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Significance of fingernail and toenail mercury concentrations as biomarkers for prenatal methylmercury exposure in relation to segmental hair mercury concentrations
Environmental Research 136 (2015) 289–294
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Significance of fingernail and toenail mercury concentrations as biomarkers for prenatal methylmercury exposure in relation to segmental hair mercury concentrations Mineshi Sakamoto a,n, Hing Man Chan b, José L Domingo c, Ricardo Boas Oliveira d, Shoichi Kawakami e, Katsuyuki Murata f a
Department of Environmental Sciences and Epidemiology, National Institute for Minamata Disease, Minamata, Japan Center for Advanced Research in Environmental Genomics, Department of Biology, University of Ottawa, Ottawa, ON, Canada Laboratory of Toxicology and Environmental Health, School of Medicine, IISPV, Universitat "Rovira i Virgili", Reus, Spain d Laboratório de Bioprospecção e Biologia Experimental, Campus Universitário de Santarém, Universidade Federal do Oeste do Pará, Santarém, Brazil e Department of Obstetrics and Gynecology, Fukuda Hospital, Kumamoto, Japan f Department of Environmental Health Sciences, Akita University School of Medicine, Akita, Japan b c
art ic l e i nf o
a b s t r a c t
Article history: Received 1 August 2014 Received in revised form 16 September 2014 Accepted 18 September 2014
Objective: To investigate the appropriateness of mercury (Hg) concentrations in fingernails and toenails at parturition for detecting prenatal exposure to methylmercury (MeHg). Methods: Total Hg concentrations were measured in 54 paired samples of fingernails, toenails, maternal blood, and maternal hair (1 cm incremental segments from the scalp toward the tip) collected at 4th weeks of (early) pregnancy, and the same specimens and cord blood collected at parturition. Results: Strong correlations were observed between Hg concentrations in fingernails and toenails at early pregnancy (r ¼0.923, p o0.01) and at parturition (r ¼ 0.895, po 0.01). At early pregnancy, Hg concentrations in fingernails and toenails showed the strongest correlations with those in hair 3–4 cm from the scalp (r ¼0.818 and r ¼ 0.747, po0.01, respectively) among the 1 cm incremental hair segments. Mercury concentrations in fingernails and toenails at parturition represented strong correlations with those in cord blood (r ¼0.803, p o0.01 for fingernails and r¼ 0.792, po 0.01 for toenails, respectively). At parturition, Hg concentrations in fingernails had the highest correlation with those in hair 0–1 cm from the scalp (r ¼0.918, po0.01), and Hg concentrations in toenails showed the highest correlation with those in hair at 2–3 cm from the scalp (r ¼ 0.872, po 0.01). Conclusions: Mercury in fingernails and toenails at early pregnancy reflected the maternal Hg body burden level approximately 5 months retroactively. At parturition, Hg levels in fingernails and toenails also showed strong correlations with those in cord blood. In addition, Hg levels in fingernails and toenails at parturition reflected more recent MeHg exposure, compared with those at early pregnancy. These results suggest that fingernails and toenails at parturition are useful biomarkers for prenatal MeHg exposure for mothers and fetuses, especially during the early third-trimester of gestation. & 2014 Elsevier Inc. All rights reserved.
Keywords: Methylmercury Mercury Fingernail Toenail Hair segment Fetus Gestation
1. Introduction Methylmercury (MeHg) is a widespread environmental neurotoxin. The organ targeted by MeHg exposure during gestation is the fetal brain, especially the developing brain during the third
Abbreviations: ; Hg, mercury; MeHg, methylmercury; CV, coefficient of variation; CVAAS, cold vapor atomic absorption spectrophotometry; ICP-MS, inductively coupled plasma mass spectrometry n Corresponding author. E-mail address: [email protected] (M. Sakamoto). http://dx.doi.org/10.1016/j.envres.2014.09.034 0013-9351/& 2014 Elsevier Inc. All rights reserved.
trimester (Rice and Barone, 2000). For this reason, biomarkers reflecting the MeHg exposure level in the fetus during the third trimester are very important for predicting the effects of MeHg on child development. In the Faroe Islands study, cord blood mercury (Hg) concentration was the preferred biomarker for MeHg exposure, although maternal hair Hg was also analyzed (Grandjean et al., 1997; Murata et al., 2004). In the Seychelles study, maternal hair Hg concentration was used as the only biomarker for fetal exposure (Davidson et al., 1998). Each biomarker has its advantages and disadvantages. Cord blood circulates in the fetal body and can directly reflect the MeHg concentrations in the fetal
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organs, including the fetal brain at birth (Cernichiari et al., 1995a; NRC, 2000). Although hair Hg analysis is associated with a number of variables such as the hair’s growth rate, density, color, waving, external contamination, and permanent treatment (WHO, 1990), segmental analysis of maternal hair is able to provide time-course information, because the average hair growth rate is commonly assumed to be about 1 cm per month (Boischio et al., 2000; Cernichiari et al., 1995a). However, because the hair follicle grows out of the skin surface after about 3 weeks, Hg concentrations in the first hair segment proximal to the scalp (0–1 cm from the scalp) will only reflect the MeHg exposure from 3 weeks before (Cernichiari et al., 1995a; Phelps et al., 1980; Yaginuma–Sakurai et al., 2012). Therefore, maternal hair Hg concentrations at parturition may not reflect the prenatal MeHg exposure during the last 3 weeks of gestation. A number of studies have employed Hg concentrations in toenails and/or fingernails as biomarkers for MeHg exposure (Alfthan, 1997; Hinners et al., 2012; Morton et al., 2004; Ohno et al., 2006; Rees et al., 2007; Suzuki et al., 1989; Yoshizawa et al., 2002). In most of these studies, toenails rather than fingernails were preferred, because toenails are often less contaminated than fingernails, especially among dental personnel and gold miners who handle Hg amalgam (Morton et al., 2004; Wickre et al., 2004). Nevertheless, the time-lag for nail growth from the nail matrix to the nail edge was not considered for analyzes in the abovementioned studies. Furthermore, the appropriateness of using fingernails and toenails as biomarkers for maternal and fetal MeHg exposure at parturition has not yet been validated. In this study, we conducted simultaneous analyzes of fingernails, toenails, maternal blood, umbilical cord blood, and maternal hair segments to investigate how Hg concentrations in nails are related to the other biomarkers, especially at parturition. We measured total Hg concentrations as a surrogate for MeHg concentrations in the samples. The overall hypothesis is that Hg concentrations in both toenails and fingernails at parturition strongly reflect the fetal exposure to MeHg during the third trimester.
