Original research paper
Prenatal molybdenum exposure and infant neurodevelopment in Mexican children Ruth Argelia Vázquez-Salas 1, Lizbeth López-Carrillo 1, José A. Menezes-Filho 2, Stephen J. Rothenberg 1, Mariano E. Cebrián 3, Lourdes Schnaas4, Gustavo Freitas de Souza Viana 2, Luisa Torres-Sánchez 1 1
National Institute of Public Health, Av. Universidad 655.Col. Sta María Ahuacatitlán, CP 62100,Cuernavaca Morelos, Mexico, 2Laboratory of Toxicology, College of Pharmacy, Federal University of Bahia, Av. Barão de Jeremoabo s/n, Salvador, Bahia, Brazil, 3Department of Toxicology, CINVESTAV, Av. IPN # 2508 Col. Instituto Politécnico Nacional, Delg. Gustavo A. Madero, 07360 Mexico City, Mexico, 4National Institute of Perinatology, Prado Sur 800 Lomas de Chapultepec, Miguel Hidalgo, 11000 Mexico City, Mexico Objective: To evaluate the association between prenatal exposure to molybdenum (Mo) and infant neurodevelopment during the first 30 months of life. Methods: We selected a random sample of 147 children who participated in a prospective cohort study in four municipalities in the State of Morelos, Mexico. The children were the products of uncomplicated pregnancies with no perinatal asphyxia, with a weight of ≥2 kg at birth, and whose mothers had no history of chronic illnesses. These women were monitored before, during, and after the pregnancy. For each of these children a maternal urine sample was available for at least one trimester of pregnancy, and urine Mo levels were determined by electrothermal atomic absorption spectrometry. Neurodevelopment was evaluated using the psychomotor (PDI) and mental development indices (MDI) of the Bayley scale. Association between prenatal exposure to Mo and infant neurodevelopment was estimated using generalized mixed effect models. Results: The average urinary concentrations of Mo adjusted for creatinine varied between 45.6 and 54.0 μg/g of creatinine at first and third trimester, respectively. For each doubling increase of Mo (μg/g creatinine) during the third trimester of pregnancy, we observed a significant reduction on PDI (β = −0.57 points; P = 0.03), and no effect on MDI (β = 0.07 points; P = 0.66). Discussion: As this is the first study that suggests a potential negative association between prenatal Mo exposure and infant neurodevelopment, these results require further confirmation. Keywords: Bayley scales, Infant neurodevelopment, Mexico, Molybdenum, Prenatal exposure
Introduction Molybdenum (Mo) is an essential trace element. It is distributed in the environment and is involved in the metabolism of aldehydes, purines, and sulfur as a cofactor of the enzymes xanthine oxidase, aldehyde oxidase, and sulfite oxidase.1,2 Owing to its strength, firmness, and anticorrosion properties, Mo is used in construction.3 Mo also reduces nitrogen to ammonium and stimulates cellular development in the plant parenchyma, which prevents the deformation and necrosis of the leaves. For this reason, it is also used as a fertilizer for various floricultural crops.4
Correspondence to: Luisa Torres-Sánchez, National Institute of Public Health, Av. Universidad 655.Col. Sta María Ahuacatitlán, CP 62100, Cuernavaca Morelos, México. Email: [email protected]
72
© W. S. Maney & Son Ltd 2014 DOI 10.1179/1476830513Y.0000000076
In humans, the main source of Mo exposure is through oral consumption of such foods as vegetables, milk, and animal organs like liver and kidneys.5 The 2007–2008 National Health and Nutrition Examination Survey of United States (NHANES) reported that the mean urinary Mo concentrations of both sexes in the general population range between 43.5 and 90.4 μg/g of creatinine in subjects aged 6 years and >20 years, respectively.6 Mo toxicity has been evaluated mainly in animals and seems to be mediated by copper (Cu) concentration.7 The main signs of Mo poisoning observed among animals are poor growth and anemia (rats, chickens, rabbits, cattle, and sheep), anorexia (rats), diarrhoea and achromotrichia (cattle and sheep), joint and bone deformities (rats, rabbits, cattle),
Nutritional Neuroscience
2014
VOL.
17
NO.
2
Vázquez-Salas et al.
central nervous system degeneration and loss of crimp in wool (sheep) and thyroidal injury, and hypothyroidism.7,8 In humans, only two studies have evaluated Mo toxicity. The first was performed in the former Soviet Union in 1966, where an excessive dietary intake of 10–15 mg/day was associated with a higher blood acid uric and an increased incidence of a gout-like syndrome.9 Recently, a study based on NHANES 2007–2008 found that higher levels of creatinineadjusted Mo were associated with a three-fold increase of the possibilities of self-reported liver problem among adults older than 20 years old.6 The state of Morelos, Mexico is the main producer of poinsettias,10 and fertilizer products containing Mo are commonly used to improve its growth. In 2001, we started a perinatal cohort study in four municipalities of the State of Morelos. The mean objective was to evaluate the association between dichlorodiphenyldichloroethylene (DDE) prenatal exposure and infant neurodevelopment. As part of this study, we analyzed maternal urine samples and, depending on the trimester of pregnancy, we found that 6–15% of maternal urine samples had higher concentrations of Mo than the 95th percentile reported by NHANES for women of all ages (152 μg/l and 139 μg/g). For this reason, the objective of this study was to evaluate the association between prenatal Mo exposure and infant neurodevelopment measured by Bayley Scales of Infant Development (BSID II) in children aged less than 30 months.
