Accepted Manuscript Title: Visual Evoked Potentials in Children Prenatally Exposed to Methylmercury Author: Takashi Yorifuji Katsuyuki Murata Kristian S. Bjerve Anna L Choi Pal Weihe Philippe Grandjean PII: DOI: Reference: To appear in: Received date: Revised date: Accepted date: S0161-813X(13)00049-1 http://dx.doi.org/doi:10.1016/j.neuro.2013.03.009 NEUTOX 1541 NEUTOX 1-1-2013 13-3-2013 21-3-2013 Please cite this article as: Yorifuji T, Murata K, Bjerve KS, Choi AL, Weihe P, Grandjean P, Visual Evoked Potentials in Children Prenatally Exposed to Methylmercury, Neurotoxicology (2013), http://dx.doi.org/10.1016/j.neuro.2013.03.009 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Visual Evoked Potentials in Children Prenatally Exposed to Methylmercury Takashi Yorifujia, Katsuyuki Muratab, Kristian S. Bjervec,d, Anna L Choie, Pal Weihef, Philippe Grandjeane,g a Department of Human Ecology, Okayama University Graduate School of Environmental Science, Okayama 700-8530, Japan b Department of Environmental Health Sciences, Akita University School of Medicine, Akita, Japan c Department of Medical Biochemistry, St. Olavs Hospital, Trondheim University Hospital, N-7021 Trondheim, Norway d Department of Laboratory Medicine, Children’s and Women’s Health, Norwegian University of Science and Technology, N-7491 Trondheim, Norway e Department of Environmental Health, Harvard School of Public Health, Boston, MA 02215, USA f Department of Occupational Medicine and Public Health, The Faroese Hospital System, FR-100 Tórshavn, Faroe Islands g Odense, Denmark _________________________________ Address correspondence to: Philippe Grandjean, MD Department of Environmental Medicine, University of Southern Denmark, DK-5000 Odense, Denmark Phone: +45 6550 3769 Fax: +45 6591 1458 E-mail: pgrandjean@health.sdu.dk Ac ce Department of Environmental Medicine, University of Southern Denmark, DK-5000 pt ed M 1 an us cr ip t Page 1 of 14 Abstract Prenatal exposure to methylmercury can cause both neurobehavioral deficits and neurophysiological changes. However, evidence of neurotoxic effects within the visual nervous system is inconsistent, possibly due to incomplete statistical adjustment for beneficial nutritional factors. We evaluated the effect of prenatal methylmercury exposure on visual evoked potential (VEP) latencies in Faroese children with elevated prenatal methylmercury exposure. A cohort of 182 singleton term births was assembled in the Faroe Islands during 1994-1995. At age 7 years, VEP tracings were obtained from 139 cohort subjects after exclusion of subjects with abnormal vision conditions. We used multiple regression analysis to evaluate the association of mercury concentrations in cord blood and maternal hair at parturition with VEP latencies after adjustment for potential confounders that included the cord-serum phospholipid concentration of n-3 polyunsaturated fatty acids (PUFAs) and the duration of breastfeeding. Unadjusted correlations between mercury exposure and VEP latencies were equivocal. Multiple regression models showed that increased mercury concentrations, especially in maternal hair, were associated with delayed latencies for VEP peak N145. After covariate adjustment, a delay of 2.22 ms (p=0.02) was seen for each doubling of the mercury present study suggests that prenatal methylmercury exposure may have an adverse effect on VEP findings despite the absence of clinical toxicity to the visual system. However, this association was apparent only after adjustment for n-3 PUFA status. Key words Evoked potentials; Food contamination; Methylmercury Compounds; Neurophysiological measures; Omega-3 fatty acids; Prenatal exposure delayed effects Ac ce concentration in maternal hair. In agreement with neuropsychological findings, the pt ed M 2 an us cr ip t Page 2 of 14 1. Introduction In a large-scale poisoning incident caused by methylmercury in Minamata, Japan, patients manifested a variety of neurological signs that included sensory disturbances, such as constriction of the visual fields (Harada, 1995). Recent attention to lower prenatal exposures to methylmercury has focused