Structural Change of Human Hair Induced by Mercury Exposure

Aug 27, 2013 - Mercury is one of the most hazardous pollutants in the environment. In this paper, the structural change of human hair induced by mercu...
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Structural Change of Human Hair Induced by Mercury Exposure Xueqing Xing,† Rong Du,†,‡ Yufeng Li,§ Bai Li,§ Quan Cai,† Guang Mo,† Yu Gong,†,‡ Zhongjun Chen,† and Zhonghua Wu*,† †

Beijing Synchrotron Radiation Facility, Institute of High Energy Physics, Chinese Academy of Sciences, Beijing, Beijing Municipality 100049, China § CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, Institute of High Energy Physics, Chinese Academy of Sciences, Beijing, Beijing Municipality 100049, China ‡ University of the Chinese Academy of Sciences, Beijing, Beijing Municipality 100049, China ABSTRACT: Mercury is one of the most hazardous pollutants in the environment. In this paper, the structural change of human hair induced by mercury exposure was studied. Human hair samples were, respectively, collected from the normal Beijing area and the Hg-contaminated Wanshan area of the Guizhou Province, China. Inductively coupled plasma mass spectroscopy was used to detect the element contents. A small angle X-ray scattering technique was used to probe the structural change. Three reflections with 8.8, 6.7, and 4.5 nm spacing were compared between the normal and the Hg-contaminated hair samples. The results confirm that the 4.5 nm reflection is from the ordered fibrillar structure of glycosaminoglycan (GAG) in proteoglycan (PG) that composes the matrix around the intermediate filaments. The increase of Ca content makes the regular oriented fibrillar structure of GAG transform to a random oriented one, broadening the angular extent of the reflection with 4.5 nm spacing. However, overdose Hg makes the core proteins where the ordered fibrils of GAG are attached become coiled, which destroys the ordered arrangements of fibrillar GAG in PG, resulting in the disappearance of the reflections with 4.5 nm spacing. The disappearance of the 4.5 nm reflection can be used as a bioindicator of overdose Hg contamination to the human body. A supercoiled-coil model of hair nanoscale structure and a possible mechanism of mercury effect in human hair are proposed in this paper.

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INTRODUCTION Human hair is a keratin-containing biological fiber. There has been some research1,2 about the structure and structural dynamics of human hair. Because human hair can be used as an indicator of human diseases such as neurologic problems, myocardial infarction, autism,3−5 and breast cancer,6,7 as well as heavy metal pollution to the environment,8−10 the structural study of human hair attracts extensive attention. It is wellknown that human hair11 contains a morphologically surface layer, intermediate layer, and central part on microscopic scale from the outside to the inside. The surface layer is called the cuticle composed mainly of lipid granules,12 forming the outer protective envelope. The intermediate layer is called the cortex, in which spindle-shaped cortical cells are aligned along the fiber axis. The central part existing in thicker hairs is called the medulla, showing usually a tube-like structure. Cortex is the main body of hair, in which cortical cells are enclosed by the cell-membrane complex. One cortical cell contains hundreds of macrofibrils with a 300 nm diameter. One macrofibril consists of about one thousand intermediate filaments (IFs) embedded in the extracellular matrix (ECM). Each IF (also called as microfibril) consists of several protofibrils. Each protofibril is a © 2013 American Chemical Society

keratin octamer. ECM is mainly composed of hydrophilic sulfur-rich proteoglycans (PG),13 forming a less organized structure and surrounding the IFs. ECM is the last protecting mantle of the ordered arrangement of IFs. The cuticle cells and the cortical cells are glued together by the cell-membrane complex consisting of cell membranes and adhesive materials. The cell-membrane complex and other nonkeratin components14,15 form the major pathway for diffusion into the fibers. Lipids were also found in the cortex and medulla of hair.12,16,17 Synchrotron radiation small-angle X-ray scattering (SAXS)18−20 demonstrated that human hair has nanoscale structures. A highly ordered structural characteristic of human hair in submicrometer periods gives rise to the diffraction patterns in small-angle region. The diameters of IFs are about 10 nm, which are assembled into an ordered supramolecular structure. The cortex presents a continuous network structure composed of α-keratin fibrils which are parallel to the axial Received: Revised: Accepted: Published: 11214

September 24, 2012 August 14, 2013 August 27, 2013 August 27, 2013 dx.doi.org/10.1021/es402335k | Environ. Sci. Technol. 2013, 47, 11214−11220