Methods We followed up a cohort of 996 women of reproductive age from January 2001 to June 2005. These women had no history of chronic (thyroid, heart, liver, kidney, or gastrointestinal) illnesses and were not being treated with anticonvulsants, nor were any of the participants breastfeeding at the time of the recruitment. All participants were residents of four municipalities in the State of Morelos, Mexico. The details of the cohort conformation have been previously reported;11 briefly, the women were identified during the premarital talks required by law before civil weddings, in the State of Morelos. Women who agreed to participate answered a structured interview about sociodemographic characteristics, reproductive and occupational history, dietary habits, smoking, and alcohol consumption. Afterwards, we followed up once every 8 weeks until the pregnancy, and then continued during and after the pregnancy ended. At each trimester of pregnancy, the evaluation included a questionnaire about the progression of the pregnancy, dietary characteristics, maternal anthropometric
Prenatal Mo exposure and infant neurodevelopment in Mexican children
measures (weight and height); also, blood samples were collected to determine serum DDE and lead concentrations, and urine samples for Mo concentrations. Once informed of the objectives of the study, each woman signed a letter of informed consent. Consent was also obtained for the children once the postnatal follow-up began. This study had the approval of the Ethics Committee of the National Institute of Public Health (CI: 515).
Study sample Fig. 1 shows the composition of the study sample. Of the total births presented during the cohort study, 353 children were products of single pregnancies, without perinatal asphyxia diagnosis, with a birth weight of ≥2 kg, and whose mothers, aged >15 years, provided urine samples at least in one trimester of their pregnancy. Among those children who had at least five of the seven visits scheduled between 1 and 30 months of age, a random selection was made to obtain a final sample of 147 children who were included in the analyses and 206 who were not included in the study.
Postnatal follow-up The postnatal follow-ups were set at first, third, and sixth months of life and after that every 6 months until 30 months of age. During the first visit, at 1 month of age, we obtained information regarding the birth characteristics and initiation of breastfeeding. In the subsequent visits, the information obtained focused on the health status of the children and on the feeding practices. After 12 months of age we asked about the number of persons living with the child, and whether or not the child attended day care. Additionally, anthropometric measures (weight, height, and cephalic circumference) were taken and infant neurodevelopment was evaluated during all visits.
Neurodevelopment evaluation The infant neurodevelopment was assessed through the Spanish version of the BSID II previously used in Mexico.12 This test can be used in children from 1 to 42 months of age and includes two indices: one evaluating psychomotor development index (PDI) and the other evaluating mental development index (MDI). The MDI measures children’s current sensory ability, memory, learning, problem solving, and verbal ability. PDI assesses gross and fine motor coordination. Among infants with biological risk (i.e. low birth weight, premature, etc.), the Bayley Scale at 12 months of age correlates with the score of verbal and motor performance at 4.5 years of age. Information about the Bayley predictability at later ages in normal children is scarce. Two trained psychologists
Nutritional Neuroscience
2014
VOL.
17
NO.
2
73
Vázquez-Salas et al.
Prenatal Mo exposure and infant neurodevelopment in Mexican children
Figure 1 Morelos cohort study.
were responsible for administering this test, and the inter-observer concordance was 0.97 for PDI and 0.99 for the MDI. Maternal intellectual coefficient (IQ) was measured through the Spanish version of the Wechsler Adult Intelligence Scale.13 The quality of the home environment was evaluated at 6 months of age through the
74
Nutritional Neuroscience
2014
VOL.
17
NO.
2
Home Observation for Measurement Environment (HOME) Scale.14
of
the
Chemical analyses From each women participant, a urine sample (10 ml) was collected during each trimester of pregnancy. The samples were refrigerated immediately and
Vázquez-Salas et al.
transported to the National Institute of Public Health, where they were kept at −70°C in plastic vials. After the random selection, the samples were sent to the Laboratory of Toxicology, Federal University of Bahia, Brazil, where the urine determinations of Mo were conducted. The analyses were made in a graphite furnace atomic absorption spectrometer AA240Z (Varian Inc., Australia). All solutions were prepared with high purity Type I water (resistivity 18.2 MΩ cm) obtained from a Milli-Q water purification system (Millipore, Bedford, MA, USA), and all materials were decontaminated with a 20% v/v solution of nitric acid. Urine samples were prepared directly in the autosampler cups, diluting 1:1 with nitric acid 0.2 and 0.5% of Triton X-100 solution with the aid of an automatic diluter to provide better accuracy. For quality control purposes, urine samples from Seronom MoU Trace elements urine (Norway, Lab Sero As, Billingstad), were used to check the reliability of the entire proposed analytical method. All the samples were above the detection limit of 12 weeks HOME scale
Mental index**
95% CI
P
β†
95% CI
P
−0.001 −0.20 −0.49
−0.5;0.5 −0.8;0.3 −1.0;0.03
0.95 0.45 0.06
0.11 0.06 −0.1
−0.2;0.4 −0.3;0.4 −0.4;0.2
0.46 0.70 0.51
0.19 0.31 −0.54
−0.3;0.7 −0.9;0.3 −1.1;−0.002
0.48 0.28 0.05
0.22 0.01 −0.06
−0.1;0.6 −0.4;0.4 −0.4;0.3
0.22 0.95 0.73
0.27 −0.50 −0.57
−0.2;0.8 −1.1;0.1 −1.1;−0.1
0.29 0.12 0.03
0.18 −0.08 0.07
−0.2;0.5 −0.5;0.3 −0.2;0.4
0.33 0.68 0.66
−7.1 −1.9 −5.1 −2.8 −0.9 −3.7 −0.4
−8.21;−6.0 −3.3;−0.7 −6.4;−3.8 −4.1;−1.6 −2.2;0.5 −5.3;−2.1 −1.5;0.7
0.00 0.004 0.00 0.00 0.21 0.00 0.51
−1.9 −1.4 −3.2 −6.1 −8.3 −5.4 −1.1
−2.7;−1.2 −2.0;−0.7 −4.3;−2.1 −7.0;−5.1 −9.5;−7.1 −6.5;−4.3 −1.8;−0.4
0.00 0.00 0.00 0.00 0.00 0.00 0.001
−1.4;2.6 0.002;0.3
0.65 0.05
1.5 0.07
0.3;2.6 −0.007; 0.1
0.01 0.08
β
Molybdenum exposure
†
0.4 0.1
*Adjusted for DDE exposure at first trimester of pregnancy, gestational age, parity, maternal age, education and IQ, birthweight, type of birth, sex of child, breastfeeding and HOME Scale. **Adjusted for gestational age, parity, maternal age, education, occupation, IQ, birthweigth, type of birth, sex of child, breastfeeding and HOME Scale. † Change by doubling of molybdenum. ‡ This model includes creatinine as covariable. § Mo levels were adjusted for creatinine.
and lead nor Mo with copper (data not included in the tables).