on subtle cognitive effects (Grandjean and Landrigan, 2006) as well as neurophysiological changes (e.g., delayed brainstem auditory evoked potential (BAEP) latencies (Murata et al. , 2007). However, although abnormalities in visual evoked potentials (VEPs) were observed in patients with methylmercury poisoning in Minamata (Imai et al. , 1991), the evidence on VEP latencies is equivocal (Murata et al. , 2007). The reason for this inconsistency may partly have been the lack of adjustment for nutritional factors (e.g., polyunsaturated fatty acids and breastfeeding) (Murata et al. , 2007). Thus, certain nutritional factors are reported to be beneficial for visual function development (Chong et al. , 2005, Decsi and Koletzko, 2005). In particular, n-3 polyunsaturated fatty acids (PUFAs), which primarily originate from fish and seafood consumption, are essential for normal brain development, especially in regard to the visual system (Innis, 1991). Such beneficial nutritional factors could be associated with et al. , 2008b). Therefore, the nutrients may cause negative confounding that leads to underestimation of the true association between methylmercury exposure and visual function (Choi et al. , 2008b). In the present study, we aim to evaluate the possible adverse effects of prenatal methylmercury exposure on VEP findings at age 7 in a Faroese birth cohort by adjusting for nutritional factors (i.e. n-3 PUFAs and duration of breastfeeding). 2. Methods 2.1. Study design and subjects 3 Page 3 of 14 Ac ce methylmercury exposure due to seafood consumption as a joint source of intake (Choi pt ed M an us cr ip t A cohort of 182 singleton term births at the National Hospital in Tórshavn, the Faroe Islands, was assembled during a 12-month period in 1994-1995 (Choi et al. , 2008a, Steuerwald et al. , 2000). Children who were born before the 36th week in gestation, or had congenital neurologic disease were excluded. At delivery, blood samples from the umbilical cord and scalp hair from the mothers (the first three cm from the root) were collected. Neurological examination of the children was carried out at age 90 months at a hospital clinic, and visual evoked potentials were recorded. The study protocol was approved by the Ethical Review Committee for the Faroe Islands, and written informed consent was obtained. At the follow-up, 25 children did not participate and therefore lacked information on VEPs. We excluded 5 births with medical risks. Moreover, we excluded 4 children with strabismus and 9 children requiring eye-glasses. Accordingly, 139 children were included in the analyses. 2.2. Measurement of Exposure We used the cord-blood and maternal hair mercury concentration as indicators of prenatal exposure to methylmercury (Grandjean et al. , 1992, Grandjean et al. , 1997). (Grandjean et al. , 1992). For subjects with a missing mercury analysis, the result was calculated from the other exposure biomarker using the average ratio between mercury concentrations in hair and cord blood. 2.3. Outcome measurements Details of the VEP recording methods are described elsewhere (Grandjean et al. , 1997, Murata et al. , 1999b). In short, pattern-reversal VEPs with binocular full-field stimulation were conducted in a darkened room. The subjects sat in a relaxed position 127 cm from the front of a 17-inch monitor screen and were asked to stare at the center 4 Page 4 of 14 Ac ce Details of the analytic methods and quality control procedures are described elsewhere pt ed M an us cr ip t of the screen. The checkerboard pattern on the screen consisted of white and black squares (mean luminance, 371 and 5 cd/m2, respectively), reversing at a rate of 2 per sec (sampling time, 0.2 ms). Two kinds of the squares were used (30 minutes and 15 minutes). The latencies of one positive and two negative peaks (P100, N75, and N145) were recorded using standard electroencephalography (EEG) electrodes fixed to the skull above the occipital cortex, the forehead, and the left mastoid (ground). 