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A New Mass Screening Method for Methylmercury Poisoning Using Mercury-Volatilizing Bacteria from Minamata Bay
Ecotoxicology and Environmental Safety 44, 100}104 (1999) Environmental Research, Section B Article ID eesa.1999.1805, available online at http://www.idealibrary.com on
A New Mass Screening Method for Methylmercury Poisoning Using Mercury-Volatilizing Bacteria from Minamata Bay Kunihiko Nakamura,* Ichiro Naruse,- and Yukio Takizawa* *National Institute for Minamata Disease, Minamata, 4058-18 Hama, Kumamoto, Japan 867-0008; and -Congenital Anomaly Research Center, Faculty of Medicine, Kyoto University, Sakyo-ku, YoshidaKonoe-cho, Kyoto 606-8315, Japan Received November 2, 1998
A simpli5ed mass screening method for methylmercury exposure was developed using methylmercury-volatilizing bacteria from Minamata Bay. Some bacteria can transform methylmercury into mercury vapor. Most mercury in the hair is methylmercury, which is readily extracted with HCl solution. Black spots are formed on X-ray 5lm due to the reduction of Agⴙ emulsion with mercury vapor produced by methylmercury-volatilizing bacteria. By exploiting these characteristics, a screening method was developed, whereby the fur of rats injected with methylmercury chloride formed clear black spots on X-ray 5lm, whereas the fur of rats injected with saline did not. Subsequently, 50 human hair samples were examined using this mass screening method. The method identi5ed people who had high mercury concentration, over 20 lg/g. A few thousand hair samples may be screened in a day using this method because it is rapid, simple, and economical. This method, therefore, enables screening of persons with methylmercury poisoning in mercury-polluted areas. 1999 Academic Press
Key Words: mercury pollution; methylmercury poisoning; mercury-volatilizing bacteria; methylmercury-volatilizing bacteria; Minamata Bay.
INTRODUCTION
Mercury pollution of the environment has become a serious problem throughout the world in recent years. Minamata disease, methylmercury poisoning, was caused by the consumption of "sh and shell"sh containing high concentrations of methylmercury in Minamata Bay and along the Agano River in Japan (Tubaki and Irukayama, 1977; Takizawa, 1979). A massive outbreak took place in Iraq as well (Bakir et al., 1973; Rustam and Hamdi, 1974) in 1971}1972. Mercury pollution has also been reported in QueH bec, Canada (Eyssen and Ruedy, 1983; Wheatley et al., 1979), the Amazon in Brazil (Malm et al., 1990; Nriagu et al., 1992), and in other areas (Baldi and D'amato, 1986; ICPS, 1990; Lopez-Artiguez et al., 1994). Identifying persons su!ering from methylmercury poisoning in mercury-polluted areas has depended on neuro-
logical examinations and determination of the mercury content in the hair and blood. A concentration of more than 50 lg/g of mercury in the hair is considered to be the threshold level for the manifestation of Minamata disease (ICPS, 1990). The amount of mercury in the hair is about 250 times that in blood (ICPS, 1990) and hair samples are much easier to obtain and store than blood or other organ samples. Thus, hair is clearly the material most suitable for mass screening of populations exposed to methylmercury. Typically, mercury concentration in the hair has been determined through the use of #ameless atomic absorption spectrometry or gas chromatography (Givanoli-Jakubczak et al., 1974; Phelps et al., 1980), but these methods are expensive and time-consuming. Moreover, these methods are not applicable for screening involving a large number of people, such as epidemiological studies. Some bacterial strains have been found to transform mercury compounds such as mercury chloride and methylmercury chloride into volatile mercury vapor (Clark et al., 1977; Nakamura et al., 1990; Summers and Silver, 1972). The bacteria detoxify the compounds by the actions of two enzymes acting sequentially. Organomarcurial lyase cleaves the carbon}mercury bond of certain organomercurials, and then mercuric reductase transforms the mercuric ion to volatile mercury vapor (Begley et al., 1986; Schiering et al., 1991). By exploiting this characteristic, a rapid and simple method for the detection of bacterial volatilization of mercury chloride has been devised, using X-ray "lm (Nakamura, and Nakahara, 1988). Most of the mercury in the hair is methylmercury, which is readily extracted with HCl solution (Suzuki et al., 1992). In the present study, a mass screening method for methylmercury exposure was developed by applying this X-ray "lm method to human hair samples. MATERIALS AND METHODS
Bacterial Strains Seven Bacillus and 10 Pseudomonas strains have been isolated from Minamata Bay. As a Bacillus subtilis strain
100 0147-6513/99 $30.00 Copyright 1999 by Academic Press All rights of reproduction in any form reserved.
101
A MASS SCREENING METHOD FOR METHYLMERCURY POISONING
had mercury-volatilizing genes (Nakamura and Silver, 1994) with the most active mercury-volatilization reaction, this strain was chosen for the present method. The basal media were Luria}Bertani medium (LB medium) and arti"cial sea water medium (Modi"ed ZoBell 2216 E medium), as described previously (Nakamura et al., 1986).