Discussion To the extent of our knowledge, this is the first study that assesses the possible association between prenatal Mo exposure and infant neurodevelopment. The findings suggest that prenatal Mo exposure during the third trimester of pregnancy may negatively affect psychomotor development over the first 30 months of life, and there is no association between Mo and mental development. Mo is an essential trace element and could be also considered a potentially toxic metal. There is not a clear mechanism of toxicity; however, in animals, symptoms associated with the toxicity of Mo appear to be mediated by copper deficiency.7 Mo promotes thiomolybdate synthesis and formation of unabsorbable copper–thiomolybdate complexes, turning risk to hypocuprosis.18 Abnormal motor behavior observed in rats exposed to copper restriction during the last two thirds of gestation and throughout lactation are consistent with changes in the cerebellar function after copper deficiency. The biological mechanisms responsible for the neuropathology of copper deficiency are not clear. A limited activity of
78
Nutritional Neuroscience
2014
VOL.
17
NO.
2
some cuproenzymes like dopamine-β-monooxygenase during critical brain development may produce a norepinephrine deficiency that results in decreased synaptic connections and alterations in the maturation of neurons, consequently affecting the motor response.19 Moreover, Mo is retained within the brain, pituitary, and adrenal glands, and adversely affects the hypothalamo-adenohypophyseal system by interfering with trophic hormone release, and thus leading to a toxic endocrinopathy.20 This is consistent with an experimental study with young rabbits, fed with molybdate 0.03% over a period of 25–31 days, in which thyroid histological alterations and reduction of the activity and concentration of serum thyroxin (T4) were observed, suggesting a possible role of Mo as an endocrine disruptor.7 In humans, the thyroid gland plays an important role in the pre- and post-natal development of the central nervous system. Throughout the pregnancy, decrease of serum T4 (hypothyroidism), is associated with neurological alterations, ranging from mild manifestations, such as learning disabilities, to severe mental retardation (cretinism).21,22 This could be considered as another potential biological mechanism that explains the observed effect of prenatal Mo exposure
Vázquez-Salas et al.
and infant motor neurodevelopment during the first 30 months of life. Although Mo exposure during pregnancy did not change substantially, developmental functions may be affected differently by exposure at different developmental stages. The cerebellum is a critical structure involved in motor skills. Cerebellum growth starts at the first trimester and reaches its maximum development at gestational week 30–40, during which time Mo may cause damage.23 The principal strengths of this study are that the infant neurodevelopment was evaluated longitudinally and that the main known risk factors associated with infant neurodevelopment were evaluated as potential confounders. Some potential sources of error must be evaluated to make an adequate interpretation of these results. The children included in the analyses were the final sample obtained from a random selection of all children with five or more evaluations over the first 30 months of life; in order to determine whether our results could be the consequence of a selection bias, we compared the children included with those not included in the analyses, and we found that during the first trimester of pregnancy the mean body mass index of the mothers of the children included was significantly lower than that of the mothers of the children not included. However, there is no evidence that body mass index could be a determinant in infant neurodevelopment. Furthermore, infant neurodevelopment was evaluated a long time before the Mo urinary determinations had been made, and the person in charge of the analyses of urinary Mo did not know the infant neurodevelopmental condition of the infants. Thus we ruled out differential measurement errors in the event or in the exposure as an explanation of these results. Even though we had a good internal quality control, the absence of an external quality control does not allow us to rule out the presence or the magnitude of an error in the assessment of the exposure. However, we believe that if error exists, this could not be differential in regard to infant neurodevelopment, and the association with psychomotor index would be underestimated. An additional source of error in the measurement of exposure could be the lack of control in the dilution of urine. Analyses of the information including Mo with and without creatinine adjustment, as well as the inclusion of creatinine as a covariable, showed similar findings, and the report of the final results about the association between prenatal Mo exposure and PDI or MDI takes into account Mo levels adjusted for creatinine. Finally, we did not find a significant interaction between Mo and dietary intake of copper. However, we could not rule out the
Prenatal Mo exposure and infant neurodevelopment in Mexican children
interaction between both metals because we only had dietary information, and the estimation of copper status using a food frequency questionnaire could be underestimated. Serum copper seems to be the most useful biomarker of copper status,24 and the correlation observed between dietary copper and its serum concentrations is weak (r = 0.17).25 In short, these results should be interpreted cautiously, in the context of an exploratory study that warrants more evaluation. The mean urinary Mo concentrations at which we observed psychomotor alterations in these children (40.4 ± 2.8 μg/l) are similar to those reported for women by the 2007–2008 National Health and Nutrition Examination Survey of the Unites Stated (40.5 μg/l).26 However, no information exists regarding the potential health impact of those levels in children. Only a recent study of adults showed a positive association between Mo and a higher prevalence of self-reported liver problems.6 The interest of Mo as an essential trace element, the lack of studies about Mo toxicity, and the absence of a toxicity limit demonstrates the necessity of further research in this area. The lack of knowledge about Mo also could be the reason for the absence of information regarding the potential toxic effects of Mo exposure on humans.
