Accumulation of Inorganic Mercury in Hair of Rats Exposed to Methylmercury or Mercuric Chloride

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1 Tohoku J. Exp. Med., 2006, 210, Inorganic Mercury Accumulation in Rat Hair 301 Accumulation of Inorganic Mercury in Hair of Rats Exposed to Methylmercury or Mercuric Chloride AKIRA YASUTAKE and NORIYUKI HACHIYA National Institute for Minamata Disease, Minamata, Japan YASUTAKE, A. and HACHIYA, N. Accumulation of Inorganic Mercury in Hair of Rats Exposed to Methylmercury or Mercuric Chloride. Tohoku J. Exp. Med., 2006, 210 (4), The concentration of methylmercury (MeHg) in human hair is an excellent marker for its exposure, since a portion of MeHg is taken up from the blood circulation to the hair protein in a dose-dependent manner. However, a small portion of the mercury in human hair is found in the inorganic form, though the mechanism of its occurrence is not well established. Here, we examined the hair uptake of inorganic mercury in the rat. Male Wistar rats were exposed to MeHg (1 μg Hg/ml) or HgCl 2 (20 μg Hg/ml) for 84 days through drinking water. The hair, grown from 49 to 84 days, was collected from the MeHg-exposed rats, and the hair samples showed 54.5 μg/g hair of the total mercury concentration, 6.1% of which was in the inorganic form. The inorganic mercury in the plasma (0.022 μg/ml), which would probably be formed from MeHg in rat tissues, accounted for as high as 29% of the total mercury (0.076 μg/ml). The hair uptake rate of inorganic mercury estimated from the hair/plasma ratio was about 1/6 lower than that of MeHg. On the other hand, the total hair mercury level in the HgCl 2 -exposed group at the same time point was 2.86 μg/g, with the inorganic portion being as high as 62%. These findings suggest that the inorganic mercury is also taken up by rat hair from the blood circulation, as is the MeHg, irrespective of the consequences of the biotransformation of MeHg or exposure to inorganic mercury itself. Accordingly, a selective quantification of inorganic mercury in human hair may be useful in detecting inorganic mercury exposure. hair mercury; blood circulation; inorganic mercury; methylmercury; rats 2006 Tohoku University Medical Press Mercury, which is widely distributed in the natural environment, is mostly found in inorganic form. Methylmercury (MeHg) is formed by saprophyte microorganisms from inorganic mercury compounds in the aquatic environment, and is accumulated in fish and shellfish through the marine food web (ATSDR 1992). Accordingly, the major route of human exposure to MeHg is the ordinary consumption of fish and shellfish. MeHg is readily absorbed from the gastrointestinal tract and distributed among various tissues including scalp hair via blood circulation. MeHg in the hair has often been used an excellent marker to estimate MeHg exposure levels (WHO 1990; Grandjean et al. 1997; NRC 2000; Yasutake et al. 2003, 2004; Nakai et al. 2004), since MeHg is highly and stably accumulated in the hair. However, besides MeHg being predominant in hair mercury, a small portion of the hair mercury was also found in the inorganic form (Phelps et al. Received July 5, 2006; revision accepted for publication October 4, Correspondence: Akira Yasutake, Biochemistry Section, National Institute for Minamata Disease, Hama, Minamata, Kumamoto , Japan. yasutake@nimd.go.jp 301

2 302 A. Yasutake and N. Hachiya 1980; NRC 2000). Although most inorganic mercury in the hair is thought to come from blood circulation, as does MeHg, detailed information on its occurrence, such as hair/blood ratio, is not available. In the present study we exposed male Wistar rats to MeHg and HgCl 2 for 84 days in their drinking water, and examined the mercury status in blood plasma and hair to determine the inorganic mercury uptake ability of hair from blood circulation. MATERIALS AND METHODS Chemicals MeHg chloride was purchased from Tokyo Chemical Industry Co. (Tokyo). HgCl 2 and glutathione (reduced form) were products of Wako Pure Chemical Ind., Ltd. (Osaka). MeHg chloride was used