approved by the Ethics Committee of the National Institute for Minamata Disease, Minamata, Japan. 2.2. Hg analytical methods The total Hg concentrations in approximately 0.5 ml of blood, 5–10 mg of hair, and 10–13 mg of nails were determined by cold vapor atomic absorption spectrophotometry (CVAAS) according to a previously described method (Akagi et al., 2000) using a mercury analyzer Model Hg-201 (Sanso Seisakusho Co. Ltd., Tokyo, Japan). The method involved sample digestion with HNO3, HClO4, and H2SO4 followed by reduction to Hg0 by SnCl2. The method detection limit was 0.01 ng/g. The accuracy of Hg analysis in blood samples was ensured using a reference blood material (Level 2, MR9067; Nycomed Co., Oslo, Norway). The mean value (n ¼5) of the determined Hg was 7.5 μg/L, which was within the recommended range of 6.8–8.5 μg/L as measured by inductively coupled plasma mass spectrometry (ICP-MS). Reference human hair (NIES CRM No. 13 Human Hairs; National Institute for Environmental Studies, Environmental Agency of Japan) was also measured as quality control. The mean value (n ¼5) of the determined total Hg was 4.32 ng/mg, which was within the certified range of 4.22– 4.62 ng/mg. A coefficient of variation (CV) of 0.8% was obtained from 5-time repeated measurements of a standard solution (50 ng Hg/ml) analysis. Hg levels were expressed as concentrations (ng/g) in blood, hair, and nail samples. 2.3. Statistical analysis Mercury concentrations for all biomarkers were expressed as geometric means and 25th–57th percentiles because the values were log-normally distributed. For the statistical analysis, logarithmically-transformed Hg concentrations were used. Differences in Hg concentrations were evaluated by a paired t-test. The strength of the relationships among the Hg concentrations in blood, hair segments, and nail samples was analyzed by the Pearson product-moment correlation coefficient. Values of pr 0.05 were considered as statistically significant.
2. Materials and methods 3. Results 2.1. Sample collection Fifty-four healthy Japanese pregnant women without any occupational exposure to Hg compounds and planning to undergo delivery at Fukuda Hospital, Kumamoto, Japan from 2006 to 2007, provided informed consent at their first pregnancy check-up to participate in the study. Their ages ranged from 21 to 41 years (mean: 29.6 74.4 years). None of the women had undergone permanent hair treatments for at least 6 months prior to participation, and they were asked not to do the treatments during gestation. Fasting venous blood samples were collected from the participants at the 4th week of (early) pregnancy in the morning and 1 day after parturition. Umbilical cord venous blood was collected at parturition. Blood samples were obtained by venipuncture and collected into heparin-Na-containing vacutainer tubes. All samples were stored at 80 °C until analysis. Approximately 50 full-length strands of maternal hair were collected at early pregnancy and parturition by cutting the strands close to the scalp in the occipital area. Each maternal hair strand was cut into 1 cm incremental segments from the scalp end toward the tip. Approximately 1 mm clippings of the free edges of fingernails and toenails were also collected at early pregnancy and at parturition. Nail polish found on one sample at early pregnancy was removed using acetone. Hair and nail samples were stored at room temperature until analysis. The study protocol was reviewed and
3.1. Hg in nails and other biomarkers at early pregnancy and parturition The geometric means and 25th–75th percentiles of the Hg concentrations (ng/g) in maternal blood, cord blood, fingernails, toenails, and 1 cm hair segments from the scalp to the tip at early pregnancy and at parturition are summarized in Table 1. Among the maternal specimens, the highest Hg concentration was observed in hair, followed by fingernails, toenails, and blood at both sampling periods. Mercury ratios of 3 specimens (hair 1–3 cm from the scalp, fingernails, and toenails) to maternal blood (mean and range in parentheses) were 307 (118–606), 130 (63.1–241), and 111 (61.6–198) at early pregnancy, and 348 (148–506), 134 (47.8–231), and 114 (35.0–224) at parturition, respectively. At parturition, Hg level in cord blood was significantly (p o0.01) higher than that in maternal blood, with an average ratio of 1.84. Mercury concentrations in maternal blood, fingernails, and toenails at parturition were significantly lower than those at early pregnancy (p o0.05 for fingernails, and p o0.01 for maternal blood and toenails). Fig. 1 depicted the correlations of Hg concentrations between fingernails and toenails at early pregnancy (A) and at parturition (B). Mercury concentrations in fingernails and toenails showed the significant correlations at early pregnancy (r ¼0.923, p o0.01) and parturition (r ¼0.895, po 0.01).
M. Sakamoto et al. / Environmental Research 136 (2015) 289–294
Table 1 Geometric means (25th–75th percentiles) of Hg concentrations (ng/g) in maternal blood, cord blood, fingernails, toenails, and hair segments from scalp to tip at early pregnancy and parturition.
Table 2 Correlations among Hg concentrations (ng/g) in maternal blood, fingernails, toenails, and hair segments from scalp to tip at early pregnancy. 54 Samples
Parturition
4.40 (3.58–5.44) 547 (425–727) 465 (358–624)
3.89 (3.25–4.69)n 7.14 (6.02–9.38)n 504 (426–585)nn 427 (350–544)n
1268 (941–1798) 1276 (928–1827) 1261 (885–1850) 1244 (892–1922) 1234 (898–1844) 1215 (851–1840) 1194 (844–1686) 1141 (844–1714) 1159 (845–1648)
1314 (1090–1709) 1257 (1030–1540) 1243 (1019–1533) 1252 (1045–1573) 1234 (1018–1605) 1241 (1032–1535) 1238 (1109–1622) 1272 (1055–1622) 1273 (1042–1744)
Differences in Hg concentrations between paired samples were determined by a paired t-test using logarithmically-transformed Hg concentrations. Hg concentrations in maternal blood, fingernails, and toenails at parturition were significantly (npo 0.05, nnp o 0.01) lower than those at early pregnancy. Hg concentration in cord blood was significantly (npo 0.01) higher than that in maternal blood at parturition. n
po 0.01. p o0.05.