Acknowledgments This study was funded by grants (41708, 31034-M, 13915) from the National Council of Science and Technology of Mexico (CONACyT) and by the South-South Collaboration Program pertaining to the Fogarty International Center of the National Institutes of Health (D43TW00640) of Mount Sinai School of Medicine/Queens College International Training and Research in Environmental and Occupational Health Program. The content is solely the responsibility of the authors and does not necessarily represent the official views of the Fogarty International Center or the National Institutes of Health. The authors are grateful to Ms Patricia Castro for her assistance in contacting the families and administering the questionnaires; also to Dr Marcia Galván Portillo and MSc Wendy Elena Becerra Romero, from the National Institute of Public Health, for their support in dietary information.
References 1 Schwarz G, Mendel RW, Ribbe M. Molybdenum cofactors, enzymes and pathways. Nature 2009;460:839–46. 2 Sardesai V. Molybdenum: an essential trace element. Nutr Clin Pract 1993;8:277–81. 3 International Molybdenum association IMOA. Molybdenum markets-an end-use analysis. Molyreview 2011:2–8. 4 Minerales del recreo SA. [Consulta 8 de Octubre 2011]. Available from: http://www.asufrar.com.ar/pdf/molibdeno.pdf 5 Food and Nutrition Board Institute of Medicine Panel on Micronutrients, Subcommittees on Upper Reference Levels of
Nutritional Neuroscience
2014
VOL.
17
NO.
2
79
Vázquez-Salas et al.
6 7 8 9 10
11
12 13 14 15
80
Prenatal Mo exposure and infant neurodevelopment in Mexican children
Nutrients and of Interpretation and Uses of Dietary Reference Intakes, and the Standing Committee on the Scientific Evaluation of Dietary Reference Intakes. Dietary reference intakes for vitamin a, vitamin k, arsenic, boron, chromium, copper, iodine, iron, manganese, molybdenum, nickel, silicon, vanadium, and zinc. Washington, DC: National Academy Press; 2001. p. 420–41. Mendy A, Gasana J, Vieira ER. Urinary heavy metals and associated medical conditions in US adult population. Int J Environ Health Res 2012;22:105–18. Widjajakusuma MCR, Basrur PK, Robinson GA. Thyroid function in molybdenotic rabbits. J. Endocr 1973;57:419–24. Pitt MA. Molybdenum toxicity: interactions between copper, molybdenum and sulphate. AgentsActions 1976;6:758–69. Kovalskiy VV, Yarovaya GA. Molybdenum-infiltratedbiogeochemicalprovinces. Agrokhimiva 1966;8:68–91. Instituto nacional de estadística y geografía INEGI [documentonthe Internet]. Actividades económicas. Available from: http://cuentame.inegi.org.mx/monografias/informacion/mor/ economia/default.aspx?tema=me&e=17#sp. Torres-Sánchez L, Rothenberg SJ, Schnaas L, Cebrián ME, Osorio E, Hernández MC, et al. In utero p,p′ -DDE exposure and infant neurodevelopment: a perinatal cohort in Mexico. Environ Health Perspect 2007;115:435–9. Bayley N. Bayley Scales of Infant Development. 2nd ed. San Antonio, TX: The Psychological Corporation; 1993. Wechsler D. WAIS-Español. Escala de inteligencia para adultos. Mexico: DF. El Manual Moderno, S.A; 1981. Caldwell B, Bradley R. Home observational of measurement of the environment. Little Rock: University of Arkansas at Little Rock; 1984. Barr D, Wilder CL, Caudill SP, Gonzalez AJ, Needham LL, Pirkle JL. Urinary creatinine concentrations in the U.S. population: implications for urinary biologic monitoring measurements. Environ Health Perspect 2005;13:192–200.
Nutritional Neuroscience
2014
VOL.
17
NO.
2
16 Hernández-Avila M, Romieu I, Parra S, Hernández-Avila J, Madrigal H, Willett W. Validity and reproducibility of a food frequency questionnaire to assess dietary intake of women living in Mexico City. Salud Publica Mex 1998;39:133–40. 17 Munoz de Chávez M, Ledesma Solano J, eds. Los alimentos y sus nutrimentos. Tablas de valor Nutritivo de Alimentos. México, DF: McGraw-Hill Interamericana; 2002. p. 25. 18 Suttle NF. Copper imbalances in ruminants and humans: unexpected common ground. Adv Nutr 2012;3:666–74. 19 Penland JG, Prohaska JR. Abnormal motor function persists following recovery from perinatal copper deficiency in rats. J Nutr 2004;134:1984–8. 20 Haywood S, Dincer Z, Jasani B, Loughran MJ. Molybdenumassociated pituitary endocrinopathy in sheep treated with ammonium tetrathiomolybdate. J Comp Pathol 2004;130:21–31. 21 Haddow J, Palomaki G, Allan W, Williams J, Knight G, Gagnon J, et al. Maternal thyroid deficiency during pregnancy and subsequent neuropsychological development of the child. N Engl J Med 1999;341:549–55. 22 Anderson GW, Schoonover CM, Jones SA. Control of thyroid hormone action in developing rat brain. Thyroid 2003;13: 1039–56. 23 Volpe JJ. Cerebellum of the premature infant: rapidly developing, vulnerable, clinically important. J Child Neurol 2009;24: 1085–104. 24 Harvey LJ, Ashton K, Hooper L, Casgrain A, Fairweather-Tait SJ. Methods of assessment of copper status in humans: a systematic review. Am J ClinNutr 2009;89:2009S–24S. 25 Ghayour-Mobarhan M, Taylor A, New SA, Lamb DJ, Ferns GA. Determinants of serum copper, zinc and selenium in healthy subjects. Ann ClinBiochem 2005;42:364–75. 26 Center for diseases control and prevention (CDC). National Report on Human Exposure to Environmental Chemicals. Urinary Molybdenum, 2011. Available from: http://www.cdc .gov/exposurereport/data_tables/URXUMO_DataTables.html.
Copyright of Nutritional Neuroscience is the property of Maney Publishing and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.
Copyright of Nutritional Neuroscience is the property of Maney Publishing and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.
