA	6 (6%)	1 (4%)	1 (2%)	4 (13%)
Allele
G	143 (70%)	36 (72%)	70 (76%)	37 (60%)
A	61 (30%)	14 (28%)	22 (24%)	25 (40%)
HWE					0.080
FOXE1 rs1867277
Genotype
GG	34(33%)	4 (16%)	19 (41%)	11 (35%)
GA	61 (60%)	16 (64%)	26 (57%)	19 (61%)	0.019
AA	7 (7%)	5 (20%)	1 (2%)	1 (3%)
Allele
G	129 (63%)	24 (48%)	64 (70%)	41 (66%)
A	75 (37%)	26 (52%)	28 (30%)	21 (34%)
HWE					0.012

The p-value corresponds to the chi-squared test. HWE = Hardy–Weinberg equilibrium.

).

Table 4. Summary of events by scenario of exposure to nitrates through drinking water.

	Exposure Scenario (NO3-N mg/L)
Parameters	Low (4.7 ± 3.3)	Medium (32.1 ± 3.7)	High (56.9 ± 14.7)	p-Value
Families	5	12	9
All individuals	25	46	31
MetHb > 1.5% n (%)	21 (84)	32 (69.5)	28 (90.3)	0.042 †
NO2− in plasma > 7 μmol/mL n (%)	22 (88)	45 (97.8)	30 (96.7)	0.097 †
NO2- Urine > 1 μmol/mL n (%)	21 (84) a	43 (93.5) b	28 (90.3)	0.001 †
SCH cases n (%)	10 (40)	21 (45.6)	15 (48.4)	0.817 ‡
FOXE1 Risk genotype AA + SCH
rs965513 n (%)	0 (0)	0 (0)	1 (3)	0.314 ‡
rs1867277 n (%)	2 (4)	0 (0)	0 (0)	0.043 ‡
TPO-Ab+ > 35 UI/mL n (%)	1 (10)	3 (14.2)	2 (13.3)	0.899 ‡

Note: The percentage of the parameters is based on the total number of subjects per exposure scenario. ^‡ chi-squared test; ^† Kruskal–Wallis test, with Bonferroni post-hoc test (p < 0.05), shown with superscript letter: ^a low–high exposure, ^b medium–high exposure.

shows a summary of the alterations observed in some variables, such as exposure and effect biomarkers for nitrates (measurement of nitrites), thyroid dysfunction (SCH), the genotypic frequency of the SNPs, and the presence of TPO-Ab as an autoimmune factor. Differences were observed in the levels of both nitrates and the exposure (nitrite in urine p = 0.001) and effect (MetHb p = 0.042) biomarkers, while SCH was observed for all levels of chronic exposure (chi-square = 0.817). The SNP rs1867277 only presented in the low exposure scenario, corresponding to 4% (chi-square = 0.043), and, finally, the TPO-Ab percentage was highest in the medium exposure scenario, with 14.2% (see Table 4).

The statistical analysis compiled the information of 16 variables for the 102 individuals included in the study, while the Pearson correlation analysis only included the continuous quantitative variables. This analysis was performed using the package RcmdrMisc version 2.7.1 [53] in the R software version 4.1.0 [52]. Positive correlations between age and BMI (r = 0.62, p ≤ 0.001), tT3 and fT3 (r = 0.51, p ≤ 0.001), and tT3 and tT4 (r = 0.50, p ≤ 0.001) were found, suggesting a moderate positive relationship. Negative correlations were observed between age and fT3 (r = −0.43, p = 0.001), BMI and fT3 (r = −0.31, p = 0.01), TSH and tT4 (r = −0.36, p = 0.001), TSH and fT4 (r = −0.41, p = 0.001), tT3 and MetHb (r = −0.43, p = 0.001), and fT3 and MetHb (r = −0.34, p = 0.001). All other correlations did not show significant differences (see Figure 2).