TABLE 2 Extractable Amount of Mercury (lg/g) in Human Hair Samples by Various Concentrations of HCl Solution (MeanⴞSD) Original mercury content in the hair 1.19
Rat Fur Samples
3.32
Fur from young adult (7 weeks old) male SLC:Wister rats was reaped from their backs, and 2}10 mg/kg of methylmercury was injected subcutaneously for 7 to 9 days with di!erent doses, as presented in Table 1. Then, newly grown fur samples were reaped again on Day 14 after the "rst injection. The mercury concentration of the fur was measured by a #ameless atomic absorption spectrometer with a mercury analysis vaporizer (Rigaku Mercury SP-1; Nippon Instrument, Tokyo, Japan). Nontreated and salinetreated control fur revealed under 1 lg/g of mercury. A maximum of 1156 lg/g of mercury was detected in the treated fur.
5.07
0.5 M HCl
1 M HCl
2 M HCl
0.67$0.04 (56.3%) 1.48$0.07 (44.6%) 1.74$0.04 (34.3%)
0.73$0.07 (61.3%) 2.42$0.03 (61.7%) 3.45$0.14 (68.0%)
0.83$0.12 (69.7%) 2.51$0.12 (77.1%) 3.98$0.07 (78.5%)
Note. n"5.
milligrams of hair sample was cut "ne with scissors and soaked in 450 ll of 0.5 M, 1 M, and 2 M HCl in a 1.5-ml microtube. The solution was then vortexed three times for 20 s. The mercury content in the supernatant was directly determined by a #ameless atomic absorption spectrometer, and recovery ratios were calculated (Table 2). Determination of Detection Limit of X-Ray Film Method
Human Hair Samples Fifty human hair samples were used in this study. Thirteen hair samples were collected from sushi restaurant workers who eat "sh frequently. In addition, 37 samples were also collected from the researchers and their families of National Institute for Minamata Disease (30 men and 7 women). The hair samples were washed with distilled water and cut "ne with scissors. The total mercury content in the hair samples was directly analyzed by a #ameless atomic absorption spectrometer. Extraction of Mercury from Human Hair Samples The extractable amount of mercury in the hair samples by various concentrations of HCl solution was examined. Fifty TABLE 1 Mercury Amount in the Rat Fur after Various Subcutaneous Injection of Methylmercury Treatment Nontreated Saline for 9 days 2 mg/kg;5 days#4 mg/kg;4 days 2 mg/kg;5 days#4 mg/kg;4 days 3 mg/kg;5 days#6 mg/kg;3 days 3 mg/kg;5 days#6 mg/kg;3 days 4 mg/kg;5 days#8 mg/kg;3 days 4 mg/kg;5 days#8 mg/kg;3 days 5 mg/kg;5 days#10 mg/kg;2 days 5 mg/kg;5 days#10 mg/kg;3 days
Mercury amount (lg/g) 0.68 0.84 369.6 455.6 400.3 512.4 641.0 670.8 990.8 1156.3
A Bacillus subtilis strain from Minamata Bay was incubated in a LB liquid medium containing 1 lg of HgCl per milliliter to induce the mercury resistance operon. After incubation for 17 h at 303C, the strain was harvested by centrifugation and washed twice with 0.5 M phosphate buffer (pH 7.0). The washed cell was resuspended at a concentration of 50 mg/ml in 0.5 M phosphate bu!er containing 0.5 M EDTA, 0.2 M magnesium acetate, and 0.1 mM thioglycolate. The cell suspension could be prepared as a stock solution and kept in the refrigerator for up to 1 week. The 1 M HCl solutions containing 0, 0.2, 1, 2, and 5 lg of CH HgCl per milliliter were neutralized with 2 M NaOH to pH 7.0. Fifty microliters of the solution was added to each well in a 96-well microplate (Cell Well 25855; Corning Glass Works), then 50 ll of the cell suspension was added to each well. The microplate was covered with X-ray "lm (Fuji Medical X-ray Film New RX; Fuji Photo Film Co. Ltd.) in a darkroom. This was set between two acrylic plastic boards and the ends were secured with clips (Double Clip No. 3; Kokuyo Co. Ltd.). The plate was then put into a dark box. The X-ray "lm was developed after reacting for 6 h at 353C. Black spots on the "lm were the result of the reduction of Ag> emulsion by mercury vapor produced by methylmercury-volatilizing bacteria. The density of black spots on the X-ray "lm was measured with a densitometer (LKB 2222 Ultra Scan XL, LKB Productor Bromma, Sweden). RESULTS
A Bacillus subtilis strain was grown in an arti"cial sea water liquid medium containing 20 lg/ml of HgCl . The
102
NAKAMURA, NARUSE, AND TAKIZAWA