without further purification, since contamination by inorganic mercury was less than 0.2%. MeHg stock solution (10 mm, 2 mg Hg/ml) was prepared by dissolving it in distilled water as a 1:1 equimolar complex with glutathione, and stored at 80 C until use. HgCl 2 stock (10 mm, 2 mg Hg/ml) was prepared as a 0.05N HCl solution, and stored at 4 C. Animals and treatment Twelve male Wistar rats (9 weeks of age) obtained from CLEA Japan (Osaka) were maintained at 23 ± 2 C and 55% humidity and divided into two groups of 6 each. All rats were exposed to mercury from drinking water ad libitum, with one group exposed to MeHg (1 μg Hg/ml), and another to HgCl 2 (20 μg Hg/ml). Since we preliminarily found about a 20-fold difference in plasma mercury concentrations (MeHg > HgCl 2 ) after oral administration of these two mercury compounds at the same dose level, the present dose levels were employed to cause parallel plasma mercury levels in both groups. Prior to exposure, about a 3 4 cm area of rat hair was shaved from the right dorsal area and used as non-exposed control hair. The hair was collected from the same area on days 21, 49 and 84. Blood samples (0.01 ml) were collected from the tail vein and hemolyzed in distilled water (0.99 ml) on days 21, 35, 49 and 70 to monitor total mercury levels. Rats were sacrificed on day 84 under ether anesthesia, and blood samples were collected from the heart using a heparinized syringe. A portion of the blood sample was hemolyzed in distilled water (1 :99, v/v) as above for total mercury analysis. An aliquot (1 ml) of each blood sample was centrifuged at 10,000 rpm for 1 min to separate plasma. The experimental protocol was approved by the Ethics Committee for Research on Animals of the National Institute for Minamata Disease. Mercury analysis The hair samples collected were thoroughly washed in detergent using an ultrasonic washer, soaked in acetone twice to remove water, and then allowed to dry on filter papers. Aliquots of hair samples (30 to 40 mg for non-exposed control, 15 to 20 mg for the mercuryexposed groups) was dissolved in 2N NaOH (0.5 ml) by heating at 60 C for 30 min, and subjected to total mercury analysis according to an oxygen combustion-gold amalgamation method using an atomic absorption mercury detector MD-1 (Nippon Instruments, Co., Ltd., Osaka). Sample solutions for inorganic mercury analysis were prepared according to the method previously reported (Yasutake and Hirayama 1990) with a slight modification. Briefly, 0.25-ml aliquots of the 2N NaOH solution in 2-ml screw-capped polypropylene tubes were acidified with 10N HCl (0.1 ml), and mixed vigorously with toluene (1 ml) in the presence of 2 zirconium particles (3 mm φ ) using a micro homogenizing system MS-100 (Tomy Digital Biology Co., Ltd., Tokyo). After the mixture was centrifuged at 12,000 rpm for 1 min, toluene at the upper phase was removed by aspiration. The toluene extraction was repeated 5 times to remove every trace of MeHg. The lower phase thus obtained, which contained inorganic mercury only, was washed with petroleum ether (1 ml) once to remove residual toluene. The residual organic solvent was removed by air-stream aspiration. 4N NaOH (0.15 ml) was added to the aqueous phase to make basic ph, since acidic samples must be avoided for oxygen combustion mercury analysis. Mercury concentrations in the samples thus prepared were analyzed as the inorganic mercury level. That analysis was qualitatively confirmed by analyzing a reference material of human hair, NIES CRM No.13, with a certified value of 4.42 ± 0.2 μ g/g. Our total mercury level from a quintuple analysis was 4.55 ± 0.05 μg/g. Total mercury levels in blood sample collected at each time point and plasma samples obtained on the final day were determined by an oxygen combustion-gold amalgamation method as above. For inorganic mercury analysis, 0.25 ml portion of the plasma sample was acidified with 6N HCl (0.1 ml), and processed similarly as the hair sample solution. The mercury level in the sample solution thus prepared was analyzed as inorganic mercury.