The PCA was performed using the packages ggplot2 version 3.3.5 [54], ggrepel version 0.9.1 [55], factoextra version 1.0.7 [56], FactoMineR version 2.4 [57], paran version 1.5.2 [58], and nFactors version 2.4.1 [59], and revealed that the three first components represent 63.3% of the variability, and the variables that most contributed to the analysis of the first component were tT3, fT3, and tT4T (see Figure 3A), while the main variables in the second component were age and BMI (see Figure 3B). Most of the individuals presenting a SHC condition were identified by increases in the NO₂^- levels in the blood, BMI, age, TSH level, and MetHb levels, while a greater relationship was found between those individuals with a non-SCH clinical condition and increases in the variables tT3, tT4, fT3, and, mainly, fT4. When the nitrate exposure scenarios were classified, the PCA analysis did not identify a clear condition but did establish that high levels of MetHb are more related to those study locations presenting high levels of nitrate in potable water (see Figure 3).

Additionally, both multivariate conglomerate and heatmap analyses were undertaken with group subjects according to the numerical variables and, at the same time, the levels of correlation were presented for each of the variables for each individual. The conglomerate analysis was carried out using the Euclidean distance index and the Ward algorithm to construct the groupings, while the heatmap analysis mapped the subject’s genotype and SCH condition, and the level of NO₃-N was carried with the package pheatmap version 1.0.12 [60] (see Figure 4).

Finally, the discriminant function analysis revealed 81% accuracy in the classification of the subjects. This contrasts with the a priori group, where discriminant function analysis showed that four out of the 95 subjects (numbers 70, 73, 74, and 76) from this group should be classified as likely to develop SCH if the levels for all the numerical variables do not decrease. The discriminant function analysis was statistically corroborated with the Wilkins test.

4. Discussion

The present study is the first to investigate evidence on the possible direct relationship between chronic nitrate exposure via the consumption of contaminated water and alterations in thyroid function in rural areas of Durango, Mexico.

As widely described in the literature, human exposure to nitrates and nitrites occurs endogenously due to the metabolism of NO [61]. It also occurs exogenously in the diet via different concentrations in drinking water and green leafy vegetables [62], food preservatives or flavorings in processed foods [63], and food supplements, such as potassium nitrate and sodium nitrate [64]. It is also present in medications that are mainly used to treat cardiovascular diseases [65]. The fact that the endogenous metabolism produces a lower amount of nitrates and nitrites than that produced by the diet [66] should be noted, as well as the subjects’ null ingestion of food supplements. Therefore, the main source of nitrate exposure in the study population is attributable to the consumption of water contaminated with these compounds.

While few studies have established base levels of nitrates in the organism, Mikiwa et al. [67] found nitrate and nitrite levels of 36.6 ± 18.5 µmol/L and 6.4 ± 2.1 µmol/L, respectively, in the plasma of a sample of ten healthy subjects of Japanese origin. Pimková et al. [68] found nitrite and nitrate levels of 1149 ± 86 nmol/L and 32.78 ± 10.33 µmol/L, respectively, in the plasma of a sample of 23 healthy subjects in the Czech Republic, while Lundberg and Weitzberg [21] reported normal plasma levels of nitrates and nitrites ranging from 20–40 µmol/L and 50–300 nmol/L, respectively. In contrast, the present study obtained blood and urine nitrite levels of 23.95 ± 9 µmol/mL and 4.90 ± 4.90 µmol/mL, respectively, which may be related to the degree of exposure to nitrate in contaminated drinking water.

The physiological level of MetHb ranges from 0–1% [69] to 2% [70]. The lactating population is more prone to developing this condition because of the low level of MetHb reductase activity, while the ease with which fetal hemoglobin is oxidized and its more acidic gastric pH enable the intestinal microbiota to reduce the amount of nitrate ingested into nitrite. High MetHb percentages were observed in this study; 79% of individuals analyzed presented levels over 1.5%, with an average of 2.80 ± 1.88.

Methemoglobinemia is a condition characterized by an abnormally high level (>40%) of MetHb, which is mainly produced by acute exposure to oxidizing compounds, pharmaceuticals, and most chemical agents [71]. However, in this study, the highest MetHb percentage was 12.35%, but the questionnaire information indicates that no prior contact with any medication or chemical agent could explain these high MetHb levels.