activity of methylmercury volatilization by this strain was determined by measuring mercury loss in a liquid culture medium containing 0.1 lg/ml of CH HgCl. The percentage of mercury lost was 94.7% after incubation for 24 h at 303C. The extractable amount of mercury in human hair samples by HCl solution is presented in Table 2. Over 60% of mercury in the hair was extracted with 1 and 2 M HCl solution. When the HCl solution was neutralized with NaOH, a large amount of NaCl was formed in the solution. The high concentrations of NaCl inhibited the bacterial volatilization of mercury (Selifonova and Barkay, 1994). The Bacillus subtilis strain from Minamata Bay used in this study could grow in a peptone water medium containing 1.2 M NaCl. For this reason, 1 M HCl was chosen for the extraction of methylmercury in the hair samples in this mass screening method. The black spots formed by the methylmercury-volatilizing bacteria in the CH HgCl solution using the X-ray "lm method are provided in Fig. 1. The densities of the black spots (Area, AU mm) measured with a densitometer are as follows: 0; 0.000, 0.1; 0.078, 0.5; 0.113, 1.0; 0.245, 2.5; 0.714. The results could be read with the naked eye semiquantitatively with the black spot formation expressing the degree of reaction. The detection limit of the method for CH HgCl was over 0.5 lg/ml. The X-ray "lm method was modi"ed for the mass screening method for methylmercury poisoning using rat fur containing methylmercury. Each 30 mg of rat fur sample was cut "ne with scissors and soaked in 270 ll of 1 M HCl in a 1.5-ml microtube to extract the methylmercury from the fur. After the sample was mixed three times for 20 s with the vortex mixture, the supernatant was transferred to a new tube and diluted with 1 M HCl to an adequate concentration. Then, 200 ll of the solution was transferred to another tube and neutralized with 2 M NaOH to pH 7.0. To each well in a 96-well microplate, 120 ll of the above solution was added. Forty microliters of the cell suspension described under Materials and Methods was then added to each well in the microplate. The plate was covered with X-ray "lm in a dark room. The plate was then left in a dark box for 6 h at 353C, until the X-ray "lm developed. The fur
FIG. 1. Black spot formation by the methylmercury-volatilizing bacteria in the methylmercury chloride solution on an X-ray "lm. Numbers represent methylmercury concentrations (lg/g) in the solution. The densities of the black spots (Area, AU mm) measured with a densitometer were as follows: 0, 0.000; 0.1, 0.078; 0.5, 0.113; 1.0, 0.245; 2.5, 0.714. Black spots formed with over 50 lg/g are detectable by the naked eye semiquantitatively.
FIG. 2. Black spot formation on an X-ray "lm by mass screening method in rat fur. Numbers represent total mercury concentrations (lg/g) in rat fur samples analyzed by #ameless atomic absorption spectrometer. Black spots formed with over 19.0 lg/g are detectable by the naked eye semiquantitatively.
of rats injected with CH HgCl formed clear black spots on the X-ray "lm, while the fur of rats treated with saline did not (Fig. 2). Fifty human hair samples, the mercury concentrations of which had already been measured by a #ameless atomic absorption spectrometer, were examined by this mass screening method. Of these samples, one from a heavy "sh-eater (sushi restaurant worker), which contained 22.1 lg/g of mercury, formed a clear black spot on the X-ray "lm (Fig. 3). Thus, this screening method allowed detection of a mercury concentration above 20 lg/g in a group of 50 people. DISCUSSION
The concentration of 50 lg/g of mercury in the hair is considered to be the threshold level for the manifestation of Minamata disease. The mercury level in the hair is the best indicator of mercury poisoning. Persons su!ering from methylmercury poisoning in mercury-polluted areas can be identi"ed by determining the amount of mercury in the hair.
FIG. 3. Black spot formation on an X-ray "lm by mass screening method in human hair. Numbers represent total mercury concentrations (lg/g) in human hair samples analyzed by #ameless atomic absorption spectrometer. Hair sample contained 22.1 lg/g formed a clear black spot on X-ray "lm.