2.4. Measurement of covariates At delivery, the midwives collected information on the course of the pregnancy and the delivery, including nutritional habits and use of alcohol and tobacco during pregnancy. A parent also completed a self-administered questionnaire, which included questions about their demographic characteristics. Fatty acid concentrations in cord-serum total phospholipids were measured using gas chromatography with a flame ionization detector after extraction, isolation and transmethylation. The concentrations were recalculated from mg phospholipid fatty acid/L serum to relative concentrations, i.e. as weight percent of all 22 phospholipid fatty acids measured (Steuerwald et al. , 2000). All results were reported as relative concentrations in percent of total phospholipids 2.5. Statistical analyses Logarithmic (base 10) transformations of the cord-blood and maternal hair mercury concentrations were conducted due to highly skewed distributions. Geometric means of these exposure biomarkers were calculated. We evaluated the effects of mercury exposure (cord-blood and maternal hair concentrations) on VEP latencies using multiple linear regression models. All of the VEP latencies approximated a Gaussian distribution, thus they were used as continuous variables without transformation. We first adjusted for children’s age at examination (as a continuous parameter) and their sex as 5 Page 5 of 14 Ac ce fatty acids. pt ed M an us cr ip t mandatory covariates (Murata et al. , 1999a, Murata et al. , 1999b). We then adjusted for nutritional factors (including cord-serum total n-3 PUFAs and duration of exclusive breastfeeding) thought to have beneficial effects on visual development (Chong et al. , 2005, Decsi and Koletzko, 2005). Finally, to examine the robustness of the beta coefficients, we adjusted for the following variables: maternal smoking during pregnancy (yes/no); previous births (0/1/at least 2); and maternal alcohol drinking during pregnancy (yes/no). By multiplying beta coefficients for the log transformed mercury concentrations by 0.301 (i.e., log2), we report the absolute change of the outcome variables for each doubling of the exposure. PASW Statistics software (SPSS Japan Inc., version 18.0J) was used for descriptive analyses and regression models. We report two-sided p-values. 3. Results Geometric averages of cord-blood and maternal hair mercury were 22.8 µg/L and 4.6 µg/g, respectively (Table 1) in accordance with the increased levels of methylmercury exposure. Cord-serum fatty acids and mercury concentrations (both in cord blood and 0.39 (p<0.01) for log transformed cord-blood mercury and 0.32 (p<0.01) for log transformed maternal hair mercury (data not shown). Table 2 shows the results of the absolute changes for the cord-blood mercury concentration as predictor of VEP latencies. In the fully-adjusted multivariate model, the delay in the P100 latency at 15 minutes was 0.62 ms (p=0.18) for each doubling of the cord-blood mercury concentration. Associations between cord-blood mercury and other latencies were in the same direction, although p-values were larger. A positive tendency was observed when we employed maternal hair mercury as an exposure biomarker (Table 3). In this case, a doubling in maternal hair mercury was 6 Page 6 of 14 Ac ce maternal hair) were positively correlated, with EPA showing correlation coefficients of pt ed M an us cr ip t associated with an increased N145 latency at 15 minutes. When we adjusted for nutritional factors as well as other potential confounders, a greater delay of 2.22 ms (p=0.02) was seen for each doubling of the maternal hair mercury concentration. 