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Mercury (Hg) is one of the most hazardous pollutants in the global environment, which can cause brain, kidney, and neurological damage. Hg accumulation in human bodies has two main routes. One is the inhalation of mercury vapor in the local environment. Another is the absorption of Hg from water and diets25 in forms of Hg0, inorganic mercury Hg2+, and methylmercury CH3Hg+.25−29 Previous studies show that metal contents in hair can be used as biological indices for exposure, like manganese, lead, mercury, and so on. Compared to other candidates30 of biological indicators such as blood and urine, hair can give a lasting record31 of metal exposure. It means that the metal distribution along the hair strands reflects the time history of exposure. A normal content of total mercury in human hair is about 0.4−0.6 μg/g, but it is considered to be poisonous32 if the Hg concentration is more than 50 μg/g. The mercury reserve in China was the third in the world. The Wanshan mercury mine located in Guizhou Province was the largest Hg production center in China. A long-term mining activity, which lasted until 2001, has caused serious Hg contamination to the local environment and adjacent ecosystem.33−36 To estimate the Hg contamination to the human body, the effect of Hg on the structure of human hair is being studied. In this paper, a comparative study for the human hairs taken from the Wanshan residents, Wanshan miners, and the Beijing (control) residents is performed. The SAXS technique is used to probe the structural difference of human hairs with and without Hg contamination. We hope this study is helpful to estimate the Hg contamination in the human body.

direction of the hair. Such assemblies are responsible for many biological functions. Therefore, the identification of the SAXS pattern of human hair could be used as a criterion for pathological transformations of the human body. The SAXS pattern of human hair is a fibrillar type diffraction pattern.18−20 A sketch map of typical SAXS patterns of hair is shown in Figure 1. Three reflections with 8.8, 6.7, and 4.5 nm

Figure 1. Sketch map of typical two-dimensional SAXS patterns of human hairs with the three main reflections. The 4.5 nm reflection is different between hairs with low-Ca content (left) and those with highCa content (right).

spacing are included in the SAXS patterns. The meridional reflections depend on the periodic spacing (∼47 nm) of αhelical bundle existing within the IFs. Such a periodic spacing is along the axial (i.e., meridional) direction of the hair. The most intensive reflection is corresponding to the spacing interval of 6.7 nm which is the seventh-order diffraction of the main period. The equatorial reflections arise from the molecular− structural packing arrangement in the cross section of the hair and correspond to the cylindrical Fourier-transform of an αhelical bundle. The 8.8 nm reflection is corresponding to an interplanar distance in the equatorial direction. In addition, there is also a diffraction arc or ring across the equator in some cases, corresponding to a molecular spacing of 4.5 nm. It should be emphasized that the reflection with 4.5 nm spacing was suggested to be a marker6,21 for diagnosis of malignant tumor of mamma tissue. However, there is also some research22,23 giving negative arguments. Evidently, whether the reflection with 4.5 nm spacing can be used as a marker for mammary cancer or not is still ambiguous. In addition, the SAXS pattern around the direct beam is often elongated and parallel to the equator, showing a double-wedge shape. Such a shape arises from the anisotropy of the scatterers with long axis in the meridional direction and short axis in the equatorial direction of human hairs. The group of Vazina studied the origin of the 4.5 nm reflection. Trace elements analysis,23,24 diffraction studies, and the structural dynamics studies of hair under stretch2 were performed. They concluded that the diffraction arc or ring with 4.5 nm spacing arose from the crystalline structure of PG molecules existing in the ECM and reflecting the periodic arrangements of the glycosaminoglycan (GAG) chain attached onto the protein core.2 At the same time, the increase in Ca content in ECM can induce the transformation of regular oriented GAG fibrils into random-coil ones. It was this configuration transformation that resulted in the increase of the angular extent in the 4.5 nm diffraction arcs.23 When Ca content increased to some extent, the diffraction arc even extended into a full diffraction ring as sketched in the right of Figure 1. Our question is as follows: what will happen when Hg content increases in human hair?

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EXPERIMENTAL SECTION Materials. Human hair samples were, respectively, collected from the normal Beijing area in 2009 and from the contaminated Wanshan area in 2003. Four of the Beijing samples were from female donors and marked as BF1, BF2, BF3, and BF4. Three of the Beijing samples were from male donors and marked as BM1, BM2, and BM3. A total of 14 Wanshan samples with relatively higher-Hg contents were, respectively, selected from lots of human hair samples for the Wanshan resident group and the Wanshan miner group. Among the 14 Wanshan samples, six were taken from the Wanshan residents and marked as WR1, WR2, WR3, WR4, WR5, and WR6. The others were from the miners of mercury mine and marked as WM1−WM8. During the hair sampling, hairs with cosmetic treatment such as hair dye, heat styling, and so on were excluded. The routine washing of hairs did not have an obvious difference between the control group from the Beijing area and the mercury group from the Wanshan area. Element Analysis. Inductively coupled plasma mass spectroscopy (ICP-MS) was used to measure the elemental content in the hair samples. Before measurement, the hair samples were repeatedly washed with deionized water, detergent, deionized water, and acetone thrice. Then, the hair samples were dried in air. Afterward, the hair samples were cut into 2−3 mm long pieces. All samples, including the standard human hair with code GBW09101 (a national gold level standard reference material of China, purchased from Chinese CRM/RM Information Center), were carefully weighed with an electronic balance with accuracy of 0.01 mg. Then, these weighed samples were, respectively, bathed in concentrated nitric acid solution (70% in volume fraction) of 10 mL overnight. The next morning, the nitric acid bathed samples were put on a hot plate with a temperature of 150−170 °C for 1 h. Then, H2O2 was gently added into the solutions to further 11215

