Kunihiko Nakai

Determination of dioxins and polychlorinated biphenyls in breast milk, maternal blood and cord blood from residents of Tohoku, Japan

Polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs) and polychlorinated biphenyls (PCBs) are bioaccumulative chemicals that are considered to be toxic contaminants based on several epidemiological studies. To elucidate exposure levels of these chemicals in the present study, concentrations of PCDD/DFs, dioxin-like PCBs (DL-PCBs) and PCBs in breast milk, maternal blood and cord blood obtained from the same participants registered in a birth cohort study in Tohoku, Japan, were measured. Congener-specific analysis revealed several differences in minor congeners of these compounds among the three specimen types, although major congeners were detected in the specimens. The toxicity equivalence quantity concentrations (1998 WHO-TEF) and PCBs in breast milk, maternal blood and cord blood on the whole and on a lipid basis were in the order of breast milk > maternal blood > cord blood. Pearsons correlation coefficients of TEQs and total PCBs among the three specimens were high, with the correlation coefficient of TEQ between breast milk and maternal blood being the highest (r=0.94, p<0.001). On the other hand, the TEQ between breast milk and cord blood was the lowest (r=0.79, p<0.001). Pearsons correlation coefficient between the TEQ and PCBs in each specimen was also high (r=0.82-0.95, p<0.001). The associations of chemical concentrations with maternal age, parity, fish intake, BMI and the rate of body weight increase during pregnancy were analyzed with multiple linear regression analysis. TEQ concentrations and PCBs were negatively associated with parity (p<0.05), and maternal age was positively associated with PCBs (p<0.05). However, the associations with BMI and fish intake during pregnancy were not significant. These results suggest that parity is an important factor affecting the concentrations of dioxins and PCBs in these specimens.

Neurobehavioral effects of prenatal exposure to methylmercury and PCBs, and seafood intake: Neonatal behavioral assessment scale results of Tohoku study of child development

As factors affecting neonatal neurodevelopment, methylmercury, polychlorinated biphenyls (PCBs), and maternal seafood intake reflecting n-3 polyunsaturated fatty acids (PUFAs) are believed to have adverse or beneficial effects, but there are a few reports addressing such factors simultaneously. We carried out a birth cohort study to clarify the effects of these three factors on the Neonatal Behavioral Assessment Scale (NBAS), administered 3 days after birth. In a total of 498 mother-neonate pairs, the total mercury level (median, 1.96microg/g) in maternal hair at parturition and the summation operatorPCB level (45.5ng/g-lipid) in cord blood were analyzed, and maternal seafood intake was estimated using a semi-quantitative food frequency questionnaire. A negative relationship between the hair mercury level and the motor cluster of NBAS was observed, even after adjusting for PCBs, maternal seafood intake, and possible confounders such as maternal age, birth weight, and parity. The summation operatorPCB level was negatively correlated with the motor cluster, but this association was attenuated after adjusting for mercury and the confounders. There was seen to be a positive association between maternal seafood intake and the motor cluster when considering the effects of mercury and PCBs. In conclusion, our data suggest that prenatal exposure to methylmercury adversely affects neonatal neurobehavioral function; in contrast, maternal seafood intake appears to be beneficial. The neurobehavioral effect of prenatal exposure to PCBs remains unclear in our study. Further research is necessary to elucidate interactive effects of methylmercury, PCBs, and n-3 PUFAs, originating from fish, on child neurodevelopment.

Characterization of DNA methylation errors in patients with imprinting disorders conceived by assisted reproduction technologies

BACKGROUND There is an increased incidence of rare imprinting disorders associated with assisted reproduction technologies (ARTs). The identification of epigenetic changes at imprinted loci in ART infants has led to the suggestion that the techniques themselves may predispose embryos to acquire imprinting errors and diseases. However, it is still unknown at what point(s) these imprinting errors arise, or the risk factors. METHODS In 2009 we conducted a Japanese nationwide epidemiological study of four well-known imprinting diseases to determine any association with ART. Using bisulfite sequencing, we examine the DNA methylation status of 22 gametic differentially methylated regions (gDMRs) located within the known imprinted loci in patients with Beckwith-Wiedemann syndrome (BWS, n=1) and also Silver-Russell syndrome (SRS, n= 5) born after ART, and compared these with patients conceived naturally. RESULTS We found a 10-fold increased frequency of BWS and SRS associated with ART. The majority of ART cases showed aberrant DNA methylation patterns at multiple imprinted loci both maternal and paternal gDMRs (5/6), with both hyper- and hypomethylation events (5/6) and also mosaic methylation errors (5/6). Although our study may have been limited by a small sample number, the fact that many of the changes were mosaic suggested that they occurred after fertilization. In contrast, few of the patients who were conceived naturally exhibited a similar pattern of mosaic alterations. The differences in methylation patterns between the patients who were conceived naturally or after ART did not manifest due to the differences in the disease phenotypes in these imprinting disorders. CONCLUSION A possible association between ART and BWS/SRS was found, and we observed a more widespread disruption of genomic imprints after ART. The increased frequency of imprinting disorders after ART is perhaps not surprising given the major epigenetic events that take place during early development at a time when the epigenome is most vulnerable.