nn
3.2. Correlations of Hg among blood, nails and segmental hair at early pregnancy Table 2 summarized the correlation coefficients matrix for Hg concentrations in maternal blood, fingernails, toenails, and all maternal hair segments from the scalp to the tip at early pregnancy. Significant correlations (p o0.01) were observed among all the biomarkers at early pregnancy. Among the 1 cm incremental maternal hair segments, both Hg concentrations in fingernails and toenails showed their highest correlation coefficients with Hg concentrations in hair at 3–4 cm from the scalp (r ¼ 0.818, p o0.01 for fingernails and r ¼0.744, p o0.01 for toenails). 3.3. Correlations of Hg among blood, nails and segmental hair at parturition
Hg in toenails (ng/g)
The matrix of correlation coefficients for Hg concentrations in maternal blood, cord blood, fingernails, toenails, and hair segments from the scalp to the tip at parturition is summarized in Table 3. Significant positive correlations (po0.01) were observed
p
Hg in fingernails (ng/g)
Correlation coefficients
Maternal blood Fingernail Toenail Hair segment (cm) from the scalp 0–1 1–2 2–3 3–4 4–5 5–6 6–7 7–8 8–9
Maternal blood
Fingernail Toenail
1 0.735 0.707
1 0.923
1
0.661 0.669 0.630 0.633 0.599 0.576 0.545 0.438 0.468
0.787 0.784 0.780 0.818 0.813 0.806 0.779 0.616 0.692
0.711 0.714 0.706 0.744 0.733 0.734 0.718 0.588 0.643
Correlation coefficients were calculated using logarithmically-transformed Hg concentrations. All correlation coefficients showed statistical significance (p o 0.01).
Table 3 Correlations among Hg concentrations (ng/g) in maternal blood, cord blood, fingernails, toenails, and hair segments from scalp to tip at parturition. 54 Samples
Correlation coefficients Maternal blood
Maternal blood 1 Cord blood 0.878 Fingernail 0.697 Toenail 0.689 Hair segment (cm) from the scalp 0–1 0.842 1–2 0.772 2–3 0.749 3–4 0.737 4–5 0.722 5–6 0.688 6–7 0.617 7–8 0.612 8–9 0.471
Cord blood
Fingernail Toenail
1 0.803 0.792
1 0.895
1
0.918 0.870 0.846 0.792 0.779 0.739 0.680 0.638 0.492
0.878 0.875 0.873 0.825 0.749 0.716 0.662 0.631 0.513
0.868 0.871 0.872 0.835 0.794 0.791 0.730 0.737 0.595
Correlation coefficients were calculated using logarithmically-transformed Hg concentrations. All correlation coefficients showed statistical significance (p o 0.01).
Hg in toenails (ng/g)
Maternal blood Cord blood Fingernail Toenail Hair segment (cm) from the scalp 0–1 1–2 2–3 3–4 4–5 5–6 6–7 7–8 8–9
Early pregnancy
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p
Hg in fingernails (ng/g)
Fig. 1. Correlations of Hg concentrations between fingernails and toenails at early pregnancy (A) and parturition (B). *p o 0.01. Dotted lines denote the 95% confidence intervals for the regression lines.
10
Hg in cord blood (ng/g)
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Hg in cord blood (ng/g)
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r = 0.803 p < 0.01 (n = 54)
5
2 1
100 200
r = 0.792 p < 0.01 (n = 54)
10 5
2 1
500 1000
100
Hg in fingernails (ng/g)
200
500 1000
Hg in toenails (ng/g)
Fig. 2. Correlations of Hg concentrations between cord blood and fingernails (A) and between cord blood and toenails (B) at parturition. *po 0.01. Dotted lines denote the 95% confidence intervals for the regression lines.
among all the biomarkers at parturition. Mercury in fingernails and toenails showed strong correlations with those in cord blood (r ¼0.803 for fingernails and r ¼ 0.792 for toenails, respectively). The correlations of Hg concentrations between fingernails and cord blood (A) and those between toenail and cord blood (B) at parturition are depicted in Fig. 2. Among the 1 cm incremental hair segments, Hg concentrations in fingernails showed the highest correlation coefficient with those in hair at 0–1 cm from the scalp (r ¼0.918, po 0.01) and Hg concentrations in toenails showed the highest correlation coefficient with Hg in hair at 2–3 cm from the scalp (r ¼0.872, p o0.01). However, the correlation coefficients of Hg concentrations between nails and hair segments at parturition were equally high among hair at 0–1, 1– 2, and 2–3 cm from the scalp. Fig. 3 depicted the correlations of Hg concentrations between fingernails and hair segments at 0–1 cm from the scalp (A) and those between toenails and hair at 3–4 cm from the scalp (B) at parturition.
4. Discussion This is the first comprehensive study investigating the appropriateness of using Hg concentrations in fingernails and toenails as biomarkers for maternal and fetal MeHg exposure at parturition, compared with those at early pregnancy. Our results indicated that Hg concentrations in both fingernails and toenails reflected the
10000
Hg in hair 2-3cm (ng/g)
Hg in hair 0-1cm (ng/g)
10000
maternal and fetal body burdens of MeHg, especially throughout the early third trimester of gestation. Keratin is the main structural protein of hair and nails, containing large amounts of sulfur-containing amino acids (Sass et al., 2004). In particular, the thiol (–SH) groups of the amino acid cysteine, and the proteins containing cysteine, have very high affinity for MeHg (Cernichiari et al., 1995b). Mercury concentrations in fingernails, toenails, hair and maternal blood showed significant correlations at early gestation and parturition. However, among the maternal specimens, the highest Hg concentration was observed in hair, followed by fingernails, toenails, and blood at both sampling periods. The higher Hg concentrations found in hair segments than in nails in the current study can be explained by the higher sulfur-containing amino acid contents in hair strands compared with nails (Baden et al., 1973; Gupchup and ZatZ, 1999). Fingernails and toenails are formed continually from the nail matrix (Gupchup and ZatZ, 1999), and the Hg concentrations in these nails reflect the MeHg concentrations in blood at the time of the nail's formation. Our results showed that Hg concentrations in fingernails were higher than those in toenail at both early pregnancy and parturition. This is consistent with findings reported in previous investigations (Goulle et al., 2009; Hislop et al., 1982; Suzuki et al., 1989). The participants in this study were ordinary Japanese pregnant women of similar age, had no known external Hg exposure, and did not use any permanent hair treatments before or during gestation. Therefore, most of the known confounding factors affecting the hair and nail Hg analysis
r = 0.878 p < 0.01 (n = 54)
1000 500 200 100 100
200
500
1000
Hg in fingernails (ng/g)
r = 0.872 p < 0.01 (n = 54)
1000 500 200 100 100
200
500
1000
Hg in toenails (ng/g)
Fig. 3. Correlations of Hg concentrations between fingernails and maternal hair 0–1 cm from the scalp (A) and between toenails and maternal hair 2–3 cm from the scalp (B) at parturition. *p o 0.01. Dotted lines denote the 95% confidence intervals for the regression lines.