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Table 1. Hg and Ca Contents (μg/g) in the Hair Samples Measured by Using ICP-MS Beijing residents

Wanshan residents

Wanshan miners

sample

Hg

Ca

sample

Hg

Ca

sample

Hg

Ca

BF1 BF2 BF3 BF4 BM1 BM2 BM3

1.7 1.3 1.4 1.3 2.5 1.8 1.9

1172.1 1092.5 1024.6 801.3 543.8 703.4 835.8

WR1 WR2 WR3 WR4 WR5 WR6

7.1 27.3 10.7 9.1 53.3 3.7

658.5 1082.2 798.0 706.1 1379.3 1289.2

WM1 WM2 WM3 WM4 WM5 WM6 WM7 WM8

127.4 527.1 184.7 148.5 186.0 175.3 246.4 550.3

1696.2 988.0 1055.1 1015.8 1177.2 987.0 1201.6 1046.1

statistical difference of the Hg contamination for males and females. The SAXS patterns of all the 21 hair samples were shown in Figure 2. It can be seen that there are three main reflections

dissolve the organic materials in the hairs. Two hours later, the hairs were completely dissolved. All these solutions were cooled to room temperature and diluted, respectively, to be 5 mL in volume. Two kinds of standard solutions were also prepared. One is the Hg standard at concentrations of 0.5, 1.0, 2.0, 5.0, 10.0, and 20.0 ppb (ng/mL), respectively. Another is Qc21 with 21 kinds of metals ions all at the same concentrations of 1.0, 5.0, 10.0, 50.0, 100.0, and 200.0 ppb, respectively. Finally, each of all these solutions was taken out 5 mL for ICP-MS measurements. The ICP-MS intensities of the unknown samples were compared with the I−C (intensity to concentration) curves of the standard solutions. Consequently, the metal contents in the hairs were obtained. For the seven Hgcontamination-free Beijing samples, their Hg contents were around 1.3 μg/g, showing normal Hg contents. However, the Hg contents were obtained to be several-10-fold higher than the normal ones for the Wanshan residents. Especially, for the Wanshan miners, their Hg contents in hair were even several hundredfold higher. In this study, six and eight hair samples were, respectively, selected from the Wanshan resident group and the Wanshan miner group for SAXS analysis. Their Hg and Ca contents in the samples are tabulated in Table 1. SAXS Measurements. The SAXS technique was used to detect the nanostructures in these hair samples. Before SAXS measurements, all hair samples were washed in the same process conditions as for ICP-MS measurements. Their SAXS patterns were collected at beamline 1W2A of Beijing Synchrotron Radiation Facility (BSRF) with wavelength of 0.154 nm. A Mar165 two-dimensional charge coupled device (CCD) detector with 2048 × 2048 pixels (each pixel size is 79 μm) was used to record the SAXS patterns. The detector-tosample distance was 1500 mm, covering a q-range of 0.31−1.76 nm−1. Here, q = 4π·sinθ/λ, where 2θ is the scattering angle and λ is the wavelength of the incoming X-ray beam. During the SAXS measurements, the hair bundles were fixed on the specimen holders. The axial direction of the hairs was put on the horizontal plane.

Figure 2. SAXS patterns of a total of 21 hair samples. Samples BF1−4 are from Beijing female donors. Samples BM1−3 are from Beijing male donors. Samples WR1−6 are from Wanshan residents. Samples WM1−8 are from Wanshan miners.

appearing on the patterns. The three reflections are, respectively, corresponding to the interplanar distances of about 8.8, 6.7, and 4.5 nm. The reflections with 8.8 and 4.5 nm spacing arise from the equatorial structure of the hairs and are located mainly in the vertical direction of Figure 2. However, a pair of faint diffraction arcs with about 6.7 nm spacing, which are from the meridional structure of hairs, can be found in the horizontal direction of Figure 2. It is corresponding to the seventh-order diffraction37 of the main periodic structure along the meridional direction of hairs. Therefore, the length of periodic structure along the meridional direction of hairs can be estimated to be about 46.9 nm. The reflection with 8.8 nm spacing is presented usually as a pair of diffraction spots. According to the hair sample stance during SAXS measurements, such a pair of diffraction spots corresponds to reflections of the crystal planes whose normal direction is along the equatorial direction of the hairs. The two diffraction spots always appear on the SAXS patterns and have strong diffraction intensity for all hair samples. Evidently, the diffraction spots with 8.8 nm spacing reflect the main structure in human hairs. That is to say, the main contents of hairs, IFs, form a highly ordered structure with interplanar spacing of about 8.8 nm. Normally, hexagonal structure is a preferential configuration of cylinders. Assuming the IFs take a cylindrical shape and have a hexagonal structure, the separation between two IFs can be estimated to be about 10.2 nm. For the normal hair samples from the Beijing area, there is a distinct diffraction ring with 4.5 nm spacing always appearing on the SAXS patterns. Obviously, the intensity distribution on

