Effect of exposure to p,p'-DDE on male hormone profile in Mexican flower growers
- Julia Blanco-Muñoz1,
- Marina Lacasaña2,3,
- Clemente Aguilar-Garduño2,
- Miguel Rodríguez-Barranco2,
- Susana Bassol4,
- Mariano E Cebrián5,
- Inmaculada López-Flores2,3,
- Isabel Ruiz-Pérez2,3
- 1Instituto Nacional de Salud Pública de México (INSP) (National Institute of Public Health of Mexico), Cuernavaca, Morelos, Mexico
- 2Escuela Andaluza de Salud Pública (EASP) (Andalusian School of Public Health), Campus Universitario de la Cartuja, Granada, Spain
- 3CIBER Epidemiología y Salud Pública (CIBERESP) (CIBER of Epidemiology and Public Health), Spain
- 4Facultad de Medicina de Torreón, Universidad Autónoma de Coahuila (School of Medicine of Torreón, Autonomous University of Coahuila), Torreón, Coahuila, Mexico
- 5Centro de Investigación y de Estudios Avanzados (CINVESTAV), Instituto Politécnico Nacional (IPN) (Center for Advanced Studies and Research of the National Polytechnic Institute), México DF, Mexico
- Correspondence to Marina Lacasaña Navarro, Escuela Andaluza de Salud Pública (EASP) (Andalusian School of Public Health), Campus Universitario de la Cartuja, c/Cuesta del Observatorio 4, CP 18180 Granada, Spain;
- Accepted 11 April 2011
- Published Online First 10 May 2011
Objectives p,p'-Dichlorodiphenyldichloroethene (p,p'-DDE) acts as an androgen receptor antagonist, however data regarding its hormonal effects in men are limited. The objective of this study was to evaluate the association between serum levels of p,p'-DDE and reproductive hormone profile in Mexican male flower growers.
Methods A longitudinal study was carried out in a population of men working in the production of flowers and ornamental plants in two Mexican states during July–October 2004 (rainy season) and December 2004–May 2005 (dry season). A questionnaire including information on socioeconomic characteristics, tobacco and alcohol use, presence of chronic and acute diseases, occupational history and anthropometry was used and blood and urine samples were obtained. Serum levels of p,p'-DDE were analysed by gas chromatography; FSH, LH, testosterone, oestradiol, inhibin B and prolactin levels were measured by enzymatic immunoassay. Urinary levels of dialkylphosphates (DAPs) were analysed by gas chromatography. Associations between serum levels of p,p'-DDE and male reproductive hormones (both transformed to their natural logarithm) were evaluated using multivariate generalised estimating equation (GEE) models.
Results Median p,p'-DDE levels were 677.2 ng/g lipid (range 9.4–12 696.5) during the rainy season and 626.7 ng/g lipid (range 9.4–13 668.1) during the dry season. After adjusting for potential confounders (age, body mass index, state of residence and DAPs), p,p'-DDE levels were negatively associated with prolactin (β=−0.04; 95% CI −0.07 to −0.008) and testosterone (β=0.04; 95% CI −0.08 to 0.005) and positively with inhibin B (β=0.11; 95% CI 0.02 to 0.21).
Conclusion These results indicate that p,p'-DDE can affect hypothalamic–pituitary–gonadal axis function in humans.
- p,p' DDE
- male hormonal profile
- flower growers
- endocrine disorders
- male reproduction
What this paper adds
p,p'-DDE, the main metabolite of DDT, blocks the union of the androgen and its receptor, acting like a potent antiandrogen.
Studies evaluating the effect of p,p'-DDE on male reproductive hormones are scarce and shown inconsistent results.
This paper provides additional evidence that p,p'-DDE acts as an endocrine disruptor and supports the hypothesis that exposure to this compound can interfere with hypothalamic–pituitary–gonadal axis function and alter the male reproductive hormone profile.
