Title Reconstruction of human exposure to heavy metals using synchrotron radiation microbeams in prehistoric and modern humans

OBJECTIVE
Teeth can serve as records of environmental exposure to heavy metals during their formation. We applied a new technology - synchrotron radiation microbeams (SRXRF) - for analysis of heavy metals in human permanent teeth in modern and historical samples.


METHODS
Each tooth was cut in half. A longitudinal section 200 mum in thickness was subjected to the determination of the heavy metal content by SRXRF or conventional analytical methods (ICP-MS analysis or reduction-aeration atomic absorption spectrometry). The relative concentrations of Pb, Hg, Cu and Zn measured by SRXRF were translated in concentrations (in g of heavy metal/g of enamel) using calibration curves by the two analytical methods.


RESULTS
Concentrations in teeth in the modern females (n = 5) were 1.2 +/- 0.5 mug/g (n = 5) for Pb; 1.7 +/- 0.2 ng/g for Hg; 0.9 +/- 1.1 mug/g for Cu; 150 +/- 24.6 mug/g for Zn. The levels of Pb were highest in the teeth samples obtained from the humans of the Edo era (1603-1868 AD: ) (0.5-4.0 mug/g, n = 4). No trend was observed in this study in the Hg content in teeth during 3,000 years. The concentrations of Cu were highest in teeth of two medieval craftsmen (57.0 and 220 mug/g). The levels of Zn were higher in modern subjects (P < 0.05) than those in the Jomon (~1000 BC: ) to Edo periods [113.2 +/- 27.4 (mug/g, n = 11)]. Reconstruction of developmental exposure history to lead in a famous court painter of the Edo period (18th century) revealed high levels of Pb (7.1-22.0 mug/g) in his childhood.


CONCLUSIONS
SRXRF is useful a method for reconstructing human exposures in very long trends.


57
Many toxic heavy metals are found in the environment, and certain levels of 58 exposure are inevitable for the inhabiting human populations. The industrial release of 59 some heavy metals, such as lead and chromium, to the environment is significantly 60 larger than the natural sources of these metals, while the levels of other heavy metals, 61 such as cadmium and mercury, from either natural or industrial sources are the same (1).

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Human beings as Homo sapiens, have been exposed to various heavy metals from stone 64 age (2). Rapid increase in exposure to levels of the heavy metals in the modern samples (C1-C3) were excavated from the cemetery, which was continuously used for 129 burials from 10 th C to 16 th C, AD. People, who lived in a town across from the cemetery, 130 were buried in this place. No information was available for individuals from the 10 th C, 131 AD. However, the 14 th C, AD subjects, from whom the teeth samples were collected,    Each tooth was cut into half by longitudinal section (Fig. 1). From one piece, a 151 longitudinal section 200 μm in thickness was cut using a diamond saw-cutter. To prevent contamination, diamond wire was immersed in distilled deionized water in a 153 plastic container and the water was replaced after each sample was cut. Fresh water was 154 used for each tooth when grinding and polishing of the samples and all samples were 155 rinsed well with water prior to analysis.   Long-term trend and data interpretation 223 Limited number of tooth samples made it impossible to draw any definitive conclusions on the long-term trend in the concentration of these four heavy metals in human teeth. 225 However, data presented in Table 2 suggests some very interesting exposure profiles.   Therefore, subjects C1 and C2 might be exposed to copper through dust or fumes.   (Table 2), he was exposed to high levels of lead at the neonatal and early 259 infantile periods and to moderate levels in his childhood period. It should be also 260 pointed out that he was heavily exposed to Cu. On the other hand, his enamel contained 261 only trace amounts of Zn.

263
In the present study, we have established a method using SRXRF to determine heavy has been employed traditionally (9,10). In the present study we also confirmed 271 usefulness of this method.
The dental enamel has been thought to be an ideal material for reconstruction of the 274 exposure histories, because heavy metals incorporated into the enamel are encapsulated 275 as they are chronologically absorbed during the subject's growth (6,7). Therefore, this 276 ability of the enamel can be fully utilized only by in situ analysis of the enamel metal 277 content with a high resolution method. For this purpose, a laser abraded method coupled 278 with ICP-MS or SRXRF seems to be promising. SRXRF has some advantages, since 279 this method enables detection of the distribution of heavy metals with high resolution.

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As an example, in our study this method showed that enamels in K's two teeth,  This study lacked solid standard reference materials that are matrix matched for 294 calibration purposes. Alternatively, we calibrated using ICP-MS or AAS, which lost 295 information of special distribution for each heavy metal. This is the major limitation of this study. Thus at present, we cannot fully utilize the advantages of SRXRF. This 297 drawback will be recovered in future.

299
In conclusion, we have developed a quantitative method using SRXRF with a 300 calibration by ICP-MS and AAS. This method allowed us spatial high sensitivity with 301 high resolution with appropriate external standards. We have applied this method to the