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RESULTS AND DISCUSSION In previous research, the Hg concentrations in male and female hairs were extensively compared and quite conflicting evidence was reported. Some claimed that females had lower Hg concentration than males, but others reported an opposite trend with females having higher concentrations of Hg in hair than males. The differences in methyl-Hg uptake and elimination between male and female mice were also reported. However, we cannot find the obvious difference of Hg contents between female and male hairs for the Beijing samples as well as for the Wanshan ones. It can be concluded that there is no 11216

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the 4.5 nm diffraction ring is inhomogeneous. The maximum intensity of this diffraction ring is located at the equatorial direction of the SAXS pattern, which is from the contribution of the equatorial directions of the hairs. In some cases, this diffraction ring is degenerated to a pair of diffraction arcs. In fact, the inhomogeneous intensity distribution on the 4.5 nm diffraction ring demonstrates that the corresponding structure is not isotropic. Such a location of maximum intensity on the diffraction ring implies that the equatorial direction of the hairs is the preferred orientation of the oriented structure. The increase of the angular extent in the diffraction arcs means that such an oriented structure tends to have random orientation. By checking the normal hair samples from the Beijing area, it can be found that their diffraction intensity distributions are not the same. The decrease of diffraction intensity can be attributed to the slight increase of Hg content, such as the Hg content of BM1 being up to 2.5 μg/g. In addition, the 4.5 nm diffraction ring does not originate from breast cancer6,21 in our cases, because almost all the female and male hairs show such a diffraction ring and the hair donors are in good health. There are two different opinions about the origin of the 4.5 nm reflection. Doucet and James’ groups, who followed Fraser’s viewpoint,38 thought that the lipid granules in hair gave the 4.5 nm reflection. Doucet’s group did several experiments showing the locations of lipids in hair.12,16,17 However, there was no direct evidence for the lipid granules in hair giving the 4.5 nm reflection. It is just one of the possibilities. Through SAXS studies on pure PGs and human hairs with different contents of Ca, Vazina’s group2,23 reported that the origin of the 4.5 nm reflection is most likely the PGs in ECM. They observed the change of 4.5 nm reflection with the content of Ca. When the Ca content increased, the diffraction arc with 4.5 nm spacing gradually became a diffraction ring. This was because the oriented fibrillar structure of GAG was transformed into a random coiled structure by the contact of Ca with the group −COO− in GAG. Besides, the diffraction intensity with 4.5 nm spacing increased with the increasing Ca content. That was due to the entrance of heavier Ca atoms into the GAG, which enhanced the scattering capacity of the fibrillar structure of GAG. The content of Ca in our hair samples was also detected by using ICP-MS. To validate the effect of Ca content in hairs, the Ca contents in the Beijing hair samples growing in a normal environment were checked. It is found that the angular extent in the 4.5 nm diffraction arcs and their diffraction intensity are positively correlated to the Ca content. For example, the Ca contents of samples BF1, BM3, and BM1 are, respectively, 1172, 801, and 544 μg/g. With the increase of Ca content in ECM, the diffraction ring is more perfect and the intensity is higher as shown in Figures 2 and 3. Our results about the effect of Ca content are in good agreement with Vazina’s results. Thus, we believe that it is more reliable to attribute the 4.5 nm reflection to the GAG of PGs in ECM. Generally speaking, the 4.5 nm reflections1,2,14,15,18−20,23 are always existent in the SAXS patterns of normal hairs. For these hair samples from the Wanshan area, their SAXS patterns have an obvious different characteristic from the normal samples from the Beijing area. The reflection with 4.5 nm spacing is difficult to distinguish from the SAXS patterns for these hair samples from Wanshan residents, and this reflection has completely disappeared on the SAXS patterns of the hairs from Wanshan miners. In order to compare clearly the changes of the diffraction intensities, the one-dimensional SAXS curves

Figure 3. One-dimensional SAXS curves along the equatorial direction of the SAXS patterns. A monotonously decreasing scatteringbackground with q-value (I ∝ q−2.3) was simply removed from the one-dimensional SAXS curves. BF1−4 are from Beijing female donors. BM1−3 are from Beijing male donors. WR1−6 are from Wanshan residents. WM1−8 are from Wanshan miners.