Dichlorodiphenyltrichloroethene (DDT) is a broad spectrum synthetic insecticide used since the 1940s for pest control in agriculture and in homes. Its high persistence in the environment and its known toxicity for some animal and vegetable species1 2 led to its use being generally discontinued worldwide in the 1970s and 1980s, although it has continued to be used for malaria control in countries where this problem is endemic. In Mexico, DDT was used for agricultural pest control until 1991, and for malaria control until 1999 before its use was restricted in 2000.3
Technically, DDT is a mixture of two isomeres, the p,p'-DDT (85%) isomere and the o,p'-DDT (15%) contaminant isomere, with traces of o,o′-DDT.4 DDT is almost completely metabolised and the greatest fraction is transformed into p,p'-DDE, with a small percentage remaining as o,p'-DDT.5 DDT and its metabolites are highly persistent and lipophilic compounds that bioaccumulate and increase in the food chain so that, even today, it is possible to detect residues in most human populations.6 7
Recent research has shown that DDT and its metabolites are able to act as endocrine disruptors. The o,p'-DDT isomere and its derivative, o,p'-DDE, are compounds that have oestrogenic activity,8 while the main metabolite, p,p'-DDE, behaves like a potent antiandrogen, in vitro and in vivo, since it blocks the union of the androgen and its receptor and inhibits the transcriptional activity induced by the androgen and its effect on target organs.9 10 Also, some studies have found an association between levels of p,p'-DDE and poor seminal parameters: decreased sperm motility, decreased sperm concentration and increased damage to sperm DNA,11 increased frequency of testicular cancer12 and congenital genital malformations.13 These effects constitute the so-called testicular dysgenesis syndrome and, although the underlying biological mechanisms have still not been elucidated, some authors have suggested that it is due to a hormonal imbalance in the male reproductive system due to exposure to endocrine disruptors during fetal life.14
If DDT and its metabolites can act as endocrine disruptors, it would be expected that they would interfere with the normal hypothalamic–pituitary–gonadal axis. However, studies evaluating the relationship between p,p'-DDE levels and the male hormonal profile are scarce and show inconsistent results. Thus, Martin et al15 found a 23% decrease in total testosterone levels and 22% decrease in the free androgen index in male African-American farmers with p,p'-DDE serum levels in the top 10th percentile, compared with all others. Similar results were reported in young men in a Mexican region where DDT was used intensively in campaigns against malaria.16 A negative association was also found between p,p'-DDE serum levels and oestradiol in Swedish men17 and Thai men,18 and a positive association with SHBG, LH and FSH.19 In contrast, other investigations did not corroborate these findings.20 21
The objective of this study was to evaluate the association between p,p'-DDE serum levels and the male hormonal profile in a group of Mexican men who were employed in floricultural enterprises, where agricultural practices are intensive and DDT was used until relatively recently. Levels of dialkylphosphates (DAPs) in urine were adjusted for since organophosphate pesticides used in the floricultural industry can also act as endocrine disruptors.22–24
Materials and methods
A longitudinal study was carried out in a population of men working in the production of flowers and ornamental plants in two Mexican states (Morelos and the State of Mexico) during July–October 2004 (rainy season) and December 2004–May 2005 (dry season). These periods correspond to the two main agricultural periods in which large quantities of pesticides are sprayed (rainy season) or are used less heavily (dry season). Workers were chosen using the employee records of 57 companies, most of which use traditional production systems, including the regular use of chemical pesticides, except for one company that uses organic production methods (biological pest control). These workers performed different activities with different levels of exposure to pesticides, from administrative tasks to pesticide application. The inclusion criteria were age between 18 and 52 years and having been in the job for at least 6 months. Men with a prior diagnosis of infertility and endocrine or chronic diseases (thyroid disease, diabetes mellitus, liver disease, renal insufficiency, cancer) were excluded from the study. Using a selection questionnaire, 143 eligible workers were identified during the rainy season, informed about the objectives of the study and invited to participate; of these, 136 (95%) agreed to participate and signed informed consent forms. Later, they completed the questionnaire and gave urine and blood samples. Eighty four workers (62%) provided biological samples again during the dry season.
A questionnaire was completed by floriculture workers on sociodemographic characteristics (age, education, family income, marital/cohabitation status), clinical and surgical history, alcohol and tobacco consumption, work history, characteristics of floricultural work (activity, hours/day, place of work: outdoors or in a greenhouse) and exposure to pesticides or other chemical products in the home.
Trained nursing personnel who had no knowledge of the study hypothesis administered the questionnaires and also carried out anthropometric measurements (weight and height).
The study was approved by the Ethics Committee of the National Institute of Public Health of Mexico.