Prenatal molybdenum exposure and infant neurodevelopment in Mexican children Ruth Argelia Vázquez-Salas 1, Lizbeth López-Carrillo 1, José A. Menezes-Filho 2, Stephen J. Rothenberg 1, Mariano E. Cebrián 3, Lourdes Schnaas4, Gustavo Freitas de Souza Viana 2, Luisa Torres-Sánchez 1 1
National Institute of Public Health, Av. Universidad 655.Col. Sta María Ahuacatitlán, CP 62100,Cuernavaca Morelos, Mexico, 2Laboratory of Toxicology, College of Pharmacy, Federal University of Bahia, Av. Barão de Jeremoabo s/n, Salvador, Bahia, Brazil, 3Department of Toxicology, CINVESTAV, Av. IPN # 2508 Col. Instituto Politécnico Nacional, Delg. Gustavo A. Madero, 07360 Mexico City, Mexico, 4National Institute of Perinatology, Prado Sur 800 Lomas de Chapultepec, Miguel Hidalgo, 11000 Mexico City, Mexico Objective: To evaluate the association between prenatal exposure to molybdenum (Mo) and infant neurodevelopment during the first 30 months of life. Methods: We selected a random sample of 147 children who participated in a prospective cohort study in four municipalities in the State of Morelos, Mexico. The children were the products of uncomplicated pregnancies with no perinatal asphyxia, with a weight of ≥2 kg at birth, and whose mothers had no history of chronic illnesses. These women were monitored before, during, and after the pregnancy. For each of these children a maternal urine sample was available for at least one trimester of pregnancy, and urine Mo levels were determined by electrothermal atomic absorption spectrometry. Neurodevelopment was evaluated using the psychomotor (PDI) and mental development indices (MDI) of the Bayley scale. Association between prenatal exposure to Mo and infant neurodevelopment was estimated using generalized mixed effect models. Results: The average urinary concentrations of Mo adjusted for creatinine varied between 45.6 and 54.0 μg/g of creatinine at first and third trimester, respectively. For each doubling increase of Mo (μg/g creatinine) during the third trimester of pregnancy, we observed a significant reduction on PDI (β = −0.57 points; P = 0.03), and no effect on MDI (β = 0.07 points; P = 0.66). Discussion: As this is the first study that suggests a potential negative association between prenatal Mo exposure and infant neurodevelopment, these results require further confirmation. Keywords: Bayley scales, Infant neurodevelopment, Mexico, Molybdenum, Prenatal exposure
Introduction Molybdenum (Mo) is an essential trace element. It is distributed in the environment and is involved in the metabolism of aldehydes, purines, and sulfur as a cofactor of the enzymes xanthine oxidase, aldehyde oxidase, and sulfite oxidase.1,2 Owing to its strength, firmness, and anticorrosion properties, Mo is used in construction.3 Mo also reduces nitrogen to ammonium and stimulates cellular development in the plant parenchyma, which prevents the deformation and necrosis of the leaves. For this reason, it is also used as a fertilizer for various floricultural crops.4
Correspondence to: Luisa Torres-Sánchez, National Institute of Public Health, Av. Universidad 655.Col. Sta María Ahuacatitlán, CP 62100, Cuernavaca Morelos, México. Email: [email protected]
72
© W. S. Maney & Son Ltd 2014 DOI 10.1179/1476830513Y.0000000076
In humans, the main source of Mo exposure is through oral consumption of such foods as vegetables, milk, and animal organs like liver and kidneys.5 The 2007–2008 National Health and Nutrition Examination Survey of United States (NHANES) reported that the mean urinary Mo concentrations of both sexes in the general population range between 43.5 and 90.4 μg/g of creatinine in subjects aged 6 years and >20 years, respectively.6 Mo toxicity has been evaluated mainly in animals and seems to be mediated by copper (Cu) concentration.7 The main signs of Mo poisoning observed among animals are poor growth and anemia (rats, chickens, rabbits, cattle, and sheep), anorexia (rats), diarrhoea and achromotrichia (cattle and sheep), joint and bone deformities (rats, rabbits, cattle),
Nutritional Neuroscience
2014
VOL.
17
NO.
2
Vázquez-Salas et al.
central nervous system degeneration and loss of crimp in wool (sheep) and thyroidal injury, and hypothyroidism.7,8 In humans, only two studies have evaluated Mo toxicity. The first was performed in the former Soviet Union in 1966, where an excessive dietary intake of 10–15 mg/day was associated with a higher blood acid uric and an increased incidence of a gout-like syndrome.9 Recently, a study based on NHANES 2007–2008 found that higher levels of creatinineadjusted Mo were associated with a three-fold increase of the possibilities of self-reported liver problem among adults older than 20 years old.6 The state of Morelos, Mexico is the main producer of poinsettias,10 and fertilizer products containing Mo are commonly used to improve its growth. In 2001, we started a perinatal cohort study in four municipalities of the State of Morelos. The mean objective was to evaluate the association between dichlorodiphenyldichloroethylene (DDE) prenatal exposure and infant neurodevelopment. As part of this study, we analyzed maternal urine samples and, depending on the trimester of pregnancy, we found that 6–15% of maternal urine samples had higher concentrations of Mo than the 95th percentile reported by NHANES for women of all ages (152 μg/l and 139 μg/g). For this reason, the objective of this study was to evaluate the association between prenatal Mo exposure and infant neurodevelopment measured by Bayley Scales of Infant Development (BSID II) in children aged less than 30 months.