A MASS SCREENING METHOD FOR METHYLMERCURY POISONING
However, the mercury level in a pregnant woman's hair is very low because their fetuses accumulate methylmercury through the placenta. Developmental defects were noted in children of Canadian Cree Indians prenatally exposed to methylmercury. The maximum hair mercury concentrations of their mothers were between 13 and 24 lg/g (MckeownEyssen et al., 1983). Developmental delay was also detected in the children of mothers whose hair mercury concentrations were in the range 6 to 18 lg/g in New Zealand (KjellstroK m et al., 1986, 1989). A risk analysis study in Iraq indicated that motor retardation occurred in children prenatally exposed to methylmercury. The mothers' hair concentrations of mercury were less than 50 lg/g, probably in the range 10}20 lg/g (Cox et al., 1989). From these reports, the World Health Organization concluded that a 5% risk of minimal e!ect in the o!spring may be associated with a peak mercury level of 10}20 lg/g in the maternal hair. Recently, the relationship between prenatal exposure to methylmercury in a population with high "sh consumption and the neurodevelopmental defects were further studied in Peru (Marsh et al., 1995) and Seychelles (Myers et al., 1995; Lapham et al., 1995). However, there was no distinct increase in the frequency of neurodevelopmental abnormalities in childhood. The mercury level in the hair of pregnant women is not a good indicator of mercury poisoning of their babies. So, this mass screening method is not suitable for the screening of pregnant women exposed to methylmercury, but the authors think it can be applied to newborn baby's hair. Hair absorbs metallic mercury vapor in air. Metallic mercury, for example, is used to extract metallic gold in Brazil, and the hair of gold miners absorbs a large amount of mercury vapor. Although the determination of mercury in the hair by a #ameless atomic absorption spectrometer with a mercury analysis vaporizer is simpler and faster than that by gas chromatography, it cannot distinguish methylmercury from metallic mercury. As the present method using methylmercury-volatilizing bacteria determines only the methylmercury amount in the hair, it is ideal for screening of methylmercury poisoning. The methylmercury-volatilizing Bacillus subtilis strain was obtained from Dr. K. Nakamura. CONCLUSIONS
A simpli"ed mass screening method for methylmercury exposure was developed using methylmercury-volatilizing bacteria from Minamata Bay. Fur of rats injected with methylmercury formed clear black spots on X-ray "lm, whereas fur of rats injected with saline did not. Fifty human hair samples were examined by this mass screening method. The screening method identi"ed people who had a mercury concentration over 20 lg/g.
103
A few thousand hair samples a day can be screened using this mass screening method because it is rapid, simple, and economical. This method, therefore, enables screening persons with methylmercury poisoning in mercury-polluted areas. ACKNOWLEDGMENTS This work was supported by Grants from the Science and Technology Agency and the Ministry of Education (08671325) of Japan. The authors thank Mrs. M. Kakimoto and A. Tazoe for assistance.
REFERENCES Bakir, F., Damuluji, S. F., Amin-Zaki, L., Murtadha, M., Khalid, A., AlRawi, N. Y., Tikriti, S., Dhahir, H. I., Clarkson, T. W., Smith, J. C., and Doherty, R. A. (1973). Methylmercury poisoning in Iraq. Science 181, 230}241. Baldi, F., and D'amato M. L. (1986). Mercury pollution in marine sediment cores near cinnabar deposits and a chlor-alkali plant. Sci. ¹otal Environ. 57, 111}120. Begley, T. P., Walts, A. E., and Walsh, C. T. (1986). Bacterial organomercuric lyase: Overproduction, isolation, and charaterization. Biochemistry 25, 7186}7192. Clark, D. L., Weiss, A. A., and Silver, S. (1977). Mercury and organomercurial resistances determined by plasmids in Pseudomonas. J. Bacteriol. 132, 186}196. Cox, C., Clarkson, T., Marsh, D., Amin-Zaki, L., AL Tikriti, S., and Myers, G. (1989). Dose}response analysis of infants prenatally exposed to methylmercury: An application of a single-strand hair analysis. Environ. Res. 49, 318}332. Eyssen, G. E. M., and Ruedy, J. (1983). Methylmercury exposure in Northern QueH bec: I. Neurologic "ndings in adults. Am. J. Epidemiol. 118, 461}469. Givanoli-Jakubczak, T., Greenwood, M. R., Smith, J. C., and Clarkson, T. W. (1974). Determination of total and inorganic mercury in hair by #ameless atomic absorption, and methylmercury in gas chromatography. Clin. Chem. 20, 222}229. ICPS (1990). Environmental Health Criteria. Vol. 101, Methylmercury. World Health Organization, Geneva. KjellstroK m, T., Kennedy, P., Wallis, S., and Mantell, C. (1986). Physical and mental development of children with prenatal exposure to mercury from "sh. Stage I: Preliminary tests at age 4. National Swedish Environmental Protection Board Report, 3080, Solna, Sweden. KjellstroK m T., Kennedy, P., Wallis, S., Stewart, A., Friberg, B., Witherspoon, T., and Matell, C. (1989). Physical and mental development of children with prenatal exposure to mercury from "sh. Stage II: Interviews and psychological tests at age 6. National Swedish Environmental Broad Report, 3642, Solna, Sweden. Lapham, L. W., Cernichiari, C., Cox, C., Myers, G. J., Baggs, R. B., Shamlaye, C. F., Davidson, P. W., and Clarkson, T. W. (1995). An analysis of autopsy brain tissue from infants prenatally exposed to methylmercury. Neuro. ¹oxicol. 16, 689}704. Lopez-Artiguez, M., Grilo, A., Martinez, D., Soria, L., Nunez, L., Ruano, A., Moreno, E., Fuente, G. F., and Repetto, M. (1994). Mercury and methylmercury in population risk groups on the Atlantic coast of Southern Spain. Arch. Environ. ¹oxicol. 27, 415}419. Malm, O., Pfei!er, W. C., Souza, C. M. M., and Reuther, R. (1990). Mercury pollution due to gold mining in the Medeira River Basin, Brazil. Ambio. 19, 11}15.