Blood samples (10 ml) were taken in fasting conditions (between 08:00 and 09:30 h) using non-heparinised Vacutainer tubes. Samples were centrifuged for 10 min at 2500 rpm and serum was kept at –70°C in Eppendorf phials and glass phials prewashed with hexane grade pesticide and covered with a Teflon cap (for reproductive hormones and DDT metabolites quantification, respectively) until analysis. Also, a single first morning urine sample (before 08:00 h) was self-collected by workers at home in a supplied container and kept at –20°C until analysis (for organophosphate metabolites quantification).
Levels of p,p'-DDE were measured in serum by means of gas chromatography with an electron capture detector (model 3400; Varian, Palo Alto, California, USA), following the protocol recommended by the US Environmental Protection Agency (1980). Concentrations were reported in lipid basis as ng/g and in wet basis as ng/ml; detection limits were 9.39 ng/g and 0.0125 ng/ml, respectively. Total lipids in serum were determined using a colorimetric method kit (Randox Laboratories Ltd, Antrim, UK).
For internal quality control, each serum sample was spiked with 6 ng/μl aldrin and the average recovery was 98.15±8.8%. For every 10 study samples, one sample of bovine serum with known quantities of p,p'-DDE, β-hexachlorocyclo-hexane (β-HCH), aldrin, hexachlorobenzene (HCB) and 1,1-dichloro-2,2-bis(p-chorophenyl)ethane (p,p'-DDD) was analysed, with recovery of 103.4%, 100.8%, 100.01%, 100.91% and 104.1%, respectively. Additionally, one randomly selected sample was analysed in duplicate in each batch, with a coefficient of variation of 4.37% for p,p'-DDE.
Organophosphate metabolites in urine
Exposure to organophosphate pesticides was assessed by measuring six DAPs in urine: dimethylphosphate (DMP), dimethylthiophosphate (DMTP), dimethyldithiophosphate (DMDTP), diethylphosphate (DEP), diethylthiophosphate (DETP) and diethyldithiophosphate (DEDTP). Extraction of DAPs from urine was performed according to the method proposed by Ueyama.25 Compounds were determined by gas–liquid chromatography using a flame photometric detector equipped with a filter to isolate phosphorous emissions.
The dimethyl (DMP, DMTP and DMDTP) and diethyl (DEP, DETP and DEDTP) metabolite concentrations were converted to their molar concentrations (μmol/l) and summed to produce a total DAP (Σ DAP) concentration for each sample.26 Metabolite concentrations were adjusted using creatinine concentration to correct for variable urine dilutions. Urinary creatinine concentration was determined by spectrophotometry using a commercial kit (Randox Creatinine Kit).
Analyses of p,p'-DDE and DAP metabolites were performed at the Laboratory of Toxicology of CINVESTAV.
Reproductive hormones in serum
The pituitary hormones (FSH, LH and prolactin), steroid hormones (testosterone and oestradiol) and inhibin B were quantified at the School of Medicine of Torreon, Autonomous University of Coahuila.
Testosterone was measured using the DE2300 ELISA Kit (R&D Systems, Minneapolis, Minnesota, USA), which has a sensitivity of 3.8 pg/ml and inter-assay and intra-assay coefficients of variation (CV) of 9.3 and 7.8%, respectively. Inhibin B was measured using a commercial, double antibody, enzyme-linked immunosorbent assay (DSL-10-84100 ACTIVE Inhibin B ELISA; Diagnostic Systems Laboratories, Webster, Texas, USA) with inter-assay and intra-assay CVs of 7.6 and 4.6%, respectively, with a sensitivity of 10 pg/ml. Oestradiol was also measured using a commercial double antibody enzyme-linked imnunosorbent assay (no. 2046z; Diagnostic Automation, Calabasas, California, USA) with inter-assay and intra-assay CVs of 6.6 and 4.9%, respectively, and a sensitivity of 10 pg/ml. LH, FSH and prolactin were analysed using an ELISA solid phase enzyme amplified sensitivity immunoassay, performed on BioSource (Invitrogen, Carlsbad, California, USA) microtitre plates (nos. KAQ1311, KAQ0841 and KAQ1441, respectively); the detection limits for LH, FSH and prolactin were 0.1 mIU/ml, 0.15 mIU/ml and 7.6 μIU/ml, respectively, with inter-assay CVs of 6.0, 8.9 and 7.1%; the intra-assay CVs were 4.9, 4.2 and 4.6%, respectively. All absorbances were measured spectrophotometrically using a PerkinElmer 35 (PerkinElmer, Waltham, Massachusetts, USA) and a Dynatech MR5000 spectrophotometer (Dynatech, Burlington, Massachusetts, USA).