4. Discussion In the present study, we found that higher mercury concentrations were associated with the prolonged latencies of VEP, in particular higher maternal hair mercury was associated with the prolonged N145 latency. This is consistent with the previous studies from a Madeiran fishing community and an Inuit community in Northern Québec which suggested possible adverse effects of mercury on VEP (Ethier et al. , 2012, Murata et al. , 1999b, Saint-Amour et al. , 2006). Although the previous Faroe Islands birth cohort studies did not observe adverse effects on VEP (Grandjean et al. , 1997, Murata et al. , 1999a), the possible reasons for the difference between the present and previous studies conducted in the Faroe Islands are that the previous studies failed to adjust for nutritional factors, in particular the n-3 PUFA status (Grandjean et al. , 1997, Murata et al. , 1999a). In agreement with the findings on BAEP latencies (Murata et al. , 2007), the prolonged latencies of VEP, in particular at N145. VEP responses depend on the integrity of the optic pathway from optic nerve to visual cortex (Otis and Handler, 1995) and are believed to reflect cortically generated responses (Chiappa and Hill, 1997). In particular, the wave of P100 latency is considered to be generated from the primary visual cortex involved in visual activity (Chiappa and Hill, 1997). The N145 peak of the visual evoked potentials is regarded a composite response from the visual cortex and nearby extrastriate areas involved in visual perception (Celsia, 1992), i.e. possibly the higher function of the visual system. Therefore, three latencies (N75, P100, and N145) may represent different functions of the visual system and these functions may develop 7 Page 7 of 14 Ac ce present study shows that prenatal exposure to methylmercury is associated with pt ed M an us cr ip t at different times. In fact, the present and previous studies observed adverse effects on each of the three latencies (Ethier et al. , 2012, Imai et al. , 1991, Murata et al. , 1999b, Saint-Amour et al. , 2006). While some statistical overlap was present, the reason for any discrepancy is unknown. The findings of our study support the notion that prenatal methylmercury has an adverse effect on VEP data even at exposure levels below those encountered in Minamata. However, this association was substantially strengthened after appropriate adjustment for nutritional factors, in particular the n-3 PUFA status. Such deficit in neurophysiological finding due to low-level exposure to methylmercury may be an initial sign of more severe effect on visual function as observed in Minamata and a useful tool to detect effects of low-level exposure to methylmercury at a population-level. Acknowledgments This study was supported by grants from the US National Institute of Environmental Health Sciences (ES09797 and ES11687) and the European Commission (ANEMONE, QLK4-CT-2001-00186). We thank Katherine Herz for editorial help with the Conflict of Interest Statement The authors declare that there are no conflicts of interest. In the interest of transparency, the author (PG) declares that he has provided paid expert testimony on mercury toxicology for the U.S. Department of Justice in a legal case concerning environmental pollution from coal-fired power plants. Ac ce manuscript. pt ed M 8 Page 8 of 14 an us cr ip t References Celsia GG. Visual Evoked Potentials in Clinical Neurology In Electrodiagnosis in clinical neurology Aminoff MJ. (ed). 3rd ed. New York: Churchill Livingstone; 1992. Chiappa KH, Hill RA. Pattern-shift visual evoked potentials: interpretation In Evoked potentials in clinical medicine, 3d. Chiappa K.H. (ed). 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Grandjean P, Weihe P, Jørgensen PJ, Clarkson T, Cernichiari E, Videro T. Impact of maternal seafood diet on fetal exposure to mercury, selenium, and lead. Arch Environ Health. 1992;47:185-95. Grandjean P, Weihe P, White RF, Debes F, Araki S, Yokoyama K, et al. Cognitive deficit in 7-year-old children with prenatal exposure to methylmercury. Neurotoxicol Teratol. 1997;19:417-28. 9 Page 9 of 14 Ac ce environmental contaminant exposure on visual brain development: a prospective pt ed M an us cr ip t Harada M. Minamata disease: methylmercury poisoning in Japan caused by environmental pollution. Crit Rev Toxicol. 1995;25:1-24. Imai H, Inayoshi S, Okajima T. Visual evoked potentials in Minamata disease. Jpn J EEG EMG. 1991;19:353-63 (in Japanese). Innis SM. Essential fatty acids in growth and development. Progress in lipid research. 