Methods We followed up a cohort of 996 women of reproductive age from January 2001 to June 2005. These women had no history of chronic (thyroid, heart, liver, kidney, or gastrointestinal) illnesses and were not being treated with anticonvulsants, nor were any of the participants breastfeeding at the time of the recruitment. All participants were residents of four municipalities in the State of Morelos, Mexico. The details of the cohort conformation have been previously reported;11 briefly, the women were identified during the premarital talks required by law before civil weddings, in the State of Morelos. Women who agreed to participate answered a structured interview about sociodemographic characteristics, reproductive and occupational history, dietary habits, smoking, and alcohol consumption. Afterwards, we followed up once every 8 weeks until the pregnancy, and then continued during and after the pregnancy ended. At each trimester of pregnancy, the evaluation included a questionnaire about the progression of the pregnancy, dietary characteristics, maternal anthropometric
Prenatal Mo exposure and infant neurodevelopment in Mexican children
measures (weight and height); also, blood samples were collected to determine serum DDE and lead concentrations, and urine samples for Mo concentrations. Once informed of the objectives of the study, each woman signed a letter of informed consent. Consent was also obtained for the children once the postnatal follow-up began. This study had the approval of the Ethics Committee of the National Institute of Public Health (CI: 515).
Study sample Fig. 1 shows the composition of the study sample. Of the total births presented during the cohort study, 353 children were products of single pregnancies, without perinatal asphyxia diagnosis, with a birth weight of ≥2 kg, and whose mothers, aged >15 years, provided urine samples at least in one trimester of their pregnancy. Among those children who had at least five of the seven visits scheduled between 1 and 30 months of age, a random selection was made to obtain a final sample of 147 children who were included in the analyses and 206 who were not included in the study.
Postnatal follow-up The postnatal follow-ups were set at first, third, and sixth months of life and after that every 6 months until 30 months of age. During the first visit, at 1 month of age, we obtained information regarding the birth characteristics and initiation of breastfeeding. In the subsequent visits, the information obtained focused on the health status of the children and on the feeding practices. After 12 months of age we asked about the number of persons living with the child, and whether or not the child attended day care. Additionally, anthropometric measures (weight, height, and cephalic circumference) were taken and infant neurodevelopment was evaluated during all visits.
Neurodevelopment evaluation The infant neurodevelopment was assessed through the Spanish version of the BSID II previously used in Mexico.12 This test can be used in children from 1 to 42 months of age and includes two indices: one evaluating psychomotor development index (PDI) and the other evaluating mental development index (MDI). The MDI measures children’s current sensory ability, memory, learning, problem solving, and verbal ability. PDI assesses gross and fine motor coordination. Among infants with biological risk (i.e. low birth weight, premature, etc.), the Bayley Scale at 12 months of age correlates with the score of verbal and motor performance at 4.5 years of age. Information about the Bayley predictability at later ages in normal children is scarce. Two trained psychologists
Nutritional Neuroscience
2014
VOL.
17
NO.
2
73
Vázquez-Salas et al.
Prenatal Mo exposure and infant neurodevelopment in Mexican children
Figure 1 Morelos cohort study.
were responsible for administering this test, and the inter-observer concordance was 0.97 for PDI and 0.99 for the MDI. Maternal intellectual coefficient (IQ) was measured through the Spanish version of the Wechsler Adult Intelligence Scale.13 The quality of the home environment was evaluated at 6 months of age through the
74
Nutritional Neuroscience
2014
VOL.
17
NO.
2
Home Observation for Measurement Environment (HOME) Scale.14
of
the
Chemical analyses From each women participant, a urine sample (10 ml) was collected during each trimester of pregnancy. The samples were refrigerated immediately and
Vázquez-Salas et al.
transported to the National Institute of Public Health, where they were kept at −70°C in plastic vials. After the random selection, the samples were sent to the Laboratory of Toxicology, Federal University of Bahia, Brazil, where the urine determinations of Mo were conducted. The analyses were made in a graphite furnace atomic absorption spectrometer AA240Z (Varian Inc., Australia). All solutions were prepared with high purity Type I water (resistivity 18.2 MΩ cm) obtained from a Milli-Q water purification system (Millipore, Bedford, MA, USA), and all materials were decontaminated with a 20% v/v solution of nitric acid. Urine samples were prepared directly in the autosampler cups, diluting 1:1 with nitric acid 0.2 and 0.5% of Triton X-100 solution with the aid of an automatic diluter to provide better accuracy. For quality control purposes, urine samples from Seronom MoU Trace elements urine (Norway, Lab Sero As, Billingstad), were used to check the reliability of the entire proposed analytical method. All the samples were above the detection limit of 12 weeks HOME scale
Mental index**
95% CI
P
β†
95% CI
P
−0.001 −0.20 −0.49
−0.5;0.5 −0.8;0.3 −1.0;0.03
0.95 0.45 0.06
0.11 0.06 −0.1
−0.2;0.4 −0.3;0.4 −0.4;0.2
0.46 0.70 0.51
0.19 0.31 −0.54
−0.3;0.7 −0.9;0.3 −1.1;−0.002
0.48 0.28 0.05
0.22 0.01 −0.06
−0.1;0.6 −0.4;0.4 −0.4;0.3
0.22 0.95 0.73
0.27 −0.50 −0.57
−0.2;0.8 −1.1;0.1 −1.1;−0.1
0.29 0.12 0.03
0.18 −0.08 0.07
−0.2;0.5 −0.5;0.3 −0.2;0.4
0.33 0.68 0.66
−7.1 −1.9 −5.1 −2.8 −0.9 −3.7 −0.4
−8.21;−6.0 −3.3;−0.7 −6.4;−3.8 −4.1;−1.6 −2.2;0.5 −5.3;−2.1 −1.5;0.7
0.00 0.004 0.00 0.00 0.21 0.00 0.51
−1.9 −1.4 −3.2 −6.1 −8.3 −5.4 −1.1
−2.7;−1.2 −2.0;−0.7 −4.3;−2.1 −7.0;−5.1 −9.5;−7.1 −6.5;−4.3 −1.8;−0.4
0.00 0.00 0.00 0.00 0.00 0.00 0.001
−1.4;2.6 0.002;0.3
0.65 0.05
1.5 0.07
0.3;2.6 −0.007; 0.1
0.01 0.08
β
Molybdenum exposure
†
0.4 0.1
*Adjusted for DDE exposure at first trimester of pregnancy, gestational age, parity, maternal age, education and IQ, birthweight, type of birth, sex of child, breastfeeding and HOME Scale. **Adjusted for gestational age, parity, maternal age, education, occupation, IQ, birthweigth, type of birth, sex of child, breastfeeding and HOME Scale. † Change by doubling of molybdenum. ‡ This model includes creatinine as covariable. § Mo levels were adjusted for creatinine.
and lead nor Mo with copper (data not included in the tables).