The general characteristics of the studied population were described with number and proportions of individuals in each category of selected variables. The χ2 test, Fisher's test and Mann–Whitney non-parametric test were used to determine differences between data from workers who provided information and biological samples in both agricultural seasons and those who withdrew from the study during the dry season.
Distributions of p,p'-DDE, total DAP and serum FSH, LH, prolactin, testosterone, inhibin B and oestradiol levels were compared between the agricultural periods (rainy vs dry season) by means of Wilcoxon's test. Percentiles are presented to characterise data dispersion.
The Kolmogorov–Smirnov test showed that the distribution of the six evaluated hormones was not normal, so they were transformed into their natural log (ln) for the subsequent statistical analyses. The independent variable (p,p'-DDE) was also log-transformed because it had a strongly asymmetric distribution. Associations between individual p,p'-DDE and hormone levels were estimated by means of a generalised estimating equation (GEE) model.
Potential confounders including age (as a continuous variable), body mass index (BMI; as a continuous variable), tobacco consumption (non-smoker, past-smoker, current smoker), alcohol consumption (<30 g/day, >30 g/day, non-drinker), years as a floriculture worker (<5, 5–10, 11–20, >20), residential pesticide exposure (yes or no), living near an industrial site (yes or no), place of residence (the State of Mexico or Morelos) and total DAP urine levels (as a continuous variable), were selected based on their biological plausibility and on current scientific knowledge. Univariate GEE models were used to evaluate associations between potential confounders and serum hormone concentrations. Multivariate adjusted GEE models were further calculated and potential confounders were considered in the adjusted models if they were related to the outcome, based on Wald tests, with a p value of <0.20, or if their inclusion changed the parameter estimated for the association between p,p'-DDE and hormone concentration by >10%. Finally, to evaluate non-linear relationships between p,p'-DDE and hormones, we regressed the hormones on quartiles of p,p'-DDE. To improve interpretability of the association, β regression coefficients were expressed as percentages of change in the hormone concentrations for interquartile increase in p,p'-DDE levels.
All statistical analyses were conducted with Stata v 8.2 and SPSS v 15.0. We rejected the null hypothesis when the p value was <0.05.
Information and biological samples were provided by 136 floriculture workers during the rainy season, and by 84 during the rainy and dry seasons. Most of workers were from Morelos (76%) and more than 50% had completed junior high school, had a history of working in floriculture for more than 5 years and reported different amounts of tobacco or alcohol consumption. No difference was observed in age distribution, BMI, tobacco and alcohol consumption, years as a floriculture worker or the condition of living near an industrial site, between the men providing samples in the rainy and dry seasons. Residential paint application was higher during the dry season, while residential pesticide use was lower (see online supplemental file 1).
There were no significant differences between the 84 workers who provided biological samples during the two seasons and those who withdrew from the study during the dry season (52 individuals) for most of the general characteristics, except regarding years spent as a floricultural worker. Moreover, there were no differences in the levels of biomarkers of exposure (p,p'-DDE and DAPs) and effect (FSH, LH, prolactin, testosterone, inhibin B) except in the serum levels of oestradiol (table 1).
Concentrations of p,p'-DDE were similar across the two seasons, whereas concentrations of total DAP metabolites (Σ DAPs) were higher in the rainy season than in the dry season. Also, levels of FSH, prolactin and oestradiol were significantly higher during the rainy season than during the dry season, whereas testosterone and inhibin B were significantly lower during the rainy season. There was no difference between seasons in LH serum level (table 2).
In the univariate analysis, the floriculturists from the State of Mexico had significantly lower levels of prolactin and inhibin B than those from Morelos; age was associated with an increase in FSH and with a decrease in prolactin and inhibin B levels; BMI was associated with decreased inhibin B levels; current smoking was associated with increased testosterone; years working in floriculture were associated with decreased prolactin levels; residential paint was associated with increased testosterone and decreased prolactin, while residential pesticide usage increased prolactin and decreased inhibin B levels; DAP urine levels were associated with an increase in FSH and prolactin and with a decrease in testosterone and inhibin B levels. Age was positively associated with p,p'-DDE serum levels (table 3).