Inhibition of endothelium-dependent relaxation by hemoglobin in rabbit aortic strips : Comparison between acellular hemoglobin derivatives and cellular hemoglobins

Hemoglobin (Hb)-based artificial oxygen carriers are supposed to induce vasoconstriction through the inactivation of endothelium-derived relaxing factor (EDRF). We examined the vasoconstrictive activity of acellular Hb and cellular Hb solutions in rabbit aortic strips. Unmodified Hb, pyridoxalated Hb, bovine unmodified Hb, haptoglobin-Hb complex (Hp-Hb), and polyoxyethylene glycol-conjugated Hb (PEG-Hb) were used as acellular Hbs having different molecular masses. Cellular Hbs included liposome-encapsulated Hb and red blood cells (RBC). In the first experiment, Hb (10 ng/ml to 1 mg/ml) was cumulatively added to the tissues in which steady-state relaxation was evoked by acetylcholine (ACh) after precontraction induced by phenylephrine. Although all Hb solutions induced a dose-dependent reversal of ACh-induced relaxation, the most potent vasoconstrictive effect was noted with acellular Hbs, and their contractile activities were almost the same independent of molecular mass. On the other hand, liposome-Hb and RBC showed reduced potencies in this order. These results indicate the importance of cellularity as the major factor determining Hb-related EDRF inactivation. In another experiment, the tissues were exposed to Hb at 0.01, 0.1, or 1 mg/ml for 30 min and ACh-induced relaxation was recorded after the complete removal of Hb in an organ bath chamber. Exposure to unmodified Hb at > 0.1-mg/ml concentrations significantly reduced the ACh-induced relaxation, whereas the relaxation was not affected by PEG-Hb, Hp-Hb, liposome-Hb, or RBC. These results suggest that unmodified Hb might be persistently associated with tissues and thereby inhibit ACh-induced relaxation. From these findings, we propose two attributes of Hb-related inhibition of endothelium-dependent relaxation: Acellular Hbs inhibit EDRF more efficiently in the luminal space than cellular Hbs, and unmodified Hb can also inhibit it adluminally and/or adventitially.

Mercury and heavy metal profiles of maternal and umbilical cord RBCs in Japanese population

Mercury (Hg) and other heavy metal profiles, namely, lead (Pb), arsenic (As), cadmium (Cd), and selenium (Se) were investigated in maternal and umbilical cord (cord) red blood cells (RBCs) at parturition in Japanese population. Correlation coefficients of Hg, Pb, As, Cd, and Se between maternal and cord RBCs were 0.91, 0.79, 0.89, 0.31, and 0.76, respectively, and the respective means of cord/maternal RBCs ratios were 1.63, 0.52, 0.62, 0.12, and 1.18. These results indicate that fetal exposure to these metals (excluding Cd) strongly reflected each maternal exposure level. Among these metals, the placental transfer of methylmercury (MeHg) seemed to be extremely high but that of Cd to be limited. Hg showed positive correlations with Se in maternal RBCs but not in cord RBCs, and the Se/Hg molar ratio was lower in the latter, suggesting that the protective effects of Se against MeHg are less expected in fetuses than in mothers.

Comparison of neurobehavioral changes in three inbred strains of mice prenatally exposed to methylmercury

Pregnant mice of three inbred strains (BALB/c, C57BL/6J, C57BL/6Cr) were orally given methylmercury (MMC; 3 x 3 mg/kg body weight) or the equivalent volume of phosphate-buffered saline during days 12-14 of gestation and allowed to deliver. The behaviors of their male offspring were evaluated in an open field and their home cage and in a Morris water maze. In the open field test, the BALB/c and C57BL/6Cr MMC groups exhibited less total locomotor activity than did their respective control groups. However, there was no significant difference observed between the MMC and control C57BL/6J strain. In the BALB/c strain, the MMC group exhibited significantly more central locomotion and significantly less peripheral locomotion than did the control group. These results indicated that the prenatal exposure to MMC caused decreases in open-field activity in the C57BL/6Cr and BALB/c strains, concomitantly with a change in emotional status in BALB/c strain. For spontaneous activity in their home cage, all groups moved more actively in the dark phase than in the light phase except BALB/c MMC group. The BALB/c MMC group moved in the light phase as much as in the dark phase, indicating a disturbance of nocturnal rhythm of spontaneous activity. In the Morris water maze, the C57BL/6Cr and C57BL/6J control groups perform very well over the 5 consecutive days. The prenatal exposure to MMC caused significantly prolonged latency in the C57BL/6Cr and C57BL/6J, but not in BALB/c strain. This result indicated that the prenatal exposure to MMC impaired the performance in the Morris water maze differently among the strains. This study provides a basis for evaluating strain-specific neurobehavioral changes when the widely used three inbred strains of mice are chronically exposed to MMC.