1991;30:39-103. Murata K, Grandjean P, Dakeishi M. Neurophysiological evidence of methylmercury neurotoxicity. Am J Ind Med. 2007;50:765-71. Murata K, Weihe P, Araki S, Budtz-Jørgensen E, Grandjean P. Evoked potentials in Faroese children prenatally exposed to methylmercury. Neurotoxicol Teratol. 1999a;21:471-2. Murata K, Weihe P, Renzoni A, Debes F, Vasconcelos R, Zino F, et al. Delayed evoked potentials in children exposed to methylmercury from seafood. Neurotoxicol Teratol. 1999b;21:343-8. Otis J, Handler J. Evoked potentialtesting in clinical neurotoxicology In Neurotoxicology : approaches and methods. Chang LW and Slikker W. (eds). San Diego: Academic Press; 1995. of visual evoked potentials in preschool Inuit children exposed to methylmercury and polychlorinated biphenyls from a marine diet. Neurotoxicology. 2006;27:567-78. Steuerwald U, Weihe P, Jørgensen PJ, Bjerve K, Brock J, Heinzow B, et al. Maternal seafood diet, methylmercury exposure, and neonatal neurologic function. J Pediatr. 2000;136:599-605. Ac ce Saint-Amour D, Roy MS, Bastien C, Ayotte P, Dewailly E, Despres C, et al. Alterations pt ed M 10 an us cr ip t Page 10 of 14 Table 1. Characteristics of 139 Faroese birth cohort members participating in the clinical examination. Age at examination (years) Sex of child (boy/girl) (%) Total n-3 polyunsaturated fatty acids (Weight % of 117 total in cord serum phospholipids)* Total n-3 polyunsaturated fatty acids (Weight % of 134 total in maternal serum phospholipids)* Duration of exclusive breast-feeding (months)* 139 Previous births (0/1/at least 2) (%) 139 Smoking during pregnancy (no/yes) (%) 139 Alcohol consumption during pregnancy (no/yes) (%) 139 125 Cord blood mercury (µg/L) † Maternal hair mercury (µg/L) † 133 a VEP latency (30 minutes) (ms) N75 138 P100 138 N145 138 a VEP latency (15 minutes) (ms) N75 139 P100 139 N145 139 a arithmetic mean ± SD; † geometric mean (interquartile range) VEP: Visual evoked potential a M 11 an us ed Ac ce pt cr ip t 10.9 ± 1.9 11.8 ± 2.0 3.5 ± 2.1 28.8/28.8/42.4 70.5/29.5 85.6/14.4 22.8 (13.7 - 41.2 ) 4.6 (2.7 - 8.2 ) 72.8 ± 3.1 105.9 ± 4.7 138.3 ± 10.8 77 ± 3.7 109.6 ± 5.6 146.1 ± 10.3 Page 11 of 14 N 139 139 Result 7.5 ± 0.1 48.2/51.8 Table 2. Difference (ms) in visual evoked potential latencies associated with a doubling in the cord blood mercury concentration. Additionally adjusted for cord-serum total n-3 polyunsaturated fatty acids and duration of exclusive breast-feeding 0.31 (0.22) 0.14 (0.73) -0.22 (0.80) 0.07 (0.83) 0.42 (0.35) 0.95 (0.27) Adjusted for age at examination and sex Additionally adjusted for other covariatesa 30 minutes N75 P100 N145 15 minutes N75 P100 N145 a b 0.16 (0.57) 0.46 (0.28) 0.69 (0.39) us cr 0.33 (0.15) b 0.20 (0.58) -0.44 (0.60) Ac ce pt ed M 12 Page 12 of 14 an Previous births, smoking during pregnancy, and alcohol consumption. P-values are shown in the parentheses. ip t 0.32 (0.22) 0.20 (0.62) -0.13 (0.88) 0.13 (0.69) 0.62 (0.18) 1.01 (0.26) Table 3. Difference (ms) in visual evoked potential latencies associated with a doubling in the maternal hair mercury concentration at parturition. Additionally adjusted for cord-serum total n-3 polyunsaturated fatty acids and duration of exclusive breast-feeding 0.22 (0.41) 0.16 (0.70) 0.83 (0.39) a b Ac ce pt ed 13 Page 13 of 14 M Previous births, smoking during pregnancy, and alcohol consumption. P-values are shown in the parentheses. an N75 P100 N145 0.01 (0.98) 0.35 (0.45) 1.58 (0.06) -0.13 (0.71) 0.29 (0.55) 2.05 (0.03) us N75 P100 N145 15 minutes 0.24 (0.34)b 0.16 (0.67) 0.43 (0.64) cr 30 minutes ip t 0.23 (0.42) 0.25 (0.58) 1.09 (0.27) -0.08 (0.83) 0.57 (0.26) 2.22 (0.02) Adjusted for age at examination and sex Additionally adjusted for other covariatesa Highlights An international research group from the US, Japan, Norway, Denmark, and the Faroe Islands carried out a prospective study on a Faroese birth cohort. Exposure to methylmercury from maternal seafood diets was increased Visual evoked potentials showed increased methylmercury-associated latencies on visual evoked potentials. These delays became statistically significant only after adjustment for negative confounding due to essential fatty acids from fish oil. Ac ce pt ed 14 Page 14 of 14 M an us cr ip t