Discussion To the extent of our knowledge, this is the first study that assesses the possible association between prenatal Mo exposure and infant neurodevelopment. The findings suggest that prenatal Mo exposure during the third trimester of pregnancy may negatively affect psychomotor development over the first 30 months of life, and there is no association between Mo and mental development. Mo is an essential trace element and could be also considered a potentially toxic metal. There is not a clear mechanism of toxicity; however, in animals, symptoms associated with the toxicity of Mo appear to be mediated by copper deficiency.7 Mo promotes thiomolybdate synthesis and formation of unabsorbable copper–thiomolybdate complexes, turning risk to hypocuprosis.18 Abnormal motor behavior observed in rats exposed to copper restriction during the last two thirds of gestation and throughout lactation are consistent with changes in the cerebellar function after copper deficiency. The biological mechanisms responsible for the neuropathology of copper deficiency are not clear. A limited activity of
78
Nutritional Neuroscience
2014
VOL.
17
NO.
2
some cuproenzymes like dopamine-β-monooxygenase during critical brain development may produce a norepinephrine deficiency that results in decreased synaptic connections and alterations in the maturation of neurons, consequently affecting the motor response.19 Moreover, Mo is retained within the brain, pituitary, and adrenal glands, and adversely affects the hypothalamo-adenohypophyseal system by interfering with trophic hormone release, and thus leading to a toxic endocrinopathy.20 This is consistent with an experimental study with young rabbits, fed with molybdate 0.03% over a period of 25–31 days, in which thyroid histological alterations and reduction of the activity and concentration of serum thyroxin (T4) were observed, suggesting a possible role of Mo as an endocrine disruptor.7 In humans, the thyroid gland plays an important role in the pre- and post-natal development of the central nervous system. Throughout the pregnancy, decrease of serum T4 (hypothyroidism), is associated with neurological alterations, ranging from mild manifestations, such as learning disabilities, to severe mental retardation (cretinism).21,22 This could be considered as another potential biological mechanism that explains the observed effect of prenatal Mo exposure
Vázquez-Salas et al.
and infant motor neurodevelopment during the first 30 months of life. Although Mo exposure during pregnancy did not change substantially, developmental functions may be affected differently by exposure at different developmental stages. The cerebellum is a critical structure involved in motor skills. Cerebellum growth starts at the first trimester and reaches its maximum development at gestational week 30–40, during which time Mo may cause damage.23 The principal strengths of this study are that the infant neurodevelopment was evaluated longitudinally and that the main known risk factors associated with infant neurodevelopment were evaluated as potential confounders. Some potential sources of error must be evaluated to make an adequate interpretation of these results. The children included in the analyses were the final sample obtained from a random selection of all children with five or more evaluations over the first 30 months of life; in order to determine whether our results could be the consequence of a selection bias, we compared the children included with those not included in the analyses, and we found that during the first trimester of pregnancy the mean body mass index of the mothers of the children included was significantly lower than that of the mothers of the children not included. However, there is no evidence that body mass index could be a determinant in infant neurodevelopment. Furthermore, infant neurodevelopment was evaluated a long time before the Mo urinary determinations had been made, and the person in charge of the analyses of urinary Mo did not know the infant neurodevelopmental condition of the infants. Thus we ruled out differential measurement errors in the event or in the exposure as an explanation of these results. Even though we had a good internal quality control, the absence of an external quality control does not allow us to rule out the presence or the magnitude of an error in the assessment of the exposure. However, we believe that if error exists, this could not be differential in regard to infant neurodevelopment, and the association with psychomotor index would be underestimated. An additional source of error in the measurement of exposure could be the lack of control in the dilution of urine. Analyses of the information including Mo with and without creatinine adjustment, as well as the inclusion of creatinine as a covariable, showed similar findings, and the report of the final results about the association between prenatal Mo exposure and PDI or MDI takes into account Mo levels adjusted for creatinine. Finally, we did not find a significant interaction between Mo and dietary intake of copper. However, we could not rule out the
Prenatal Mo exposure and infant neurodevelopment in Mexican children
interaction between both metals because we only had dietary information, and the estimation of copper status using a food frequency questionnaire could be underestimated. Serum copper seems to be the most useful biomarker of copper status,24 and the correlation observed between dietary copper and its serum concentrations is weak (r = 0.17).25 In short, these results should be interpreted cautiously, in the context of an exploratory study that warrants more evaluation. The mean urinary Mo concentrations at which we observed psychomotor alterations in these children (40.4 ± 2.8 μg/l) are similar to those reported for women by the 2007–2008 National Health and Nutrition Examination Survey of the Unites Stated (40.5 μg/l).26 However, no information exists regarding the potential health impact of those levels in children. Only a recent study of adults showed a positive association between Mo and a higher prevalence of self-reported liver problems.6 The interest of Mo as an essential trace element, the lack of studies about Mo toxicity, and the absence of a toxicity limit demonstrates the necessity of further research in this area. The lack of knowledge about Mo also could be the reason for the absence of information regarding the potential toxic effects of Mo exposure on humans.