Another health aspect of interest to the present study is the state of thyroid function in the presence of chronic nitrate exposure. Nitrate, together with other sodium–iodide symporter inhibitors, is able to alter thyroid function [72], leading to it being suggested as a possible thyroid disruptor in humans [24,36]. In contrast to the findings obtained by Van Maanen et al. [32], Tájtaková et al. [33], Gatseva and Argirova [31], and Ward et al. [29], our results do not provide data on abnormal growth, thyroid nodules, or the presence of any type of thyroid carcinoma. Moreover, while they do not concur with the reduced TSH levels and increased thyroid hormone levels found by the foregoing studies, they do show altered thyroid function after chronic nitrate exposure via potable water, ranging from 4.7 ± 3.3 mg/L to 56.9 ± 14.7 mg/L. The alteration found in the present study corresponds to SCH and occurs due to the increased levels of TSH (up to 45%) and a reduction in tT4 and tT3 (up to 49% and 19.6%, respectively), a finding concurring with that reported by Aschebrook-Kilfoy et al. [34]. Another significant finding of the present study is the 85% urinary nitrite detected, which indicates that the study population exceeds the baseline (1 µmol/mL) and, thus, that high levels of these nitrogenated compounds are excreted by the kidney in the presence of high nitrate concentrations in drinking water. Van Maanen et al. [32] observed an increase in urinary nitrate, which corresponded to the increased nitrate concentration in the drinking water of the study population, thus showing a dose–response relationship.

While previous studies have indicated the relationship between high levels of nitrates in potable water and altered thyroid function, others have shown that the relationship is not completely established, and structural or functional changes in the thyroid gland have not been found [73]. In contrast, the present study identified nitrate concentrations in potable water of up to 56.9 ± 14.7 mg/L, which is considered to be chronic exposure (≥1 year), and further, identified the presence of other conditioning factors for the development of SCH.

The prevalence of SCH found by the present study was 45%, which exceeds the national and global prevalence of 8% and 10%, respectively [45,74], and remains high even under the scenarios of low (40%), medium (45.6%), and high (48.4%) exposure. Hashimoto’s thyroiditis (a form of chronic lymphocytic thyroiditis or chronic autoimmune thyroiditis) is characterized by the autoimmune destruction of the thyroid gland, which leads to epithelial cell apoptosis and diffuse lymphocytic infiltration due to the action of specific B and T cells. Moreover, it also causes follicular destruction [75], which leads to reduced levels of thyroid hormone synthesis. This chronic autoimmune thyroiditis is the main cause of both clinical and subclinical primary hypothyroidism in areas with sufficient or excessive iodine content [74,76]. This is accompanied by the presence of anti-thyroid antibodies, mainly TPO and anti-thyroglobulin antibodies, which are considered biomarkers of thyroid gland damage [77]. In contrast to the 60% established by Bromińska et al. [78] and Malathi et al. [79] and the 50% established by Jayashankar et al. [80], the present study found an SCH level of 15.7% in the presence of TPO-Ab (>35 UI/mL). Therefore, autoimmune disorders would not be the main cause of SCH in the families of the study population. Although the exact etiology of the development of Hashimoto’s thyroiditis remains to be established, the interaction among genetic susceptibility factors, nutritional factors, and environmental triggers may be involved [81].