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Marsh, D. O., Turner, M. D., Smith, J. C., Alloen, P., and Richdale, N. (1995). Fetal methylmercury study in a Peruvian "sh-eating population. Neuro. ¹oxicol. 16, 717}726. Mckeown-Eyssen, G. E., Ruedy, J., and Neims, S. (1983). Methylmercury exposure in Northern QueH bec: II. Neurological "ndings in children. Am. J. Epidemiol. 118, 470}479. Myers, G. J., Davidson, P. W., Cox, C., Shamlaye, C. F., Tanner, M. A., Marsh, D. O., Cernichiari, E., Lapham, L. W., Berlin, M., and Clarkson, T. W. (1995). Summary of the Sychelles child development study on the relationship of fetal methylmercury exposure to neurodevelopment. Neuro. ¹oxicol. 16, 711}716. Nakamura, K., Fujisaki, T., and Tamashiro, H. (1986). Characteristics of Hg-resistant bacteria isolated from Minamata Bay sediment. Environ. Res. 40, 58}67. Nakamura, K., and Nakahara, H. (1988). Simpli"ed X-ray "lm method for detection of bacterial volatilization of mercury chloride by Esherichia coli. Appl. Environ. Microbiol. 54, 2871}2873. Nakamura, K., Sakamoto, M., Uchiyama, H., and Yagi, O. (1990). Organomercurial-volatilizing bacteria in the mercury-polluted sediment of Minamata Bay, Japan. Appl. Environ. Microbiol. 56, 304}305. Nakamura, K., and Silver, S. (1994). Molecular analysis of mercury-resistant Bacillus isolates from sediment of Minamata Bay, Japan. Appl. Environ. Microbiol. 60, 4596}4599. Nriagu, J. O., Pfei!er, W. C., Malm, O., de Souza, C. M. M., and Mierle, G. (1992). Mercury pollution in Brazil. Nature 356, 389.
Phelps, R. W., Clarkson, T. W., Kreshaw, T. G., and Wheatley, B. (1980). Interrelationships of blood and hair mercury concentrations in a North American population exposed to methylmercury. Arch. Environ. Health. 35, 161}168. Rustam, H., and Hamdi, T. (1974). Methylmercury poisoning in Iraq. Brain 97, 499}510. Schiering, N., Kabsch, W., Moore, M. J., Distefano, M. D., Walsh, C. T., and Pai, E. F. (1991). Structure of the detoxi"cation catalyst mercuric ion reductase from Bacillus sp. strain RC 607. Nature 352, 168}171. Selifonova, O. V., and Barkay, T. (1994). Role of Na> in transport of Hg> and induction of the Tn 21 mer operon. Appl. Environ. Microbiol. 60, 3503}3507. Summers, A. O., and Silver, S. (1972). Mercury resistance in a plasmidbearing strain of Escherichia coli. J. Bacteriol. 112, 1228}1236. Suzuki, T., Takemoto, T., and Miyama, T. (1992). Extractability of mercury with hydrochloric acid in human hair. Ind. Health 10, 118}121. Takizawa, Y. (1979). Epidemiology of mercury poisoning. In ¹he Biogeochemistry of Mercury in the Environment (J. O. Nriagu, Ed.), pp. 325}363. Elsevier/North-Holland Biomedical Press, Amsterdam. Tubaki, T., and Irukayama, K. (Eds.) (1977). Minamata disease: Methylmercury poisoning in Minamata and Niigata, Japan. Kodansha, Ltd., Tokyo, and Elsevier Scienti"c, Amsterdam. Wheatley, B., Barbeau, A., Clarkson, T. W., and Lapham, L. (1979). Methylmercury poisoning in Canada Indians*The elusive diagnosis. Can. J. Neurol. Sci. 6, 417}422.
A New Mass Screening Method for Methylmercury Poisoning Using Mercury-Volatilizing Bacteria from Minamata Bay Kunihiko Nakamura,* Ichiro Naruse,- and Yukio Takizawa* *National Institute for Minamata Disease, Minamata, 4058-18 Hama, Kumamoto, Japan 867-0008; and -Congenital Anomaly Research Center, Faculty of Medicine, Kyoto University, Sakyo-ku, YoshidaKonoe-cho, Kyoto 606-8315, Japan Received November 2, 1998
A simpli5ed mass screening method for methylmercury exposure was developed using methylmercury-volatilizing bacteria from Minamata Bay. Some bacteria can transform methylmercury into mercury vapor. Most mercury in the hair is methylmercury, which is readily extracted with HCl solution. Black spots are formed on X-ray 5lm due to the reduction of Agⴙ emulsion with mercury vapor produced by methylmercury-volatilizing bacteria. By exploiting these characteristics, a screening method was developed, whereby the fur of rats injected with methylmercury chloride formed clear black spots on X-ray 5lm, whereas the fur of rats injected with saline did not. Subsequently, 50 human hair samples were examined using this mass screening method. The method identi5ed people who had high mercury concentration, over 20 lg/g. A few thousand hair samples may be screened in a day using this method because it is rapid, simple, and economical. This method, therefore, enables screening of persons with methylmercury poisoning in mercury-polluted areas. 1999 Academic Press
Key Words: mercury pollution; methylmercury poisoning; mercury-volatilizing bacteria; methylmercury-volatilizing bacteria; Minamata Bay.