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were controlled. The only a few factors that can affect the Hg concentrations in hair and nails are their growth rates and MeHg incorporation into their matrices during gestation. Mercury concentrations in fingernails and toenails showed significant positive correlations with those in maternal and cord blood and all hair segments at parturition, indicating that both fingernails and toenails are equally useful as biomarkers for maternal and fetal exposure to MeHg in subjects who are free from external pollution to Hg compounds. At parturition, cord blood Hg was 1.84 times higher than maternal blood Hg. The higher MeHg accumulation in cord blood, as compared with maternal blood, has been previously reported (Sakamoto et al., 2002, 2013; Stern and Smith, 2003). This can be explained by the high MeHg transfer across the placenta, via the active neutral amino acid transport system (Aschner and Clarkson, 1988; Kajiwara et al., 1996). This high placental transfer is a specific characteristic of MeHg, differing from other metals (Sakamoto et al., 2010, 2013). The lower Hg concentrations in maternal blood, fingernails, and toenails at parturition compared to those at early pregnancy can be explained by the high MeHg transfer from the mother to the fetus during gestation. Although the reason is not clear, a significant decline of Hg concentration was not observed in the first hair segment proximal to the scalp. Both Hg concentrations in fingernails and toenails collected at early pregnancy showed the highest correlation coefficients with those in hair 3–4 cm from the scalp. These findings suggest that there was a time lag of approximately 5 months between MeHg incorporation from blood into the nail matrix and its appearance in the free edge, including a delay of approximately 3 weeks between MeHg incorporation from blood into growing hair strands and its appearance above the scalp (Cernichiari et al., 1995a; Phelps et al., 1980; Yaginuma–Sakurai et al., 2012). Hislop et al. (1982) observed the first peak of Hg in blood, followed by peaks in hair, fingernails, and toenails after fish consumption by 20 volunteers. The fingernail growth rate is approximately 3 mm per month, and fingernails require approximately 6 months to regrow completely (Gupchup and ZatZ, 1999; Hamilton et al., 1955). The growth rate of toenails is approximately 1 mm per month, being 30–50% of the growth rate of fingernails. However, toenails are shorter than fingernails, except for the lengths of the big toe and thumb, and the growth rates of nails are largely dependent on their length from the nail bed (Gupchup and ZatZ, 1999). These results will partly explain the reason why Hg concentrations in fingernails and toenails at early pregnancy showed equally high correlation coefficients with Hg concentrations in hair 3–4 cm from the scalp. However, further studies are needed to confirm the exact kinetics. The most significant findings of this study are the strong positive relationships between Hg concentrations in nails and cord blood collected at parturition, and the relationships between Hg concentrations in nails and 1 cm incremental hair segments grown during gestation. The changes in the amino acid metabolism in the maternal body start at early pregnancy, and the resulting protein accumulation is accelerated by the growth of the fetus (Kalhan, 2000). However, changes in growth rates and MeHg incorporation in hair and nails will also occur during gestation. Both Hg concentrations in fingernails and toenails showed their highest correlation coefficients with those in hair 3–4 cm from the scalp at early pregnancy. In contrast to the results for samples collected from early pregnancy, correlation coefficients of Hg concentrations between nails and hair segments at parturition were equally high among hair 0–1, 1–2, and 2–3 cm from the scalp. Consequently, fingernails and toenails can indicate MeHg exposure to the mother and fetus throughout the 2–4 months prior to parturition, including the delay of approximately 3 weeks between MeHg incorporation from blood into growing
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hair strands and its appearance above the scalp. The entire period 2–4 months prior to parturition corresponds to the early thirdtrimester of gestation. The migration of the peak correlation coefficient, between Hg concentrations in nails and hair segments 3–4 cm from the scalp at early pregnancy to the hair at 0–1, 1–2, 3–4 cm from the scalp at parturition, can be explained either by a decrease in the hair growth rate at late gestation (Pecoraro et al., 1971), or an increase in the growth rate of fingernails from mid to late gestation (Hewitt and Hillman, 1966). However, no study yet has examined the differences in toenail growth rates during gestation. In conclusion, Hg concentrations in fingernails and toenails at early pregnancy reflected the maternal MeHg body burden level approximately 5 months retroactively. Moreover, Hg concentrations in fingernails and toenails at parturition can be useful biomarkers for prenatal MeHg exposure to mothers and fetuses, especially throughout the early third trimester of gestation.
Acknowledgments This work was supported by a grant for Comprehensive Research of Minamata Disease from the Ministry of the Environment, Japan. It was also supported by JSPS KAKENHI Grant Number 23510085. We express our deep gratitude to the women who generously participated in the study.