Alterations in the synthesis and secretion of thyroid hormones may be a consequence of a perturbation in the expression of the genes encoding thyroid transcription factors or the presence of genetic variations [82]. The gene FOXE1 plays an important role in the growth and development of thyroid glands and the proliferation and differentiation of follicular thyroid cells and acts as a regulator of cell function, growth, and differentiation [83]. Two of its polymorphisms, rs965513 [A] and rs1867277 [A], contribute independently to establishing a predisposition for the development of papillary thyroid cancer [84]. Similarly, the presence of the allele of the rs965513 polymorphism impacts thyroid function, reducing the levels of TSH and fT4 and increasing fT3 levels [85], and is associated with the development of hypothyroidism and goiter [86]. Furthermore, the presence of the allele of the rs965513 polymorphism participates in the recruitment of the USF1/2 factors by the FOXE1 promoter, resulting in an alteration in the state of the FOXE1 gene expression [87]. Furthermore, it has been found that genetic variations in FOXE1, including rs965513 and rs1867277, are risk factors associated with increased susceptibility to differentiated thyroid cancer [88]. In terms of the results of the present study, the frequency of the occurrence of the polymorphic allele in the study population was 30% for rs965513 [A] and 37% for rs1867277 [A], which is in line with the global (rs965513 [A] = 20% and rs1867277 [A] = 31%) and national (rs965513 [A] = 26% and rs1867277 [A] = 29%) levels. Considering that only the AA genotype of both polymorphisms could be the cause of thyroid function alterations, the present study found the presence of the genetic variants rs965513 and rs2200733 in only 3 and 4%, respectively, of the SCH cases found, thus reducing the impact of genetic factors in the study population.

Increased BMI is one correlation plausibly connected to the presence of SCH, in that hypothyroidism and SCH provide the conditions for reduced metabolic function, alterations in thermogenesis, and processes involving the metabolism of both glucose and lipids. This translates into weight gain over the long term once the level of thyroid hormone synthesis begins to decrease [89]. Given the foregoing, it is possible that the correlation between age and BMI found in the present study may reveal that the older a subject, the greater their exposure to nitrates and, consequently, the greater the alteration in metabolic processes, thus resulting in weight gain. However, we also consider that the correlation would be affected by other factors, such as nutritional variation, sedentarism, and even the inherent relation with growth.

It should be noted that some studies indicate a relationship between an increased BMI and increased thyroid hormone (fT3) levels [90,91], while others maintain that changes in the serum thyroid hormone (f4T) may cause increases in BMI [92]. The foregoing is the opposite of the negative correlation found in the present study between BMI and fT3 levels. We consider that high nitrate exposure would play an important role in the thyroid profile and requires attention in the study population.

In terms of the relationship observed between age and thyroid profile, the results of the present study provide evidence of a negative and statistically significant correlation. However, the production of hormones regulated by the endocrine system, including the thyroid hormones, decreases due to aging, which causes general morphological and physiological changes [93]. Nevertheless, the importance of the chronic presence of nitrate as an inhibitor of iodine uptake in the thyroid gland cannot be understated.

It is of the utmost importance to highlight that the following environmental characteristics of the rural region in which the present study was carried out are problems with real impacts on human health: intermittent rainfall; soil/aquifer contamination via inorganic nitrate and other compounds; over-fertilization; over-use of land for forage crops; intensive livestock and agricultural practices. These problems are not only present in rural areas of Durango, Mexico, they are also public health and environmental problems in other regions of the world [94].

As discussed previously, the development of SCH may have a multifactorial origin. However, this study suggests that the SCH observed in the population may be due to chronic exposure to high levels of nitrate in water for human consumption, which alters thyroid function.

Some of the limitations of this study include the lack of urinary iodine quantification; even though it is considered a good indicator of the nutritional iodine status in humans, it does not provide direct information on thyroid function. In addition, the distribution of iodized salt in the general population and the absence of congenital hypothyroidism would indicate that the population has an adequate source of iodine intake. It is necessary to designate more robust study types, such as case-controls or cohorts, in order to investigate and specify the factors that condition the presence and development of SCH in the analyzed population.

5. Conclusions

The results of this study suggest that environmental factors have a direct and significant influence on thyroid dysfunction due to chronic exposure to nitrates in drinking water. Therefore, the high frequency of SCH observed in the study population could be due to nitrate contamination of drinking water and not a response to an additive effect and other causal factors. This study analyzed some concentrations of nitrate, within permissible limits, that can be found in drinking water and suggests that exposure to high concentrations of nitrates during critical periods of development can have significant long-term consequences. It is necessary to know the role that nitrate in drinking water plays as an endocrine disruptor. Our findings contribute to the implementation of public health measures that seek to guarantee access to water resources of adequate quality, in addition to raising awareness in the rural population of Durango about the risk of consuming water with a high content of nitrates.