INTRODUCTION
Mercury pollution of the environment has become a serious problem throughout the world in recent years. Minamata disease, methylmercury poisoning, was caused by the consumption of "sh and shell"sh containing high concentrations of methylmercury in Minamata Bay and along the Agano River in Japan (Tubaki and Irukayama, 1977; Takizawa, 1979). A massive outbreak took place in Iraq as well (Bakir et al., 1973; Rustam and Hamdi, 1974) in 1971}1972. Mercury pollution has also been reported in QueH bec, Canada (Eyssen and Ruedy, 1983; Wheatley et al., 1979), the Amazon in Brazil (Malm et al., 1990; Nriagu et al., 1992), and in other areas (Baldi and D'amato, 1986; ICPS, 1990; Lopez-Artiguez et al., 1994). Identifying persons su!ering from methylmercury poisoning in mercury-polluted areas has depended on neuro-
logical examinations and determination of the mercury content in the hair and blood. A concentration of more than 50 lg/g of mercury in the hair is considered to be the threshold level for the manifestation of Minamata disease (ICPS, 1990). The amount of mercury in the hair is about 250 times that in blood (ICPS, 1990) and hair samples are much easier to obtain and store than blood or other organ samples. Thus, hair is clearly the material most suitable for mass screening of populations exposed to methylmercury. Typically, mercury concentration in the hair has been determined through the use of #ameless atomic absorption spectrometry or gas chromatography (Givanoli-Jakubczak et al., 1974; Phelps et al., 1980), but these methods are expensive and time-consuming. Moreover, these methods are not applicable for screening involving a large number of people, such as epidemiological studies. Some bacterial strains have been found to transform mercury compounds such as mercury chloride and methylmercury chloride into volatile mercury vapor (Clark et al., 1977; Nakamura et al., 1990; Summers and Silver, 1972). The bacteria detoxify the compounds by the actions of two enzymes acting sequentially. Organomarcurial lyase cleaves the carbon}mercury bond of certain organomercurials, and then mercuric reductase transforms the mercuric ion to volatile mercury vapor (Begley et al., 1986; Schiering et al., 1991). By exploiting this characteristic, a rapid and simple method for the detection of bacterial volatilization of mercury chloride has been devised, using X-ray "lm (Nakamura, and Nakahara, 1988). Most of the mercury in the hair is methylmercury, which is readily extracted with HCl solution (Suzuki et al., 1992). In the present study, a mass screening method for methylmercury exposure was developed by applying this X-ray "lm method to human hair samples. MATERIALS AND METHODS
Bacterial Strains Seven Bacillus and 10 Pseudomonas strains have been isolated from Minamata Bay. As a Bacillus subtilis strain
100 0147-6513/99 $30.00 Copyright 1999 by Academic Press All rights of reproduction in any form reserved.
101
A MASS SCREENING METHOD FOR METHYLMERCURY POISONING
had mercury-volatilizing genes (Nakamura and Silver, 1994) with the most active mercury-volatilization reaction, this strain was chosen for the present method. The basal media were Luria}Bertani medium (LB medium) and arti"cial sea water medium (Modi"ed ZoBell 2216 E medium), as described previously (Nakamura et al., 1986).
TABLE 2 Extractable Amount of Mercury (lg/g) in Human Hair Samples by Various Concentrations of HCl Solution (MeanⴞSD) Original mercury content in the hair 1.19
Rat Fur Samples
3.32
Fur from young adult (7 weeks old) male SLC:Wister rats was reaped from their backs, and 2}10 mg/kg of methylmercury was injected subcutaneously for 7 to 9 days with di!erent doses, as presented in Table 1. Then, newly grown fur samples were reaped again on Day 14 after the "rst injection. The mercury concentration of the fur was measured by a #ameless atomic absorption spectrometer with a mercury analysis vaporizer (Rigaku Mercury SP-1; Nippon Instrument, Tokyo, Japan). Nontreated and salinetreated control fur revealed under 1 lg/g of mercury. A maximum of 1156 lg/g of mercury was detected in the treated fur.
5.07
0.5 M HCl
1 M HCl
2 M HCl
0.67$0.04 (56.3%) 1.48$0.07 (44.6%) 1.74$0.04 (34.3%)
0.73$0.07 (61.3%) 2.42$0.03 (61.7%) 3.45$0.14 (68.0%)
0.83$0.12 (69.7%) 2.51$0.12 (77.1%) 3.98$0.07 (78.5%)
Note. n"5.