In the univariate analysis, serum levels of p,p'-DDE were significantly associated with a decrease in testosterone (β=−0.04; 95% CI −0.08 to −0.0001) and prolactin (β=−0.04; 95% CI −0.08 to −0.009) levels, and with a marginally significant increase in levels of inhibin B (β=0.09; 95% CI −0.007 to 0.19). Similar results were obtained after adjusting for age, BMI, state of residence and total levels of DAPs in urine, although the negative association between p,p'-DDE and testosterone was marginally significant (β=−0.04; 95% CI −0.08 to 0.005) and the positive association between p,p'-DDE and inhibin B reached statistical significance (β=0.11; 95% CI 0.02 to 0.21). No significant associations were found between p,p'-DDE and FSH, LH and oestradiol (table 4).
When we performed the analysis in which serum hormone levels (untransformed) were regressed on quartiles of p,p'-DDE, a decreasing trend in testosterone (figure 1A), a non-significant decreasing trend in prolactin (figure 1B) and an increasing trend in inhibin B (figure 1C) were observed. Compared with workers with p,p'-DDE levels in the first quartile, workers with p,p'-DDE in the fourth quartile showed a decrease of 17% in testosterone serum levels, a decrease of 9% in prolactin serum levels and an increase of 60% in inhibin B serum levels (figure 1A–C).
We found that serum p,p'-DDE levels were negatively associated with serum levels of prolactin and testosterone, and positively associated with levels of inhibin B. The results of this study support the hypothesis that DDT and its metabolites may act as endocrine disruptors in humans, affecting the serum levels of male hormones.
Studies carried out in vitro and in animal models suggested that the effects of exposure to organochlorinated compounds on the male reproductive system and on seminal parameters could be mediated, at least partially, by their endocrine disruptive ability.10 In the case of p,p'-DDE, these effects would be mainly mediated by their anti-androgenic potential, since p,p'-DDE binds to the androgen receptor. In humans, p,p'-DDE also behaves like an antagonist of the androgen receptor by blocking the hormone effect on target organs. Therefore, taking into account the physiology of the hypothalamus–hypophysis–gonadal axis, an increase in the secretion of gonadotropin-releasing hormone by the hypothalamus could be expected, which in turn would increase the secretion of hypophysiary gonadotropins, specifically LH, which would stimulate the Leydig cells in the testes to increase testosterone secretion.15 However, our results do not support this hypothesis, since we observed a decrease in total testosterone levels and did not observe an effect on LH. These results are consistent with those reported by Martin et al15 who found that higher levels of DDE were associated with lower levels of total testosterone in African-American farmers. Ayotte et al16 found a negative association between p,p'-DDE levels and bioavailable/total testosterone ratio in Mexican men living in the State of Chiapas, a region where malaria is endemic and where DDT was widely used in campaigns to eradicate the Anopheles mosquito until 1999. However, other authors have not found an association between p,p'-DDE levels and the male hormonal profile.20 21
The inconsistencies in the results obtained from the different studies may be due to differences in levels of exposure. In our population, p,p'-DDE levels (median 643 ng/g) were higher than those reported by Hagmar et al20 and Cocco et al21 with medians of 240 ng/g and 396 ng/g, respectively, who did not observe an association, and lower than those reported by Ayotte et al16 and Martin et al15 with 77 900 ng/g and 1213 ng/g, respectively. Thus, p,p'-DDE effects on the male hormonal profile may be dose-dependent as show by Giwercman et al19 who investigated the effect of p,p'-DDE exposure on the male hormonal profile in four cohorts (Greenland, Sweden, Kharkiv and Warsaw) and found that p,p'-DDE levels were associated with an increase in free testosterone in men in Greenland and Kharkiv (where p,p'-DDE levels were higher), while a non-significant decrease was found in this hormone in cohorts in Sweden and Warsaw. In the same study, the authors found a negative association with inhibin B levels in Kharkiv and a positive association, like ours, in Greenland. Inhibin B secretion seems to be a useful marker to explore potential Sertoli cell toxicants, however there is very limited information available on the effect of exposure to DDT and its metabolites on serum inhibin B; the in vitro response of immature rat Sertoli cells to DDT shows no effect on inhibin B secretion,27 while p,p'-DDE could inhibit the expression of this hormone.28
Consistent with our results, previous studies did not find any association between p,p'-DDE and oestradiol serum levels in adult men.19 21 However, two studies among Swedish and Thai men reported decreased oestradiol levels with increasing p,p'-DDE,17 18 while You et al29 found that hepatic aromatase, the enzyme involved in the conversion of testosterone to oestradiol, in adult male rats treated with DDE was greatly increased although the difference in serum 17β-oestradiol between treated animals and controls was not statistically significant.