3 Inorganic Mercury Accumulation in Rat Hair 303 RESULTS AND DISCUSSION Hair mercury is well documented as an excellent marker of MeHg exposure in humans (WHO 1990; Grandjean et al. 1997; NRC 2000; Yasutake et al. 2003, 2004; Nakai et al. 2004). In addition to MeHg, however, low levels of inorganic mercury (Hg 2+ ) have also been found in human hair (Phelps et al. 1980; NRC 2000). The hair inorganic mercury is thought to come from the blood circulation as does MeHg due to the following: i) the biotransformation of MeHg in the circulation and/or various tissues, ii) the direct absorption of this mercury species from the gastrointestinal tract, since a small portion of inorganic mercury is absorbed (WHO 1991), and iii) the pulmonary absorption of elemental mercury vapor, followed by oxidation to Hg 2+. However, hair total mercury is not considered to measurably reflect exposure to inorganic mercury (NRC 2000; Lindberg et al. 2004). Thus, sufficient information on the hair inorganic mercury is currently unavailable. To examine the occurrence of inorganic mercury in hair, male Wistar rats were exposed for 84 days to MeHg (1 μ g Hg/ml) and HgCl 2 (20 μ g Hg/ml) from drinking water. Although the hair uptake of mercury develops from blood circulation, the mercury in plasma, not in RBC, directly contributes to it. In our preliminary experiment, we found about a 20-fold difference in plasma mercury concentrations (MeHg > HgCl 2 ) after oral administration of these two mercury compounds at the same dose level. Accordingly, a 20-fold higher concentration of HgCl 2 than of MeHg in the drinking water was employed to induce comparable plasma concentrations in both exposure groups. In the present study, total mercury levels in the plasma on day 84 were and μ g/ml in MeHg and HgCl 2 -exposed rats, respectively (Table 1). Those dose levels of both mercury compounds caused no adverse effects throughout the experiment (data not shown). Exposure to MeHg caused a continuous time-dependent increase in the blood mercury levels of rats through the experimental period, though the rate of increase rate declined markedly after 35 days (Fig. 1). The final blood mercury levels in this group were 14.3 μg/ml, only 0.5% (0.076 μ g/ml) of which was distributed in the plasma (Fig. 1, Table 1). This indicated that rat hemoglobin had a very strong affinity for MeHg due to high number of cysteine residues on the protein surface compared to other animal species including humans (Doi and Tagawa 1983). Since TABLE 1. Total and inorganic mercury levels in rat plasma and hair (μg/ml or μg/g) collected on day 84 of MeHg (1 μg Hg/ml) or HgCl 2 (20 μg Hg/ml) exposure. Total Hg Inorganic Hg % of inorganic Hg Plasma mercury Control ± MeHg-exposed ± ± HgCl 2 -exposed ± ± Hair mercury Control ± ± MeHg-exposed 54.5 ± ± HgCl 2 -exposed 2.86 ± ± Hair/plasma ratio MeHg-exposed HgCl 2 -exposed Control hair was collected prior to mercury exposure, and hair grown from 49 to 84 days was collected for exposed samples. Values show are mean ± S.D. obtained from 6 rats.