milligrams of hair sample was cut "ne with scissors and soaked in 450 ll of 0.5 M, 1 M, and 2 M HCl in a 1.5-ml microtube. The solution was then vortexed three times for 20 s. The mercury content in the supernatant was directly determined by a #ameless atomic absorption spectrometer, and recovery ratios were calculated (Table 2). Determination of Detection Limit of X-Ray Film Method
Human Hair Samples Fifty human hair samples were used in this study. Thirteen hair samples were collected from sushi restaurant workers who eat "sh frequently. In addition, 37 samples were also collected from the researchers and their families of National Institute for Minamata Disease (30 men and 7 women). The hair samples were washed with distilled water and cut "ne with scissors. The total mercury content in the hair samples was directly analyzed by a #ameless atomic absorption spectrometer. Extraction of Mercury from Human Hair Samples The extractable amount of mercury in the hair samples by various concentrations of HCl solution was examined. Fifty TABLE 1 Mercury Amount in the Rat Fur after Various Subcutaneous Injection of Methylmercury Treatment Nontreated Saline for 9 days 2 mg/kg;5 days#4 mg/kg;4 days 2 mg/kg;5 days#4 mg/kg;4 days 3 mg/kg;5 days#6 mg/kg;3 days 3 mg/kg;5 days#6 mg/kg;3 days 4 mg/kg;5 days#8 mg/kg;3 days 4 mg/kg;5 days#8 mg/kg;3 days 5 mg/kg;5 days#10 mg/kg;2 days 5 mg/kg;5 days#10 mg/kg;3 days
Mercury amount (lg/g) 0.68 0.84 369.6 455.6 400.3 512.4 641.0 670.8 990.8 1156.3
A Bacillus subtilis strain from Minamata Bay was incubated in a LB liquid medium containing 1 lg of HgCl per milliliter to induce the mercury resistance operon. After incubation for 17 h at 303C, the strain was harvested by centrifugation and washed twice with 0.5 M phosphate buffer (pH 7.0). The washed cell was resuspended at a concentration of 50 mg/ml in 0.5 M phosphate bu!er containing 0.5 M EDTA, 0.2 M magnesium acetate, and 0.1 mM thioglycolate. The cell suspension could be prepared as a stock solution and kept in the refrigerator for up to 1 week. The 1 M HCl solutions containing 0, 0.2, 1, 2, and 5 lg of CH HgCl per milliliter were neutralized with 2 M NaOH to pH 7.0. Fifty microliters of the solution was added to each well in a 96-well microplate (Cell Well 25855; Corning Glass Works), then 50 ll of the cell suspension was added to each well. The microplate was covered with X-ray "lm (Fuji Medical X-ray Film New RX; Fuji Photo Film Co. Ltd.) in a darkroom. This was set between two acrylic plastic boards and the ends were secured with clips (Double Clip No. 3; Kokuyo Co. Ltd.). The plate was then put into a dark box. The X-ray "lm was developed after reacting for 6 h at 353C. Black spots on the "lm were the result of the reduction of Ag> emulsion by mercury vapor produced by methylmercury-volatilizing bacteria. The density of black spots on the X-ray "lm was measured with a densitometer (LKB 2222 Ultra Scan XL, LKB Productor Bromma, Sweden). RESULTS
A Bacillus subtilis strain was grown in an arti"cial sea water liquid medium containing 20 lg/ml of HgCl . The
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NAKAMURA, NARUSE, AND TAKIZAWA
activity of methylmercury volatilization by this strain was determined by measuring mercury loss in a liquid culture medium containing 0.1 lg/ml of CH HgCl. The percentage of mercury lost was 94.7% after incubation for 24 h at 303C. The extractable amount of mercury in human hair samples by HCl solution is presented in Table 2. Over 60% of mercury in the hair was extracted with 1 and 2 M HCl solution. When the HCl solution was neutralized with NaOH, a large amount of NaCl was formed in the solution. The high concentrations of NaCl inhibited the bacterial volatilization of mercury (Selifonova and Barkay, 1994). The Bacillus subtilis strain from Minamata Bay used in this study could grow in a peptone water medium containing 1.2 M NaCl. For this reason, 1 M HCl was chosen for the extraction of methylmercury in the hair samples in this mass screening method. The black spots formed by the methylmercury-volatilizing bacteria in the CH HgCl solution using the X-ray "lm method are provided in Fig. 1. The densities of the black spots (Area, AU mm) measured with a densitometer are as follows: 0; 0.000, 0.1; 0.078, 0.5; 0.113, 1.0; 0.245, 2.5; 0.714. The results could be read with the naked eye semiquantitatively with the black spot formation expressing the degree of reaction. The detection limit of the method for CH HgCl was over 0.5 lg/ml. The X-ray "lm method was modi"ed for the mass screening method for methylmercury poisoning using rat fur containing methylmercury. Each 30 mg of rat fur sample was cut "ne with scissors and soaked in 270 ll of 1 M HCl in a 1.5-ml microtube to extract the methylmercury from the fur. After the sample was mixed three times for 20 s with the vortex mixture, the supernatant was transferred to a new tube and diluted with 1 M HCl to an adequate concentration. Then, 200 ll of the solution was transferred to another tube and neutralized with 2 M NaOH to pH 7.0. To each well in a 96-well microplate, 120 ll of the above solution was added. Forty microliters of the cell suspension described under Materials and Methods was then added to each well in the microplate. The plate was covered with X-ray "lm in a dark room. The plate was then left in a dark box for 6 h at 353C, until the X-ray "lm developed. The fur
FIG. 1. Black spot formation by the methylmercury-volatilizing bacteria in the methylmercury chloride solution on an X-ray "lm. Numbers represent methylmercury concentrations (lg/g) in the solution. The densities of the black spots (Area, AU mm) measured with a densitometer were as follows: 0, 0.000; 0.1, 0.078; 0.5, 0.113; 1.0, 0.245; 2.5, 0.714. Black spots formed with over 50 lg/g are detectable by the naked eye semiquantitatively.