Acknowledgments This study was funded by grants (41708, 31034-M, 13915) from the National Council of Science and Technology of Mexico (CONACyT) and by the South-South Collaboration Program pertaining to the Fogarty International Center of the National Institutes of Health (D43TW00640) of Mount Sinai School of Medicine/Queens College International Training and Research in Environmental and Occupational Health Program. The content is solely the responsibility of the authors and does not necessarily represent the official views of the Fogarty International Center or the National Institutes of Health. The authors are grateful to Ms Patricia Castro for her assistance in contacting the families and administering the questionnaires; also to Dr Marcia Galván Portillo and MSc Wendy Elena Becerra Romero, from the National Institute of Public Health, for their support in dietary information.
References 1 Schwarz G, Mendel RW, Ribbe M. Molybdenum cofactors, enzymes and pathways. Nature 2009;460:839–46. 2 Sardesai V. Molybdenum: an essential trace element. Nutr Clin Pract 1993;8:277–81. 3 International Molybdenum association IMOA. Molybdenum markets-an end-use analysis. Molyreview 2011:2–8. 4 Minerales del recreo SA. [Consulta 8 de Octubre 2011]. Available from: http://www.asufrar.com.ar/pdf/molibdeno.pdf 5 Food and Nutrition Board Institute of Medicine Panel on Micronutrients, Subcommittees on Upper Reference Levels of
Nutritional Neuroscience
2014
VOL.
17
NO.
2
79
Vázquez-Salas et al.
6 7 8 9 10
11
12 13 14 15
80
Prenatal Mo exposure and infant neurodevelopment in Mexican children
Nutrients and of Interpretation and Uses of Dietary Reference Intakes, and the Standing Committee on the Scientific Evaluation of Dietary Reference Intakes. Dietary reference intakes for vitamin a, vitamin k, arsenic, boron, chromium, copper, iodine, iron, manganese, molybdenum, nickel, silicon, vanadium, and zinc. Washington, DC: National Academy Press; 2001. p. 420–41. Mendy A, Gasana J, Vieira ER. Urinary heavy metals and associated medical conditions in US adult population. Int J Environ Health Res 2012;22:105–18. Widjajakusuma MCR, Basrur PK, Robinson GA. Thyroid function in molybdenotic rabbits. J. Endocr 1973;57:419–24. Pitt MA. Molybdenum toxicity: interactions between copper, molybdenum and sulphate. AgentsActions 1976;6:758–69. Kovalskiy VV, Yarovaya GA. Molybdenum-infiltratedbiogeochemicalprovinces. Agrokhimiva 1966;8:68–91. Instituto nacional de estadística y geografía INEGI [documentonthe Internet]. Actividades económicas. Available from: http://cuentame.inegi.org.mx/monografias/informacion/mor/ economia/default.aspx?tema=me&e=17#sp. Torres-Sánchez L, Rothenberg SJ, Schnaas L, Cebrián ME, Osorio E, Hernández MC, et al. In utero p,p′ -DDE exposure and infant neurodevelopment: a perinatal cohort in Mexico. Environ Health Perspect 2007;115:435–9. Bayley N. Bayley Scales of Infant Development. 2nd ed. San Antonio, TX: The Psychological Corporation; 1993. Wechsler D. WAIS-Español. Escala de inteligencia para adultos. Mexico: DF. El Manual Moderno, S.A; 1981. Caldwell B, Bradley R. Home observational of measurement of the environment. Little Rock: University of Arkansas at Little Rock; 1984. Barr D, Wilder CL, Caudill SP, Gonzalez AJ, Needham LL, Pirkle JL. Urinary creatinine concentrations in the U.S. population: implications for urinary biologic monitoring measurements. Environ Health Perspect 2005;13:192–200.
Nutritional Neuroscience
2014
VOL.
17
NO.
2
16 Hernández-Avila M, Romieu I, Parra S, Hernández-Avila J, Madrigal H, Willett W. Validity and reproducibility of a food frequency questionnaire to assess dietary intake of women living in Mexico City. Salud Publica Mex 1998;39:133–40. 17 Munoz de Chávez M, Ledesma Solano J, eds. Los alimentos y sus nutrimentos. Tablas de valor Nutritivo de Alimentos. México, DF: McGraw-Hill Interamericana; 2002. p. 25. 18 Suttle NF. Copper imbalances in ruminants and humans: unexpected common ground. Adv Nutr 2012;3:666–74. 19 Penland JG, Prohaska JR. Abnormal motor function persists following recovery from perinatal copper deficiency in rats. J Nutr 2004;134:1984–8. 20 Haywood S, Dincer Z, Jasani B, Loughran MJ. Molybdenumassociated pituitary endocrinopathy in sheep treated with ammonium tetrathiomolybdate. J Comp Pathol 2004;130:21–31. 21 Haddow J, Palomaki G, Allan W, Williams J, Knight G, Gagnon J, et al. Maternal thyroid deficiency during pregnancy and subsequent neuropsychological development of the child. N Engl J Med 1999;341:549–55. 22 Anderson GW, Schoonover CM, Jones SA. Control of thyroid hormone action in developing rat brain. Thyroid 2003;13: 1039–56. 23 Volpe JJ. Cerebellum of the premature infant: rapidly developing, vulnerable, clinically important. J Child Neurol 2009;24: 1085–104. 24 Harvey LJ, Ashton K, Hooper L, Casgrain A, Fairweather-Tait SJ. Methods of assessment of copper status in humans: a systematic review. Am J ClinNutr 2009;89:2009S–24S. 25 Ghayour-Mobarhan M, Taylor A, New SA, Lamb DJ, Ferns GA. Determinants of serum copper, zinc and selenium in healthy subjects. Ann ClinBiochem 2005;42:364–75. 26 Center for diseases control and prevention (CDC). National Report on Human Exposure to Environmental Chemicals. Urinary Molybdenum, 2011. Available from: http://www.cdc .gov/exposurereport/data_tables/URXUMO_DataTables.html.
Copyright of Nutritional Neuroscience is the property of Maney Publishing and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.
Copyright of Nutritional Neuroscience is the property of Maney Publishing and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.