in the equatorial direction were extracted with Fit2D software.39 To give prominence to the diffraction peaks, a monotonously decreasing scattering background (I ∝ q−2.3) was simply subtracted14 from the one-dimensional SAXS curves. The resulting diffraction peaks are shown in Figure 3. Obviously, the 4.5 nm reflections (q ≈ 1.38 nm−1) have almost disappeared. When the changes of Ca and Hg contents are compared, as listed in Table 1, it can be seen that the Ca contents are almost at the same level for the Wanshan and Beijing samples; even the average Ca content is a little higher in the former than in the latter. According to the effect of Ca content in hair as observed by Vazina’s group for normal hairs, the 4.5 nm diffraction intensity of Wanshan samples should be the same or a little higher than the Beijing samples. However, not only does the 4.5 nm diffraction intensity not increase but also it disappears in Wanshan samples. Evidently, there must be another overwhelming factor disturbing the ordered arrangements of GAG fibrils in PGs. By checking the SAXS patterns, it can be found that the reflections with 4.5 nm spacing exist in SAXS patterns as the Hg content is lower, as in Beijing samples, but they are absent as the Hg content is higher as in almost all the Wanshan samples. A remarkably higher Hg content can be found in Wanshan samples than in the Beijing ones as listed in Table 1. Therefore, the disappearance of the 4.5 nm reflections in the Wanshan samples can be attributed to the increase of Hg content in these hair samples. At least, the key factor influencing the diffraction intensity of the 4.5 nm reflection is the Hg content in the hairs. It is roughly evaluated that the reflection with 4.5 nm spacing will disappear as the Hg content exceeds 10 μg/g. From the above discussion, it can be concluded that the Ca content increasing in hair makes the 4.5 nm diffraction intensity increase, but the Hg content increasing makes it decrease. Perhaps, the normal structure of human hairs could have a slight difference due to different genetics and age. Unfortunately, we have not found the relevant research reports. In this study, all the hair samples were donated by the Chinese people in the same ethnic group. Therefore, the genetic difference is negligible. In addition, all the hair samples were taken from people with black hairs. The influence of age on the structure of 11217

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Figure 4. Schematic representation of structure model of IFs (A), ECM (B), and mercury effect on PG (C) in human hair. (A). IF structure model: An arrangement of coiled-coils in the cross section of the hair. Two α-helices form a coiled-coil dimer with diameter of about 2 nm. Four closely packed coiled-coils form a protofibril (octamer) with diameter of about 4.8 nm. Three protofibrils form an IF. The cylindrical IFs form approximately a hexagonal structure with an interplanar spacing of 8.8 nm. The separation between two IFs is about 10.2 nm. The space among the IFs is filled with ECM. ECM consists of PGs. (B). A sketch map of partial ECM structure. The PGs are bonded to the hyaluronate through the link protein (not shown). The GAGs are bonded to the aggregate core protein. (C). A sketch map of the PG with the entrance of Hg species. Hg is bonded to sulfur atoms from cysteine in the core protein, which results in the configuration deformation of the protein.

bondings make the core protein become coiled, which results in the further dilapidation of the ordered arrangements of GAG chains attached on the core proteins. Consequently, the corresponding 4.5 nm reflection disappeared. Besides the Hg−S bonding, whether Hg−C bonding also exists in our hair samples is, at present, ambiguous. The transport mechanisms to hair of inorganic mercury and methylmercury species are likely to be different. The previous XAFS study44 was on hair taken from residents of the Republic of the Seychelles who had lifelong dietary exposure to relatively high levels of methylmercury compounds through consumption of marine fish on a daily basis. Due to the different sources of mercury exposure, the previous XAFS results are therefore not likely to be directly relevant to the hair investigated here. Combining our results with the previous research,18,23,45 a supercoiled-coil model of hair and a possible mechanism of the mercury effect in human hair are proposed as shown in Figure 4. Two α-helices form a coiled-coil dimer with diameter of about 2 nm. Four closely packed coiled-coils form a protofibril (octamer) with diameter of about 4.8 nm. Three protofibrils form an IF. The cylindrical IFs form approximately a hexagonal structure with an interplanar spacing of 8.8 nm. The separation between two IFs is about 10.2 nm. IFs are embedded in the ECM as shown in Figure 4A. The inflexible IFs play the role of skeleton to stabilize the structure. Therefore, the X-ray diffraction with 8.8 nm spacing always appears on the scattering patterns, and the 8.8 nm reflection is almost not affected by the entrance of Hg species, which demonstrates that Hg has no effect to the configuration of IFs. However, Hg species can enter the flexible ECM. A sketch map of partial ECM structure is shown in Figure 4B. The PGs are bonded to the hyaluronate through the link protein (not shown in Figure 4B). The GAGs are bonded to the aggregate core protein. The reflection with 4.5 nm spacing is just from the oriented fibrillar structure of GAG in ECM. When Hg is transported to ECM, Hg is bonded to S-containing and/or other closely related species from cysteine and other residuals in the core protein, forming the S− Hg−S bridge bonds between two sites of the core protein. This results in the configuration deformation of the aggregate core protein as shown in Figure 4C. Due to the interaction between positive charged metal (for example, Ca) and multiple anion groups, the configuration of polysaccharide chains has been changed, which causes the broadening of the reflection with 4.5 nm spacing from an ideal spot to an arc covering some angular