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Rice, D., Barone Jr., S., 2000. Critical periods of vulnerability for the developing nervous system: evidence from humans and animal models. Environ. Health Perspect. 108 (Suppl. 3), S511–S533. Sakamoto, M., Kakita, A., Wakabayashi, K., Takahashi, H., Nakano, A., Akagi, H., 2002. Evaluation of changes in methylmercury accumulation in the developing rat brain and its effects: a study with consecutive and moderate dose exposure throughout gestation and lactation periods. Brain Res. 949, 51–59. Sakamoto, M., Murata, K., Kubota, M., Nakai, K., Satoh, H., 2010. Mercury and heavy metal profiles of maternal and umbilical cord RBCs in Japanese population. Ecotoxicol. Environ. Saf. 73, 1–6. Sakamoto, M., Yasutake, A., Domingo, J.L., Chan, H.M., Kubota, M., Murata, K., 2013. Relationships between trace element concentrations in chorionic tissue of placenta and umbilical cord tissue: potential use as indicators for prenatal exposure. Environ. Int. 60, 106–111. Sass, J.O., Skladal, D., Zelger, B., Romani, N., Utermann, B., 2004. Trichothiodystrophy: quantification of cysteine in human hair and nails by application of sodium azide-dependent oxidation to cysteic acid. Arch. Dermatol. Res. 296, 188–191. Stern, A.H., Smith, A.E., 2003. An assessment of the cord blood: maternal blood methylmercury ratio: implications for risk assessment. Environ. Health Perspect. 111, 1465–1470. Suzuki, T., Watanabe, S., Matsuo, N., 1989. Comparison of hair with nail as index media for biological monitoring of mercury. Jpn. J. Ind. Health 31, 235–238. WHO, 1990. Methylmercury. Environmental Health Criteria. 101. World Health Organization, Geneva. Yaginuma-Sakurai, K., Murata, K., Iwai-Shimada, M., Nakai, K., Kurokawa, N., Tatsuta, N., Satoh, H., 2012. Hair-to-blood ratio and biological half-life of mercury: experimental study of methylmercury exposure through fish consumption in humans. J. Toxicol. Sci. 37, 123–130. Yoshizawa, K., Rimm, E.B., Morris, J.S., Spate, V.L., Hsieh, C.C., Spiegelman, D., Stampfer, M.J., Willett, W.C., 2002. Mercury and the risk of coronary heart disease in men. N. Engl. J. Med. 347, 1755–1760.
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Significance of fingernail and toenail mercury concentrations as biomarkers for prenatal methylmercury exposure in relation to segmental hair mercury concentrations Mineshi Sakamoto a,n, Hing Man Chan b, José L Domingo c, Ricardo Boas Oliveira d, Shoichi Kawakami e, Katsuyuki Murata f a
Department of Environmental Sciences and Epidemiology, National Institute for Minamata Disease, Minamata, Japan Center for Advanced Research in Environmental Genomics, Department of Biology, University of Ottawa, Ottawa, ON, Canada Laboratory of Toxicology and Environmental Health, School of Medicine, IISPV, Universitat "Rovira i Virgili", Reus, Spain d Laboratório de Bioprospecção e Biologia Experimental, Campus Universitário de Santarém, Universidade Federal do Oeste do Pará, Santarém, Brazil e Department of Obstetrics and Gynecology, Fukuda Hospital, Kumamoto, Japan f Department of Environmental Health Sciences, Akita University School of Medicine, Akita, Japan b c
art ic l e i nf o
a b s t r a c t
Article history: Received 1 August 2014 Received in revised form 16 September 2014 Accepted 18 September 2014
Objective: To investigate the appropriateness of mercury (Hg) concentrations in fingernails and toenails at parturition for detecting prenatal exposure to methylmercury (MeHg). Methods: Total Hg concentrations were measured in 54 paired samples of fingernails, toenails, maternal blood, and maternal hair (1 cm incremental segments from the scalp toward the tip) collected at 4th weeks of (early) pregnancy, and the same specimens and cord blood collected at parturition. Results: Strong correlations were observed between Hg concentrations in fingernails and toenails at early pregnancy (r ¼0.923, p o0.01) and at parturition (r ¼ 0.895, po 0.01). At early pregnancy, Hg concentrations in fingernails and toenails showed the strongest correlations with those in hair 3–4 cm from the scalp (r ¼0.818 and r ¼ 0.747, po0.01, respectively) among the 1 cm incremental hair segments. Mercury concentrations in fingernails and toenails at parturition represented strong correlations with those in cord blood (r ¼0.803, p o0.01 for fingernails and r¼ 0.792, po 0.01 for toenails, respectively). At parturition, Hg concentrations in fingernails had the highest correlation with those in hair 0–1 cm from the scalp (r ¼0.918, po0.01), and Hg concentrations in toenails showed the highest correlation with those in hair at 2–3 cm from the scalp (r ¼ 0.872, po 0.01). Conclusions: Mercury in fingernails and toenails at early pregnancy reflected the maternal Hg body burden level approximately 5 months retroactively. At parturition, Hg levels in fingernails and toenails also showed strong correlations with those in cord blood. In addition, Hg levels in fingernails and toenails at parturition reflected more recent MeHg exposure, compared with those at early pregnancy. These results suggest that fingernails and toenails at parturition are useful biomarkers for prenatal MeHg exposure for mothers and fetuses, especially during the early third-trimester of gestation. & 2014 Elsevier Inc. All rights reserved.
Keywords: Methylmercury Mercury Fingernail Toenail Hair segment Fetus Gestation
1. Introduction Methylmercury (MeHg) is a widespread environmental neurotoxin. The organ targeted by MeHg exposure during gestation is the fetal brain, especially the developing brain during the third
Abbreviations: ; Hg, mercury; MeHg, methylmercury; CV, coefficient of variation; CVAAS, cold vapor atomic absorption spectrophotometry; ICP-MS, inductively coupled plasma mass spectrometry n Corresponding author. E-mail address: [email protected] (M. Sakamoto). http://dx.doi.org/10.1016/j.envres.2014.09.034 0013-9351/& 2014 Elsevier Inc. All rights reserved.
trimester (Rice and Barone, 2000). For this reason, biomarkers reflecting the MeHg exposure level in the fetus during the third trimester are very important for predicting the effects of MeHg on child development. In the Faroe Islands study, cord blood mercury (Hg) concentration was the preferred biomarker for MeHg exposure, although maternal hair Hg was also analyzed (Grandjean et al., 1997; Murata et al., 2004). In the Seychelles study, maternal hair Hg concentration was used as the only biomarker for fetal exposure (Davidson et al., 1998). Each biomarker has its advantages and disadvantages. Cord blood circulates in the fetal body and can directly reflect the MeHg concentrations in the fetal
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organs, including the fetal brain at birth (Cernichiari et al., 1995a; NRC, 2000). Although hair Hg analysis is associated with a number of variables such as the hair’s growth rate, density, color, waving, external contamination, and permanent treatment (WHO, 1990), segmental analysis of maternal hair is able to provide time-course information, because the average hair growth rate is commonly assumed to be about 1 cm per month (Boischio et al., 2000; Cernichiari et al., 1995a). However, because the hair follicle grows out of the skin surface after about 3 weeks, Hg concentrations in the first hair segment proximal to the scalp (0–1 cm from the scalp) will only reflect the MeHg exposure from 3 weeks before (Cernichiari et al., 1995a; Phelps et al., 1980; Yaginuma–Sakurai et al., 2012). Therefore, maternal hair Hg concentrations at parturition may not reflect the prenatal MeHg exposure during the last 3 weeks of gestation. A number of studies have employed Hg concentrations in toenails and/or fingernails as biomarkers for MeHg exposure (Alfthan, 1997; Hinners et al., 2012; Morton et al., 2004; Ohno et al., 2006; Rees et al., 2007; Suzuki et al., 1989; Yoshizawa et al., 2002). In most of these studies, toenails rather than fingernails were preferred, because toenails are often less contaminated than fingernails, especially among dental personnel and gold miners who handle Hg amalgam (Morton et al., 2004; Wickre et al., 2004). Nevertheless, the time-lag for nail growth from the nail matrix to the nail edge was not considered for analyzes in the abovementioned studies. Furthermore, the appropriateness of using fingernails and toenails as biomarkers for maternal and fetal MeHg exposure at parturition has not yet been validated. In this study, we conducted simultaneous analyzes of fingernails, toenails, maternal blood, umbilical cord blood, and maternal hair segments to investigate how Hg concentrations in nails are related to the other biomarkers, especially at parturition. We measured total Hg concentrations as a surrogate for MeHg concentrations in the samples. The overall hypothesis is that Hg concentrations in both toenails and fingernails at parturition strongly reflect the fetal exposure to MeHg during the third trimester.