4 304 A. Yasutake and N. Hachiya Fig. 1. Time-dependent changes in total mercury concentrations in rat blood during exposure to MeHg (1 μg Hg/ml, ) or HgCl 2 (20 μg Hg/ml, ) through drinking water. Blood samples were collected from the tail vein on days 21, 35, 49 and 70, and from the heart on day 84. Plasma mercury levels on day 84 were also shown. Each value represents mean ± S.D. obtained from 6 rats. MeHg binds strongly to sulfhydryl group (Simpson 1961), the number of cysteine residue on hemoglobin surface would largely affect MeHg distribution in the erythrocytes. The rat hair collected on day 84, which grew from 49 to 84 days, contained 54.5 μ g/g of total mercury (Table 1). The hair total mercury was calculated to reach concentrations of 3.8 and 717 times the whole blood and plasma concentrations, respectively. Although an average hair/blood ratio of total mercury concentration in humans was documented to be 250 (NRC 2000), the ratio in the rats obtained here was as low as 3.8, probably due to the very high distribution of MeHg in RBC as mentioned above. Inorganic mercury occupied 6.08 and 29.4% of hair and plasma total mercury, respectively, in the MeHg-exposed rats (Table 1). When limited to inorganic mercury, the hair showed a 150-fold higher concentration than that in plasma. Since the hair inorganic mercury level in non-exposed control rats (0.021 μ g/g) was much lower (159 times) than in MeHg-exposed rats (Table 1), inorganic mercury formed from MeHg in rat tissues would accumulate in the hair via the circulation. Furthermore, if the remainder of the total and inorganic mercury was considered to be MeHg in hair and plasma, the hair/plasma ratio of MeHg was as high as 952, which was 6.4 times higher than that of inorganic mercury. In contrast to the MeHg-exposed rats, the blood mercury levels in the HgCl 2 -exposed group had already attained a maximum of μg/ml by day 35, after which the concentration gradually declined to μg/ml at 84 days, despite the sustained exposure (Fig. 1). Such an early maximum in the HgCl 2 -exposed rat blood would be due to a shorter biological half life of this mercury species compared to MeHg. The plasma mercury in this group on day 84 was μg/ml, about 1/3 of the whole blood mercury was distributed there, and most of which (97.7%) was in inorganic form (Table 1). The hair total mercury concentration in this group (2.86 μg/g) was 12 and 36 times higher than whole blood and plasma concentrations, respectively (Fig. 1, Table 1). Among the hair mercury, the inorganic portion was shown to be 62%, though the plasma mercury was mostly shown by this form. Thus, a significant portion (38%) of the hair mercury was MeHg, though its ratio in plasma was as low as 2.3%. This might also indicate the much higher efficiency of MeHg hair uptake than that of inorganic mercury as observed in MeHg-exposed rats. The total mercury levels in the hair samples grown during days 21 to 49 in MeHg (54 ± 3.7 μg/g) and HgCl 2 -exposed rats (3.11 ± 0.39 μg/g) were quite similar to the samples of 49 to 84 days mentioned above. This might indicate that the mercury uptake from the blood circulation to the hair occurred under a steady state, at least after day 49. It should be noted that the MeHg portion (38% of total mercury) in the hair of HgCl 2 - exposed rats was 8.3 times higher than in nonexposed control rats (Table 1). It might be possible that some of the inorganic mercury was converted to MeHg in rat body. The conversion might occur in rat tissue via some enzymatic reaction or in the intestinal tube by the action of gut flora. Although the de novo formation of MeHg was difficult to detect from plasma analysis, hair analysis would make easier detection due to hair s