hairs is also negligible. The effect of cosmetic treatment on the hairs was also excluded in the hair sampling process. Another important factor that can change the structure of hairs is diet. Due to the uptake of some metals from foods, one route of metal excretion is via the deposition of metals into hair as it grows. However, the metal contents can be evaluated by the element analysis. Except the prominent difference of Hg contents between the Beijing hair samples and the Wanshan hair samples, the statistical difference of other metal contents is also negligible. During the mining process, a mass of mercury was exposed to the earth’s surface. Because of the lower melting point (−38.8 °C) and the higher saturated vapor pressure (0.37 Pa @ 30 °C), mercury is easily evaporated to the surrounding environment at room temperature. The high level of Hg content in the hairs of the Wanshan miners is due to the inhalation of mercury vapor40 and the consumption of Hgcontaminated foodstuffs since the local soil, water, rice, fish, and meat were found to be contaminated.33,41−43 Therefore, the structural difference of hairs between the normal Beijing samples and the Hg-contaminated Wanshan samples can be ascribed to the different Hg contents. The X-ray absorption fine structure (XAFS) technique is a sensitive tool to detect the local atomic species, coordination number, and bond length. A previous XAFS study44 on human hairs validates that the chemical forms of mercury in human hair is predominantly methylmercury−cysteine (acting as Hg− S and Hg−C) or closely related species. They found that the XAFS spectra of Hg-contaminated hairs can be fitted well by combining 77% of a two-coordinate CH3HgS species and 23% of a three-coordinate [Hg(SR)3]− species. This result implies that about 1/3 of the bondings around the central Hg atoms are Hg−C bonds and 2/3 are Hg−S bonds. For our hair samples from individuals exposed in a heavily contaminated elemental mercury mine site area, Hg−S bondings are also the main existing form of mercury in human hair. As we know, the PGs in ECM are sulfur-rich. Therefore, we think that the mercury− cysteine is concentrated in the ECM of hair. When mercury is transported to human hair, it can form the bondings of S−Hg− S with the cysteines at different sites of core proteins in PGs. A possible biochemical transformation is predicted as follows: R−SH + Hg + HS−R → R−S−Hg−S−R

Here, HS−R represents HS−CH2CH(NH2)COOH (cysteine). Hg is bonded to the S from cysteine. These Hg−S 11218

dx.doi.org/10.1021/es402335k | Environ. Sci. Technol. 2013, 47, 11214−11220

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fetal exposure to methylmercury in the Japanese population. Environ. Res. 2008, 106, 270−276. (11) Robbins, C. R. Chemical and physical behavior of human hair, 4th, ed.; Springer-Verlag: New York, 2002. (12) Briki, F.; Busson, B.; Kreplak, L.; Dumas, P.; Doucet, J. Exploring a biological tissue from atomic to macroscopic scale using synchrotron radiation: Example of hair. Cell Mol. Biol. 2000, 46, 1005− 1016. (13) Zahn, H.; Föhles, J.; Nienhaus, M.; Schwan, A.; Spel, M. Wool as a biological composite structure. Ind. Eng. Chem. Prod. Dev. 1980, 19, 496−501. (14) Kreplak, L.; Mérigoux, C.; Briki, F.; Flot, D.; Doucet, J. Investigation of human hair cuticle structure by microdiffraction: Direct observation of cell membrane complex swelling. Biochim. Biophys. Acta 2001, 1547, 268−274. (15) Ohta, N.; Oka, T.; Inoue, K.; Yagi, N.; Kato, S.; Hatta, I. Structural analysis of cell membrane complex of a hair fibre by microbeam X-ray diffraction. J. Appl. Crystallogr. 2005, 38, 274−279. (16) Dumas, P.; Miller, L. The use of synchrotron infrared microspectroscopy in biological and biomedical investigations. Vib. Spectrosc. 2003, 32, 3−21. (17) Kreplak, L.; Briki, F.; Duvault, Y.; Doucet, J.; Mérigoux, C.; Leroy, F.; Lévêque, J. L.; Miller, L.; Carr, G. L.; Williams, G. P.; Dumas, P. Profiling lipids across Caucasian and Afro-American hair transverse cuts, using synchrotron infrared microspectrometry. Int. J. Cosmet. Sci. 2001, 23, 369−374. (18) Wilk, K. E.; James, V. J.; Amemiya, Y. The intermediate filament structure of human hair. Biochim. Biophys. Acta 1995, 1245, 392−396. (19) Rafik, M. E.; Doucet, J.; Briki, F. The intermediate filament architecture as determined by X-ray diffraction modeling of hard alpha-keratin. Biophys. J. 2004, 86, 3893−3904. (20) Rafik, M. E.; Briki, F.; Burghammer, M.; Doucet, J. In vivo formation steps of the hard alpha-keratin intermediate filament along a hair follicle: Evidence for structural polymorphism. J. Struct. Biol. 2006, 154, 79−88. (21) James, V. False-positive results in studies of changes in fiber diffraction of hair from patients with breast cancer may not be false. J. Natl. Cancer Inst. 2003, 95, 170−171. (22) Briki, F.; Busson, B.; Salicru, B.; Estève, F.; Doucet, J. Breastcancer diagnosis using hair. Nature 1999, 400, 226−226. (23) Aksirov, A. M.; Gerasimov, V. S.; Kondratyev, V. I.; Korneev, V. N.; Kulipanov, G. N.; Lanina, N. F.; Letyagin, V. P.; Mezentsev, N. A.; Sergienko, P. M.; Tolochko, B. P.; Trounova, V. A.; Vazina, A. A. Biological and medical application of SR from the storage rings of VEPP-3 and “Siberia-2”. The origin of specific changes of small-angle X-ray diffraction pattern of hair and their correlation with the elemental content. Nucl. Instrum. Methods Phys. Res., Sect. A 2001, 470, 380−387. (24) Trounova, V. A.; Vazina, A. A.; Lanina, N. F.; Sergienko, P. M.; Kondratyev, V. I. Correlation between element concentrations and Xray diffraction patterns in hair. X-Ray Spectrom. 2002, 31, 314−318. (25) Hall, B. D.; Baron, L. A.; Somers, C. M. Mercury concentrations in surface water and harvested waterfowl from the Prairie Pothole region of Saskatchewan. Environ. Sci. Technol. 2009, 43, 8759−8766. (26) Liu, J.; Feng, X.; Qiu, G.; Anderson, C. W. N.; Yao, H. Prediction of methyl mercury uptake by rice plants (Oryza sativa L.) using the diffusive gradient in thin films technique. Environ. Sci. Technol. 2012, 46, 11013−11020. (27) Schartup, A. T.; Mason, R. P.; Balcom, P. H.; Hollweg, T. A.; Chen, C. Y. Methylmercury production in estuarine sediments: Role of organic matter. Environ. Sci. Technol. 2013, 47, 695−700. (28) Li, P.; Feng, X.; Qiu, G.; Shang, L.; Wang, S. Mercury pollution in Wuchuan mercury mining area, Guizhou, Southwestern China: The impacts from large scale and artisanal mercury mining. Environ. int. 2012, 42, 59−66. (29) Tsui, M. T. K.; Blum, J. D.; Kwon, S. Y.; Finlay, J. C.; Balogh, S. J.; Nollet, Y. H. Sources and transfers of methylmercury in adjacent river and forest food webs. Environ. Sci. Technol. 2012, 46, 10957− 10964.