FIG. 2. Black spot formation on an X-ray "lm by mass screening method in rat fur. Numbers represent total mercury concentrations (lg/g) in rat fur samples analyzed by #ameless atomic absorption spectrometer. Black spots formed with over 19.0 lg/g are detectable by the naked eye semiquantitatively.
of rats injected with CH HgCl formed clear black spots on the X-ray "lm, while the fur of rats treated with saline did not (Fig. 2). Fifty human hair samples, the mercury concentrations of which had already been measured by a #ameless atomic absorption spectrometer, were examined by this mass screening method. Of these samples, one from a heavy "sh-eater (sushi restaurant worker), which contained 22.1 lg/g of mercury, formed a clear black spot on the X-ray "lm (Fig. 3). Thus, this screening method allowed detection of a mercury concentration above 20 lg/g in a group of 50 people. DISCUSSION
The concentration of 50 lg/g of mercury in the hair is considered to be the threshold level for the manifestation of Minamata disease. The mercury level in the hair is the best indicator of mercury poisoning. Persons su!ering from methylmercury poisoning in mercury-polluted areas can be identi"ed by determining the amount of mercury in the hair.
FIG. 3. Black spot formation on an X-ray "lm by mass screening method in human hair. Numbers represent total mercury concentrations (lg/g) in human hair samples analyzed by #ameless atomic absorption spectrometer. Hair sample contained 22.1 lg/g formed a clear black spot on X-ray "lm.
A MASS SCREENING METHOD FOR METHYLMERCURY POISONING
However, the mercury level in a pregnant woman's hair is very low because their fetuses accumulate methylmercury through the placenta. Developmental defects were noted in children of Canadian Cree Indians prenatally exposed to methylmercury. The maximum hair mercury concentrations of their mothers were between 13 and 24 lg/g (MckeownEyssen et al., 1983). Developmental delay was also detected in the children of mothers whose hair mercury concentrations were in the range 6 to 18 lg/g in New Zealand (KjellstroK m et al., 1986, 1989). A risk analysis study in Iraq indicated that motor retardation occurred in children prenatally exposed to methylmercury. The mothers' hair concentrations of mercury were less than 50 lg/g, probably in the range 10}20 lg/g (Cox et al., 1989). From these reports, the World Health Organization concluded that a 5% risk of minimal e!ect in the o!spring may be associated with a peak mercury level of 10}20 lg/g in the maternal hair. Recently, the relationship between prenatal exposure to methylmercury in a population with high "sh consumption and the neurodevelopmental defects were further studied in Peru (Marsh et al., 1995) and Seychelles (Myers et al., 1995; Lapham et al., 1995). However, there was no distinct increase in the frequency of neurodevelopmental abnormalities in childhood. The mercury level in the hair of pregnant women is not a good indicator of mercury poisoning of their babies. So, this mass screening method is not suitable for the screening of pregnant women exposed to methylmercury, but the authors think it can be applied to newborn baby's hair. Hair absorbs metallic mercury vapor in air. Metallic mercury, for example, is used to extract metallic gold in Brazil, and the hair of gold miners absorbs a large amount of mercury vapor. Although the determination of mercury in the hair by a #ameless atomic absorption spectrometer with a mercury analysis vaporizer is simpler and faster than that by gas chromatography, it cannot distinguish methylmercury from metallic mercury. As the present method using methylmercury-volatilizing bacteria determines only the methylmercury amount in the hair, it is ideal for screening of methylmercury poisoning. The methylmercury-volatilizing Bacillus subtilis strain was obtained from Dr. K. Nakamura. CONCLUSIONS
A simpli"ed mass screening method for methylmercury exposure was developed using methylmercury-volatilizing bacteria from Minamata Bay. Fur of rats injected with methylmercury formed clear black spots on X-ray "lm, whereas fur of rats injected with saline did not. Fifty human hair samples were examined by this mass screening method. The screening method identi"ed people who had a mercury concentration over 20 lg/g.
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A few thousand hair samples a day can be screened using this mass screening method because it is rapid, simple, and economical. This method, therefore, enables screening persons with methylmercury poisoning in mercury-polluted areas. ACKNOWLEDGMENTS This work was supported by Grants from the Science and Technology Agency and the Ministry of Education (08671325) of Japan. The authors thank Mrs. M. Kakimoto and A. Tazoe for assistance.
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