5 Inorganic Mercury Accumulation in Rat Hair 305 high accumulation rate. Alternatively, the hair uptake of MeHg might be enhanced in the presence of excess amounts of plasma inorganic mercury, since modified distribution of MeHg was documented in inorganic mercury-treated mice (Yamamoto et al. 1980). Further study including tissue MeHg analysis must be conducted to elucidate it in more detail. Since the tissue uptake of MeHg from blood circulation was shown to occur via the active transporter for neutral amino acids (Hirayama 1980; Aschner 1989; Kajiwara et al. 1996), its hair uptake would also occur via a similar, though as yet undefined, mechanism. The occurrence of inorganic mercury in rat hair might indicate the presence of some transporter(s) for this mercury species. The hair uptake rates, estimated from hair/plasma ratios, were 6.3 times higher for MeHg than for inorganic mercury in MeHgexposed rats. However, it might not necessarily reflect the activities of both transporters, since the hair uptake rate would depend not only on the transporter activities, but also on the chemical forms (population of low molecular weight forms) of mercury in the plasma. Interestingly, the hair/plasma ratio of inorganic mercury concentration in the HgCl 2 - exposed rats was 23.2, which was 6.5 times lower than in the MeHg-exposed rats. Although the hair uptake of inorganic mercury might be enhanced in the presence of excess amounts of MeHg in the circulation, further study will have to be conducted to verify that. The present study demonstrated that not only MeHg, but also inorganic mercury, irrespective of a consequence of biotransformation of MeHg or exposure to inorganic mercury itself, accumulated in rat hair via the circulation, though its concentration rate (hair/plasma ratio) was somewhat lower than MeHg. Lindberg et al. (2004) reported that total mercury levels in human hair reflected MeHg exposure but not inorganic mercury exposure from dental amalgam, even in non-fish-eating people. However, a selective hair analysis of inorganic mercury and MeHg (or total mercury) may be useful in detecting inorganic mercury exposure, especially in cases of occupational exposure at the high doses often occurring in gold mining areas (Lodenius and Malm 1998). Acknowledgments The authors wish to thank Ms. M. Ogata for her technical assistance with the animal treatment and mercury analysis. References Agency for Toxic Substances and Disease Registry (ATSDR) (1992) Mercury toxicity. Am. Fam. Physician, 46, Aschner, M. (1989) Brain, liver and kidney 203Hg-methylmercury uptake in the rat: relationship to the neutral amino acid carrier. Pharmacol. Toxicol., 65, Doi, R. & Tagawa, M. (1983) A study on the biochemical and biological behavior of methylmercury. Toxicol. Appl. Pharmacol., 69, Grandjean, P., Weihe, P., White, R.F., Debes, F., Araki, S., Yokoyama, K., Murata, K., Sørensen, N., Dahl, R. & Jørgensen, P.J. (1997) Cognitive deficit in 7-year-old children prenatally exposed to methylmercury. Neurotoxicology, 19, Hirayama, K. (1980) Effects of amino acids on brain uptake of methylmercury. Toxicol. Appl. Pharmacol., 55, Kajiwara, Y, Yasutake, A., Adachi, T. & Hirayama, K. (1996) Methylmercury transport across the placenta via neutral amino acid carrier. Arch. Toxicol., 70, Lindberg, A., Björnberg, K.A., Vahter, M. & Berglund, M. (2004) Exposure to methylmercury in non-fish-eating people in Sweden. Environ. Res., 96, Lodenius, M. & Malm, O. (1998) Mercury in the Amazon. Rev. Environ. Contam. Toxicol., 157, Nakai, K., Suzuki, K., Oka, T., Murata, K., Sakamoto, M., Okamura, K., Hosokawa, T., Sakai, T., Nakamura, T., Saito, Y., Kurokawa, N., Kameo, S. & Satoh, H. (2004) The Tohoku study of child development: A cohort study of effects of perinatal exposure to methylmercury and environmentally persistent organic pollutants on neurobehavioral development in Japanese children. Tohoku J. Exp. Med., 202, National Research Council (NRC). Committee on the Toxicology Effects of Methylmercury (2000) Toxicological Effects of Methylmercury, National Academy Press, Washington, D.C. Phelps, R.W., Clarkson, T.W., Kershaw, T.G. & Wheatley, B. (1980) Interrelationships of blood and hair mercury concentrations in a North American population exposed to methylmercury. Arch. Environ. Health, 35, Simpson, R.B. (1961) Association constants of methylmercury with sufhydryl and other bases. J. Am. Chem. Soc., 83, WHO (1990) IPCS Environmental Health Criteria 101 Methylmercury. World Health Organization, Geneva. WHO (1991) IPCS Environmental Health Criteria 118 Inorganic Mercury. World Health Organization, Geneva. Yamamoto, R., Satoh, H., Suzuki, T. & Nobunaga, T. (1980) Modified distribution of methylmercury by additional exposure to elemental mercury or mercuric chloride in mice fed methylmercuric chloride. J. Pharmacobiodyn., 3,

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