On other hand, prior epidemiological studies that have evaluated the effect of p,p'-DDE on the male hormonal profile in humans, do not report information on prolactin. In rats, exposure to chlordecone, an organochlorinated pesticide, was associated with a decrease in prolactin levels.30 Also, in the male glaucous gull, a predator whose natural habitat is the Arctic, and who due to its diet has high levels of organic halogenated compounds (mainly organochlorinated and organobrominated compounds), it was found that basal prolactin levels tended to vary negatively when levels of these contaminants were increased.31 We found that the increase in p,p'-DDE serum levels was associated with a significant decrease in prolactin serum levels. This hormone allows the expression of LH in its receptors at the Leydig cells and a decrease in its levels could explain, at least partially, the negative association found between p,p'-DDE and testosterone. The effect of p,p'-DDE on prolactin could be mediated by its action at the central nervous system, specifically on the dopaminergic system, which is involved in the suppression of prolactin secretion by the anterior hypophysis. Studies performed in animal models have shown that exposure to some organochlorinated compounds, such as DDT, aroclor or aldrin, is associated with alterations in the secretion or transport of dopamine.32–34 Prolactin's sensitivity to diverse toxins has led some authors to propose it as an early indicator of neurological damage.35 However, the effects of toxins on prolactin may be biphasic, depending on the magnitude of the exposure,36 so we can find decreases as well as increases. This reflects the complexity of the control of prolactin secretion, which is not only modulated by dopamine but also by several other neurotransmitters.37
Regarding the limitations of this study, a selection bias is improbable since there were no differences in sociodemographics or chemical exposure, including biomarkers of exposure, between workers who participated during both seasons and those who did not participate during the dry season. However, it is possible that some workers were not included in the study because they were not working at the time of selection due to illness caused by their higher susceptibility to pesticides. In this hypothetical case, our results could have been biased to the null due to the healthy worker effect.
Another potential limitation is the fact that two urine and two blood samples were analysed for only 84 individuals. For workers who withdrew from the study during the dry season, only one urine and one blood sample were available. In men with stable weight, p,p'-DDE concentrations tend to remain constant over time; for this reason we consider that using two measures for estimating exposure status is highly reliable. However, as organophosphates are not persistent compounds, measurement of their urine levels represents exposure during the previous 24–48 h and does not reflect exposure during extended periods. However, some authors have postulated that a single urine sample could correctly classify subjects according to their exposure level during the 3 months prior to the taking of the sample. Thus, Meeker et al38 when comparing TCPY (a metabolite of chlorpyriphos) values obtained from a urine sample with a geometric mean corresponding to nine samples obtained over 3 months, found that the sensitivity and specificity in the case of TCPY fluctuated between 0.44 and 0.84. Because DAPs are unspecific metabolites of organophosphate pesticides we expect their behaviour to be similar to that described by Meeker.38
On the one hand, as secretion of LH and testosterone follows a pulsing pattern, diurnal fluctuations in the levels of these hormones cannot be evaluated through a single determination; however, a single determination is sufficiently reliability for population studies.39 40 On the other hand, the floriculturists' participation would have decreased considerably if they had been asked to provide a series of blood samples. To minimise the effect of diurnal fluctuation, all samples were taken between 08:00 and 09:30 h.
Summarising, the results of this study show that there are significant associations between p,p'-DDE serum levels and the male hormonal profile, and support the hypothesis that DDT and its derivatives can act as endocrine disruptors in humans. The effects were more evident at the level of prolactin and inhibin B. Larger studies are warranted to confirm these findings and to address their biological and clinical relevance.
The authors thank the participants in this study. We also thank Rosa María García Hernández for her help in laboratory analysis and Kellogg's of Mexico for their contribution of products.
Funding This study was supported by the Consejo Nacional de Ciencia y Tecnología of Mexico CONACYT (National Council of Science and Technology), project number SALUD-2002-C01-7574 and by the Fondo Sectorial de Investigación para la Educación SEP-CONACYT, project number 49793.
Competing interests None.
Ethics approval This study was conducted with the approval of the Instituto Nacional de Salud Pública (National Institute of Public Health), Mexico.
Provenance and peer review Not commissioned; externally peer reviewed.