extent. The configuration deformation of the protein induced by the entrance of overdose mercury further exacerbates the orientation of fibrillar GAG in ECM. Consequently, the structure of fibrillar GAG in ECM becomes disordered. It is the entrance and bonding of the overdose mercury to the PGs in ECM that result in the breakage of the ordered structure and the reflection disappearance related to the 4.5 nm spacing. The above results demonstrate that the ordered structure of PGs in the Wanshan hair samples was completely destroyed by the overdose Hg contamination. It can be concluded that the ordered structures of PGs in ECM of human hairs are degradative with the increase of Hg content. The disappearance of the reflections with 4.5 nm spacing can be used as a bioindicator of overdose Hg contamination to the human body.

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AUTHOR INFORMATION

Corresponding Author

*Tel: 86-10-88235982; fax: 86-10-88235982; e-mail: wuzh@ ihep.ac.cn. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is supported by National Natural Scientific Foundation (Nos. U1232203, 10385008, 11205168) of China and the Knowledge Innovation Program of Chinese Academy of Sciences (Grant No. kjcx3.syw.n8) and the Momentous Equipment Program of Chinese Academy of Sciences (Grant No. YZ200829).

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REFERENCES

(1) Kreplak, L.; Franbourg, A.; Briki, F.; Leroy, F.; Dalle, D.; Doucet, J. A new deformation model of hard alpha-keratin fibers at the nanometer scale: Implications for hard alpha-keratin intermediate filament mechanical properties. Biophys. J. 2002, 82, 2265−2274. (2) Vazina, A. A.; Bras, W.; Dolbnya, I. P.; Korneev, V. N.; Lanina, N. F.; Matyushin, A. M.; Sergienko, P. M.; Zabelin, A. V. Peculiarities of human hair structural dynamics. Nucl. Instrum. Methods Phys. Res., Sect. A 2005, 543, 153−157. (3) Bernard, S.; Enayati, A.; Roger, H.; Binstock, T.; Redwood, L. The role of mercury in the pathogenesis of autism. Mol. Psychiatry 2002, 7, S42−S43. (4) Clarkson, T. W.; Magos, L.; Myers, G. J. The toxicology of mercury - current exposures and clinical manifestations. N. Engl. J. Med. 2003, 349, 1731−1737. (5) Yoshizawa, K.; Rimm, E. B.; Morris, J. S.; Spate, V. L.; Hsieh, C. C.; Spiegelman, D.; Stampfer, M. J.; Willett, W. C. Mercury and the risk of coronary heart disease in men. N. Engl. J. Med. 2002, 347, 1755−1760. (6) James, V.; Kearsley, J.; Irving, T.; Amemiya, Y.; Cookson, D. Using hair to screen for breast cancer. Nature 1999, 398, 33−34. (7) Saengkaew, P.; Ussawawongaraya, W.; Khaweerat, S.; Rugmai, S.; Ouajai, S.; Luengviriya, J.; Sanorpim, S.; Tirarattanasompot, M.; Rhianphumikarakit, S. A preliminary X-ray study on human-hair microstructures for a health-state indicator. World Acad. Sci., Eng. Technol. 2011, 59, 1945−1949. (8) Foo, S. C.; Khoo, N. Y.; Heng, A.; Chua, L. H.; Chia, S. E.; Ong, C. N.; Ngim, C. H.; Jeyaratnam, J. Metals in hair as biological indices for exposure. Int. Arch. Occup. Environ. Health 1993, 65, S83−S86. (9) Li, P.; Feng, X.; Qiu, G.; Shang, L.; Li, G. Human hair mercury levels in the Wanshan mercury mining area, Guizhou Province, China. Environ. Geochem. Health 2009, 31, 683−691. (10) Sakamoto, M.; Kubota, M.; Murata, K.; Nakai, K.; Sonoda, I.; Satoh, H. Changes in mercury concentrations of segmental maternal hair during gestation and their correlations with other biomarkers of 11219