approved by the Ethics Committee of the National Institute for Minamata Disease, Minamata, Japan. 2.2. Hg analytical methods The total Hg concentrations in approximately 0.5 ml of blood, 5–10 mg of hair, and 10–13 mg of nails were determined by cold vapor atomic absorption spectrophotometry (CVAAS) according to a previously described method (Akagi et al., 2000) using a mercury analyzer Model Hg-201 (Sanso Seisakusho Co. Ltd., Tokyo, Japan). The method involved sample digestion with HNO3, HClO4, and H2SO4 followed by reduction to Hg0 by SnCl2. The method detection limit was 0.01 ng/g. The accuracy of Hg analysis in blood samples was ensured using a reference blood material (Level 2, MR9067; Nycomed Co., Oslo, Norway). The mean value (n ¼5) of the determined Hg was 7.5 μg/L, which was within the recommended range of 6.8–8.5 μg/L as measured by inductively coupled plasma mass spectrometry (ICP-MS). Reference human hair (NIES CRM No. 13 Human Hairs; National Institute for Environmental Studies, Environmental Agency of Japan) was also measured as quality control. The mean value (n ¼5) of the determined total Hg was 4.32 ng/mg, which was within the certified range of 4.22– 4.62 ng/mg. A coefficient of variation (CV) of 0.8% was obtained from 5-time repeated measurements of a standard solution (50 ng Hg/ml) analysis. Hg levels were expressed as concentrations (ng/g) in blood, hair, and nail samples. 2.3. Statistical analysis Mercury concentrations for all biomarkers were expressed as geometric means and 25th–57th percentiles because the values were log-normally distributed. For the statistical analysis, logarithmically-transformed Hg concentrations were used. Differences in Hg concentrations were evaluated by a paired t-test. The strength of the relationships among the Hg concentrations in blood, hair segments, and nail samples was analyzed by the Pearson product-moment correlation coefficient. Values of pr 0.05 were considered as statistically significant.
2. Materials and methods 3. Results 2.1. Sample collection Fifty-four healthy Japanese pregnant women without any occupational exposure to Hg compounds and planning to undergo delivery at Fukuda Hospital, Kumamoto, Japan from 2006 to 2007, provided informed consent at their first pregnancy check-up to participate in the study. Their ages ranged from 21 to 41 years (mean: 29.6 74.4 years). None of the women had undergone permanent hair treatments for at least 6 months prior to participation, and they were asked not to do the treatments during gestation. Fasting venous blood samples were collected from the participants at the 4th week of (early) pregnancy in the morning and 1 day after parturition. Umbilical cord venous blood was collected at parturition. Blood samples were obtained by venipuncture and collected into heparin-Na-containing vacutainer tubes. All samples were stored at 80 °C until analysis. Approximately 50 full-length strands of maternal hair were collected at early pregnancy and parturition by cutting the strands close to the scalp in the occipital area. Each maternal hair strand was cut into 1 cm incremental segments from the scalp end toward the tip. Approximately 1 mm clippings of the free edges of fingernails and toenails were also collected at early pregnancy and at parturition. Nail polish found on one sample at early pregnancy was removed using acetone. Hair and nail samples were stored at room temperature until analysis. The study protocol was reviewed and
3.1. Hg in nails and other biomarkers at early pregnancy and parturition The geometric means and 25th–75th percentiles of the Hg concentrations (ng/g) in maternal blood, cord blood, fingernails, toenails, and 1 cm hair segments from the scalp to the tip at early pregnancy and at parturition are summarized in Table 1. Among the maternal specimens, the highest Hg concentration was observed in hair, followed by fingernails, toenails, and blood at both sampling periods. Mercury ratios of 3 specimens (hair 1–3 cm from the scalp, fingernails, and toenails) to maternal blood (mean and range in parentheses) were 307 (118–606), 130 (63.1–241), and 111 (61.6–198) at early pregnancy, and 348 (148–506), 134 (47.8–231), and 114 (35.0–224) at parturition, respectively. At parturition, Hg level in cord blood was significantly (p o0.01) higher than that in maternal blood, with an average ratio of 1.84. Mercury concentrations in maternal blood, fingernails, and toenails at parturition were significantly lower than those at early pregnancy (p o0.05 for fingernails, and p o0.01 for maternal blood and toenails). Fig. 1 depicted the correlations of Hg concentrations between fingernails and toenails at early pregnancy (A) and at parturition (B). Mercury concentrations in fingernails and toenails showed the significant correlations at early pregnancy (r ¼0.923, p o0.01) and parturition (r ¼0.895, po 0.01).
M. Sakamoto et al. / Environmental Research 136 (2015) 289–294
Table 1 Geometric means (25th–75th percentiles) of Hg concentrations (ng/g) in maternal blood, cord blood, fingernails, toenails, and hair segments from scalp to tip at early pregnancy and parturition.