A meta-analysis of the evidence on the impact of prenatal and early infancy exposures to mercury on autism and attention deficit/hyperactivity disorder in the childhood

Although a measurable number of epidemiological studies have been conducted to clarify the associations between mercury exposure during embryo or early infancy and later incidences of autism spectrum disorders (ASD) or attention-deficit hyperactivity disorder (ADHD), the conclusion still remains unclear. Meta-analysis was conducted for two major exposure sources; i.e., thimerosal vaccines that contain ethylmercury (clinical exposure), and environmental sources, using relevant literature published before April 2014. While thimerosal exposures did not show any material associations with an increased risk of ASD or ADHD (the summary odds ratio (OR) 0.99, 95% confidence interval (CI) 0.80-1.24 for ASD; OR 0.91, 95% CI 0.70-1.13 for ADHD/ADD), significant associations were observed for environmental exposures in both ASD (OR 1.66, 95% CI 1.14-2.17) and ADHD (OR 1.60, 95% CI 1.10-2.33). The summary ORs were similar after excluding studies not adjusted for confounders. Moderate adverse effects were observed only between environmental inorganic or organic mercury exposures and ASD/ADHD. However, these results should be interpreted with caution since the number of epidemiological studies on this issue was limited and still at an early stage. Further studies focused on subjects with genetic vulnerabilities of developmental disorders are warranted for better understanding of the effects of such environmental exposures.

Intervention study on cardiac autonomic nervous effects of methylmercury from seafood.

To scrutinize whether the provisional tolerable weekly intake (PTWI, 3.4 microg/kg body weight/week) of methylmercury in Japan is safe for adults, we conducted an intervention study using heart rate variability (HRV) that has been considered to reflect cardiac events. Fifty-four healthy volunteers were recruited and divided into experimental and control groups. The experimental group was exposed to methylmercury at the PTWI level through consumption of bigeye tuna and swordfish for 14 weeks, and HRV parameters were compared between the two groups. In the experimental group, mean hair mercury levels, determined before and after the dietary methylmercury exposure and after 15-week wash-out period following the cessation of exposure, were 2.30, 8.76 and 4.90 microg/g, respectively. The sympathovagal balance index of HRV was significantly elevated after the exposure, and decreased to the baseline level at the end of this study. Still, such changes in HRV parameters were not found in the control group with a mean hair mercury level of around 2.1 microg/g. In conclusion, the PTWI does not appear to be safe for adult health, because methylmercury exposure from fish consumption induced a temporary sympathodominant state. Rather, long-term exposure to methylmercury may pose a potential risk for cardiac events involving sympathovagal imbalance among fish-consuming populations.

Permeability characteristics of hemoglobin derivatives across cultured endothelial cell monolayers

To better understand the vascular activity of hemoglobin-based (Hb-based) oxygen carriers, the endothelial permeability characteristics of Hb derivatives having various molecular masses were defined by using monolayers of bovine endothelial cells cultured on microporous membranes. The endothelial permeability of unmodified bovine Hb was almost twice that of bovine serum albumin. Intramolecularly cross-linked human Hb showed slightly but significantly reduced permeability as compared with unmodified bovine Hb. Polyethyleneglycol modification or haptoglobin binding to Hb further reduced the permeability. These properties were intensified in conditions in which the endothelial barrier function was reduced by pretreatment with either interleukin-6 (100 ng/mL, 21 hours) or lipopolysaccharide (1 microg/mL, 10 hours). In contrast, there was little permeability of liposome-encapsulated Hb, and it was almost unaffected by the pretreatments. These data provide the first information that Hb derivatives with smaller molecular masses show larger transendothelial flux. Because Hb is a potent scavenger of endothelium-derived relaxing factor (EDRF), our observations support the idea that smaller Hb-based acellular oxygen carriers are potent vasoconstrictors as a result of abluminal EDRF scavenging.

Effects of hair treatment on hair mercury-The best biomarker of methylmercury exposure?

ObjectivesExposure misclassification is a major obstacle to obtain accurate dose-response relationships. In order to solve this problem, the impact of hair treatment on total mercury in hair was assessed in Japanese women.MethodsA cross-sectional study was carried out among 327 women at age 24–49 years to determine hair mercury levels and estimate daily mercury intakes from seafood by using a food frequency questionnaire.ResultsHair mercury levels in the women and daily mercury intake ranged from 0.11 to 6.86 (median 1.63) μg/g and from 0.77 to 144.9 (median 15.0) μg/day, respectively. The hair mercury was positively correlated with the daily mercury intake (p<0.001). When the women were divided into two subgroups based on artificial hair-waving, hair coloring/dyeing, residence (non-fishing and fishing areas), and working status, a significant difference in the hair mercury level was observed between the women with and without artificial hair-waving only (p<0.001). The multiple regression analysis showed that the log-transformed hair mercury level was significantly related to the log-transformed daily mercury intake (standardized regression coefficient βs=0.307) and artificial hair-waving (βs=−0.276); but not to hair coloring/dyeing, residence, working status or age. Permanent hair treatment was estimated to reduce total mercury in hair by approximately 30%, after adjusting for daily mercury intake and other possible factors.ConclusionsThese findings suggest that hair mercury is not the best biomarker of methylmercury exposure when a study population includes women with artificial hair-waving.