dx.doi.org/10.1021/es402335k | Environ. Sci. Technol. 2013, 47, 11214−11220

Environmental Science & Technology

Article

(30) Afridi, H. I.; Kazi, T. G.; Jamali, M. K.; Kazi, G. H.; Arain, M. B.; Jalbani, N.; Shar, G. Q.; Sarfaraz, R. A. Evaluation of toxic metals in biological samples (scalp hair, blood and urine) of steel mill workers by electrothermal atomic absorption spectrometry. Toxicol. Ind. Health 2006, 22, 381−393. (31) Suzuki, T.; Yamamoto, R. Organic mercury levels in human hair with and without storage for eleven years. Bull. Environ. Contam. Toxicol. 1982, 28, 186−188. (32) Chen, W. Y.; Wang, Y. C.; Kuo, M. S. Determination of total mercury and methylmercury in human hair by graphite-furnace atomic absorption spectrophotometry using 2,3-dimercaptopropane-1-sulfonate as a complexing agent. Anal. Sci. 2002, 18, 255−260. (33) Feng, X.; Qiu, G.; Wang, S.; Shang, L. Distribution and speciation of mercury in surface waters in mercury mining areas in Wanshan, Southwestern China. J. Phys. IV France 2003, 107, 455−458. (34) Horvat, M.; Nolde, N.; Fajon, V.; Jereb, V.; Logar, M.; Lojen, S.; Jacimovic, R.; Falnoga, I.; Liya, Q.; Faganeli, J.; Drobne, D. Total mercury, methylmercury and selenium in mercury polluted areas in the province Guizhou, China. Sci. Total Environ. 2003, 304, 231−256. (35) Zhang, G.; Liu, C. Q.; Wu, P.; Yang, Y. The geochemical characteristics of mine-waste calcines and runoff from the Wanshan mercury mine, Guizhou, China. Appl. Geochem. 2004, 19, 1735−1744. (36) Qiu, G.; Feng, X.; Wang, S.; Shang, L. Mercury and methylmercury in riparian soil, sediments, mine-waste calcines, and moss from abandoned Hg mines in east Guizhou province, southwestern China. Appl. Geochem. 2005, 20, 627−638. (37) Corino, G. L.; French, P. W. Diagnosis of breast cancer by X-ray diffraction of hair. Int. J. Cancer 2008, 122, 847−856. (38) Fraser, R. D. B.; Rogers, G. E.; Filshie, B. K.; Macrae, T. P. Lipids in keratinized tissues. J. Mol. Biol. 1963, 7, 90−91. (39) Hammersley, A. P. Fit2D software. ESRF website: http://www. esrf.eu/computing/scientific/FIT2D/; V12.012, 2004-01-09/2008-0928. (40) Dai, Z. H.; Feng, X. B.; Sommar, J.; Li, P.; Fu, X. W. Spatial distribution of mercury deposition fluxes in Wanshan Hg mining area, Guizhou province, China. Atmos. Chem. Phys. 2012, 12, 6207−6218. (41) Feng, X.; Li, P.; Qiu, G.; Wang, S.; Li, G.; Shang, L.; Meng, B.; Jiang, H.; Bai, W.; Li, Z.; Fu, X. Human exposure to methylmercury through rice intake in mercury mining areas, Guizhou province, China. Environ. Sci. Technol. 2008, 42, 326−332. (42) Qiu, G.; Feng, X.; Wang, S.; Fu, X.; Shang, L. Mercury distribution and speciation in water and fish from abandoned Hg mines in Wanshan, Guizhou province, China. Sci. Total Environ. 2009, 407, 5162−5168. (43) Wang, J.; Feng, X.; Anderson, C. W. N.; Zhu, W.; Yin, R.; Wang, H. Mercury distribution in the soil-plant-air system at the Wanshan mercury mining district in Guizhou, Southwest China. Environ. Toxicol. Chem. 2011, 30, 2725−2731. (44) George, G. N.; Singh, S. P.; Myers, G. J.; Watson, G. E.; Pickering, I. J. The chemical forms of mercury in human hair: A study using X-ray absorption spectroscopy. J. Biol. Inorg. Chem. 2010, 15, 709−715. (45) Zahn, H.; Wortmann, F. J.; Höcker, H. Chemie und Aufbau der Wolle. Chem. Unserer Zeit 1997, 31, 280−290.

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