Table 2 Correlations among Hg concentrations (ng/g) in maternal blood, fingernails, toenails, and hair segments from scalp to tip at early pregnancy. 54 Samples
Parturition
4.40 (3.58–5.44) 547 (425–727) 465 (358–624)
3.89 (3.25–4.69)n 7.14 (6.02–9.38)n 504 (426–585)nn 427 (350–544)n
1268 (941–1798) 1276 (928–1827) 1261 (885–1850) 1244 (892–1922) 1234 (898–1844) 1215 (851–1840) 1194 (844–1686) 1141 (844–1714) 1159 (845–1648)
1314 (1090–1709) 1257 (1030–1540) 1243 (1019–1533) 1252 (1045–1573) 1234 (1018–1605) 1241 (1032–1535) 1238 (1109–1622) 1272 (1055–1622) 1273 (1042–1744)
Differences in Hg concentrations between paired samples were determined by a paired t-test using logarithmically-transformed Hg concentrations. Hg concentrations in maternal blood, fingernails, and toenails at parturition were significantly (npo 0.05, nnp o 0.01) lower than those at early pregnancy. Hg concentration in cord blood was significantly (npo 0.01) higher than that in maternal blood at parturition. n
po 0.01. p o0.05.
nn
3.2. Correlations of Hg among blood, nails and segmental hair at early pregnancy Table 2 summarized the correlation coefficients matrix for Hg concentrations in maternal blood, fingernails, toenails, and all maternal hair segments from the scalp to the tip at early pregnancy. Significant correlations (p o0.01) were observed among all the biomarkers at early pregnancy. Among the 1 cm incremental maternal hair segments, both Hg concentrations in fingernails and toenails showed their highest correlation coefficients with Hg concentrations in hair at 3–4 cm from the scalp (r ¼ 0.818, p o0.01 for fingernails and r ¼0.744, p o0.01 for toenails). 3.3. Correlations of Hg among blood, nails and segmental hair at parturition
Hg in toenails (ng/g)
The matrix of correlation coefficients for Hg concentrations in maternal blood, cord blood, fingernails, toenails, and hair segments from the scalp to the tip at parturition is summarized in Table 3. Significant positive correlations (po0.01) were observed
p
Hg in fingernails (ng/g)
Correlation coefficients
Maternal blood Fingernail Toenail Hair segment (cm) from the scalp 0–1 1–2 2–3 3–4 4–5 5–6 6–7 7–8 8–9
Maternal blood
Fingernail Toenail
1 0.735 0.707
1 0.923
1
0.661 0.669 0.630 0.633 0.599 0.576 0.545 0.438 0.468
0.787 0.784 0.780 0.818 0.813 0.806 0.779 0.616 0.692
0.711 0.714 0.706 0.744 0.733 0.734 0.718 0.588 0.643
Correlation coefficients were calculated using logarithmically-transformed Hg concentrations. All correlation coefficients showed statistical significance (p o 0.01).
Table 3 Correlations among Hg concentrations (ng/g) in maternal blood, cord blood, fingernails, toenails, and hair segments from scalp to tip at parturition. 54 Samples
Correlation coefficients Maternal blood
Maternal blood 1 Cord blood 0.878 Fingernail 0.697 Toenail 0.689 Hair segment (cm) from the scalp 0–1 0.842 1–2 0.772 2–3 0.749 3–4 0.737 4–5 0.722 5–6 0.688 6–7 0.617 7–8 0.612 8–9 0.471
Cord blood
Fingernail Toenail
1 0.803 0.792
1 0.895
1
0.918 0.870 0.846 0.792 0.779 0.739 0.680 0.638 0.492
0.878 0.875 0.873 0.825 0.749 0.716 0.662 0.631 0.513
0.868 0.871 0.872 0.835 0.794 0.791 0.730 0.737 0.595
Correlation coefficients were calculated using logarithmically-transformed Hg concentrations. All correlation coefficients showed statistical significance (p o 0.01).
Hg in toenails (ng/g)
Maternal blood Cord blood Fingernail Toenail Hair segment (cm) from the scalp 0–1 1–2 2–3 3–4 4–5 5–6 6–7 7–8 8–9
Early pregnancy
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p
Hg in fingernails (ng/g)
Fig. 1. Correlations of Hg concentrations between fingernails and toenails at early pregnancy (A) and parturition (B). *p o 0.01. Dotted lines denote the 95% confidence intervals for the regression lines.
10
Hg in cord blood (ng/g)
M. Sakamoto et al. / Environmental Research 136 (2015) 289–294
Hg in cord blood (ng/g)
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r = 0.803 p < 0.01 (n = 54)
5
2 1
100 200
r = 0.792 p < 0.01 (n = 54)
10 5
2 1
500 1000
100
Hg in fingernails (ng/g)
200
500 1000
Hg in toenails (ng/g)
Fig. 2. Correlations of Hg concentrations between cord blood and fingernails (A) and between cord blood and toenails (B) at parturition. *po 0.01. Dotted lines denote the 95% confidence intervals for the regression lines.
among all the biomarkers at parturition. Mercury in fingernails and toenails showed strong correlations with those in cord blood (r ¼0.803 for fingernails and r ¼ 0.792 for toenails, respectively). The correlations of Hg concentrations between fingernails and cord blood (A) and those between toenail and cord blood (B) at parturition are depicted in Fig. 2. Among the 1 cm incremental hair segments, Hg concentrations in fingernails showed the highest correlation coefficient with those in hair at 0–1 cm from the scalp (r ¼0.918, po 0.01) and Hg concentrations in toenails showed the highest correlation coefficient with Hg in hair at 2–3 cm from the scalp (r ¼0.872, p o0.01). However, the correlation coefficients of Hg concentrations between nails and hair segments at parturition were equally high among hair at 0–1, 1– 2, and 2–3 cm from the scalp. Fig. 3 depicted the correlations of Hg concentrations between fingernails and hair segments at 0–1 cm from the scalp (A) and those between toenails and hair at 3–4 cm from the scalp (B) at parturition.
