A Pre-Hispanic Head Raffaella Bianucci1*, Maria Jeziorska2, Rudy Lallo3, Grazia Mattutino 4 , Massimo Massimelli5, Genevieve Phillips6, Otto Appenzeller7 1 Laboratory of Anthropology, Department of Animal and Human Biology, University of Turin, Turin, Italy, 2 Division of Regenerative Medicine, University of Manchester, Manchester, United Kingdom, 3 Fondazione Pinna Pintor and Studio Futura, Turin, Italy, 4 Laboratory of Criminalistic Sciences, Department of Anatomy, Pharmacology and Legal Medicine, University of Turin, Turin, Italy, 5 Legal Medicine Office, Turin, Italy, 6 Fluorescence Microscopy Facility, Cancer Research and Treatment Center, University of New Mexico Albuquerque, New Mexico, United States of America, 7 New Mexico Health Enhancement and Marathon Clinics Research Foundation, Albuquerque, New Mexico, United States of America Abstract This report on a male head revealed biologic rhythms, as gleaned from hydrogen isotope ratios in hair, consistent with a South-American origin and Atomic Mass Spectrometry radiocarbon dating (AMS) compatible with the last pre-Hispanic period (1418–1491 AD, 95.4% probability). Biopsies showed exceptionally well-preserved tissues. The hair contained high levels of toxic elements (lead, arsenic and mercury) incompatible with life. There was no evidence for lead deposition in bone consistent with post-mortem accumulation of this toxic element in the hair. We propose that the high content of metals in hair was the result of metabolic activity of bacteria leading to metal complexation in extra cellular polymeric substances (EPS). This is a recognized protective mechanism for bacteria that thrive in toxic environments. This mechanism may account for the tissues preservation and gives a hint at soil composition where the head was presumably buried. Our results have implications for forensic toxicology which has, hitherto, relied on hair analyses as one means to reconstruct pre- mortem metabolism and for detecting toxic elements accumulated during life. Our finding also has implications for other archaeological specimens where similar circumstances may distort the results of toxicological studies. Citation: Bianucci R, Jeziorska M, Lallo R, Mattutino G, Massimelli M, et al (2008) A Pre-Hispanic Head. PLoS ONE 3(4): e2053. doi:10.1371/journal.pone.0002053 Editor: Ronald Beckett, Quinnipiac University, United States of America Received December 9, 2007; Accepted March 12, 2008; Published April 30, 2008 Copyright: 2008 Bianucci et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: NMHEMC Research Foundation, Albuquerque NM USA Competing Interests: The authors have declared that no competing interests exist. * E-mail: raffaella.bianucci@unito.it Introduction A South-American male (TSA nu1) whose head is now housed in Turin’s Museum of Anthropology and Ethnography was in his twenties when he died some 600 years ago. A huge scar (14.5 cm6 2.9 cm) is visible on his right cheek overlaying a fracture (Fig. 1). An earlier report on this specimen detailed the results of two series of CT scans to determine age and cause of death of this young man [1]. Subsequently an endoscopic examination of the interior of the cranium through the foramen magnum revealed the presence of a white-yellowish ‘waxy’ material filling the skull. Since these initial analyses, several additional studies confirmed and amplified evidence for the extraordinary preservation of this head. Here we present a hypothesis to explain this unusual preservation. We also amplify the evidence, previously obtained, for the preservation of the specimen using different techniques. We posit that post-mortem bacterial activity in the tissues concentrated lead, arsenic and mercury from the soil. These toxic elements, subsequently, inhibited other bacterial growths which, under ordinary circumstances, are detrimental to tissue preserva- tion. Our findings imply that the reliance of forensic toxicology on hair analyses as a reflection of pre-mortem metabolism and toxic exposure might, occasionally, be misguided. Results Radiocarbon dating places the head into the last pre-Hispanic period [2], between 1418–1491 A.D. (95% probability range). Stable isotopes were analyzed in hair. The measured d13C value was 218.1% and the measured value of d15N was +12.4 %. Biopsies examined by light and electron microscopy revealed exceptionally well-preserved tissues. At low magnification (2506), in H & E (haematoxylin-eosin), the various components of the cutaneous samples such as epidermis, dermis, pilo-sebaceous glands and sweat glands were easily identifiable. (Fig. 2) The epidermis was covered by a thick brownish layer made of an amorphous material occasionally interrupted by granulations. The epidermis showed normal structure with the different layers easily identifiable; the nuclei and the edges of cytoplasm were clearly detectable. Desmosomes were observable in the Malpighian layer as well as melanin pigment in the cytoplasm’s basal layer (Fig. 2). The underlying dermis, made up of collagen and elastic fibres, was partially decomposed and showed fungal hyphae and spores identified by periodic-acid Shiff reaction (PAS) and Grocott stains. Some fungal contaminants were also present in the epidermis and in the hypodermis. There was normal immunoreactivity for cytoker- atins of the epidermis and of the glandular epithelium (Table 1). Microanalysis performed on the material filling the skull showed the presence of barium sulphate and lead. The distribution of these metals was not homogeneous showing accumulations in some areas and dispersal in others (Fig. 3). Confocal microscopic appearances of contemporaneous, blond and black hairs, were compared with hair from the specimen and with hair obtained from a naturally mummified South-American female mummy also belonging to Turin’s Museum of Anthropol- ogy [3]. PLoS ONE | www.plosone.org 1 April 2008 | Volume 3 | Issue 4 | e2053