4. Discussion This is the first comprehensive study investigating the appropriateness of using Hg concentrations in fingernails and toenails as biomarkers for maternal and fetal MeHg exposure at parturition, compared with those at early pregnancy. Our results indicated that Hg concentrations in both fingernails and toenails reflected the
10000
Hg in hair 2-3cm (ng/g)
Hg in hair 0-1cm (ng/g)
10000
maternal and fetal body burdens of MeHg, especially throughout the early third trimester of gestation. Keratin is the main structural protein of hair and nails, containing large amounts of sulfur-containing amino acids (Sass et al., 2004). In particular, the thiol (–SH) groups of the amino acid cysteine, and the proteins containing cysteine, have very high affinity for MeHg (Cernichiari et al., 1995b). Mercury concentrations in fingernails, toenails, hair and maternal blood showed significant correlations at early gestation and parturition. However, among the maternal specimens, the highest Hg concentration was observed in hair, followed by fingernails, toenails, and blood at both sampling periods. The higher Hg concentrations found in hair segments than in nails in the current study can be explained by the higher sulfur-containing amino acid contents in hair strands compared with nails (Baden et al., 1973; Gupchup and ZatZ, 1999). Fingernails and toenails are formed continually from the nail matrix (Gupchup and ZatZ, 1999), and the Hg concentrations in these nails reflect the MeHg concentrations in blood at the time of the nail's formation. Our results showed that Hg concentrations in fingernails were higher than those in toenail at both early pregnancy and parturition. This is consistent with findings reported in previous investigations (Goulle et al., 2009; Hislop et al., 1982; Suzuki et al., 1989). The participants in this study were ordinary Japanese pregnant women of similar age, had no known external Hg exposure, and did not use any permanent hair treatments before or during gestation. Therefore, most of the known confounding factors affecting the hair and nail Hg analysis
r = 0.878 p < 0.01 (n = 54)
1000 500 200 100 100
200
500
1000
Hg in fingernails (ng/g)
r = 0.872 p < 0.01 (n = 54)
1000 500 200 100 100
200
500
1000
Hg in toenails (ng/g)
Fig. 3. Correlations of Hg concentrations between fingernails and maternal hair 0–1 cm from the scalp (A) and between toenails and maternal hair 2–3 cm from the scalp (B) at parturition. *p o 0.01. Dotted lines denote the 95% confidence intervals for the regression lines.
M. Sakamoto et al. / Environmental Research 136 (2015) 289–294
were controlled. The only a few factors that can affect the Hg concentrations in hair and nails are their growth rates and MeHg incorporation into their matrices during gestation. Mercury concentrations in fingernails and toenails showed significant positive correlations with those in maternal and cord blood and all hair segments at parturition, indicating that both fingernails and toenails are equally useful as biomarkers for maternal and fetal exposure to MeHg in subjects who are free from external pollution to Hg compounds. At parturition, cord blood Hg was 1.84 times higher than maternal blood Hg. The higher MeHg accumulation in cord blood, as compared with maternal blood, has been previously reported (Sakamoto et al., 2002, 2013; Stern and Smith, 2003). This can be explained by the high MeHg transfer across the placenta, via the active neutral amino acid transport system (Aschner and Clarkson, 1988; Kajiwara et al., 1996). This high placental transfer is a specific characteristic of MeHg, differing from other metals (Sakamoto et al., 2010, 2013). The lower Hg concentrations in maternal blood, fingernails, and toenails at parturition compared to those at early pregnancy can be explained by the high MeHg transfer from the mother to the fetus during gestation. Although the reason is not clear, a significant decline of Hg concentration was not observed in the first hair segment proximal to the scalp. Both Hg concentrations in fingernails and toenails collected at early pregnancy showed the highest correlation coefficients with those in hair 3–4 cm from the scalp. These findings suggest that there was a time lag of approximately 5 months between MeHg incorporation from blood into the nail matrix and its appearance in the free edge, including a delay of approximately 3 weeks between MeHg incorporation from blood into growing hair strands and its appearance above the scalp (Cernichiari et al., 1995a; Phelps et al., 1980; Yaginuma–Sakurai et al., 2012). Hislop et al. (1982) observed the first peak of Hg in blood, followed by peaks in hair, fingernails, and toenails after fish consumption by 20 volunteers. The fingernail growth rate is approximately 3 mm per month, and fingernails require approximately 6 months to regrow completely (Gupchup and ZatZ, 1999; Hamilton et al., 1955). The growth rate of toenails is approximately 1 mm per month, being 30–50% of the growth rate of fingernails. However, toenails are shorter than fingernails, except for the lengths of the big toe and thumb, and the growth rates of nails are largely dependent on their length from the nail bed (Gupchup and ZatZ, 1999). These results will partly explain the reason why Hg concentrations in fingernails and toenails at early pregnancy showed equally high correlation coefficients with Hg concentrations in hair 3–4 cm from the scalp. However, further studies are needed to confirm the exact kinetics. The most significant findings of this study are the strong positive relationships between Hg concentrations in nails and cord blood collected at parturition, and the relationships between Hg concentrations in nails and 1 cm incremental hair segments grown during gestation. The changes in the amino acid metabolism in the maternal body start at early pregnancy, and the resulting protein accumulation is accelerated by the growth of the fetus (Kalhan, 2000). However, changes in growth rates and MeHg incorporation in hair and nails will also occur during gestation. Both Hg concentrations in fingernails and toenails showed their highest correlation coefficients with those in hair 3–4 cm from the scalp at early pregnancy. In contrast to the results for samples collected from early pregnancy, correlation coefficients of Hg concentrations between nails and hair segments at parturition were equally high among hair 0–1, 1–2, and 2–3 cm from the scalp. Consequently, fingernails and toenails can indicate MeHg exposure to the mother and fetus throughout the 2–4 months prior to parturition, including the delay of approximately 3 weeks between MeHg incorporation from blood into growing
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hair strands and its appearance above the scalp. The entire period 2–4 months prior to parturition corresponds to the early thirdtrimester of gestation. The migration of the peak correlation coefficient, between Hg concentrations in nails and hair segments 3–4 cm from the scalp at early pregnancy to the hair at 0–1, 1–2, 3–4 cm from the scalp at parturition, can be explained either by a decrease in the hair growth rate at late gestation (Pecoraro et al., 1971), or an increase in the growth rate of fingernails from mid to late gestation (Hewitt and Hillman, 1966). However, no study yet has examined the differences in toenail growth rates during gestation. In conclusion, Hg concentrations in fingernails and toenails at early pregnancy reflected the maternal MeHg body burden level approximately 5 months retroactively. Moreover, Hg concentrations in fingernails and toenails at parturition can be useful biomarkers for prenatal MeHg exposure to mothers and fetuses, especially throughout the early third trimester of gestation.
Acknowledgments This work was supported by a grant for Comprehensive Research of Minamata Disease from the Ministry of the Environment, Japan. It was also supported by JSPS KAKENHI Grant Number 23510085. We express our deep gratitude to the women who generously participated in the study.
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