A role for iron and oxygen chemistry in preserving soft tissues, cells and molecules from deep time

Abstract

The persistence of original soft tissues in Mesozoic fossil bone is not explained by current chemical degradation models. We identified iron particles (goethite-αFeO(OH)) associated with soft tissues recovered from two Mesozoic dinosaurs, using transmission electron microscopy, electron energy loss spectroscopy, micro-X-ray diffraction and Fe micro-X-ray absorption near-edge structure. Iron chelators increased fossil tissue immunoreactivity to multiple antibodies dramatically, suggesting a role for iron in both preserving and masking proteins in fossil tissues. Haemoglobin (HB) increased tissue stability more than 200-fold, from approximately 3 days to more than two years at room temperature (25°C) in an ostrich blood vessel model developed to test post-mortem 'tissue fixation' by cross-linking or peroxidation. HB-induced solution hypoxia coupled with iron chelation enhances preservation as follows: HB + O2 > HB - O2 > -O2 >> +O2. The well-known O2/haeme interactions in the chemistry of life, such as respiration and bioenergetics, are complemented by O2/haeme interactions in the preservation of fossil soft tissues.

Keywords: Fenton chemistry; goethite; haemoglobin; iron; protein cross-linking; soft tissue preservation.

Figure 1.

TEM images of (a,c,e) T. rex (MOR 1125) vessels, (b,d) B. canadensis (MOR 2598) vessels and (f) ostrich vessels. Tyrannosaurus rex vessels show iron particles infiltrating a relatively amorphous ‘organic’ layer (arrows, a,c). Higher magnification (c) shows a structure similar in morphology to an endothelial cell nucleus seen in ostrich vessel (EN, f), protruding into the lumen of an isolated vessel. No chromatin or nuclear membrane is visible in the dinosaur structures, but these features are visible in the ostrich. (e) Higher magnification of the area within the box in (c) shows variation in texture within the ‘organic’ layer. B. candensis vessels are more completely infiltrated with iron (b), but in some views (d), an organic layer is still visible. The ostrich vessel (f) shows nuclear membrane (EN) within the endothelial cell (EC). Cytoplasmic extensions make up the bulk of the vessel wall, and a TJ uniting two endothelial cells. Scale bar, 2 µm for (a–d), 1 µm for (e,f).

Figure 2.

(a–c) µ-XRF maps of HB-incubated ostrich, B. canadensis (MOR 2598) and T. rex (MOR 1125) vessel tissues at 3 µm resolution, with locations of analysis identified by white numerical labels, illustrating the intimate association of Fe with each vessel tissue. (d–f) µ-XANES analysis of iron chemical speciation at representative locations, respectively, for each vessel tissue, where the experimental data are plotted in black and the least-square linear combination fits are in red. HB-incubated ostrich tissue was found to contain iron in the form of oxy-HB and biogenic (disordered non-crystalline) iron oxyhydroxide; by contrast, dinosaur vessels contained goethite (α-FeO(OH)) in addition to disordered biogenic-like oxide (BLO) iron oxyhydroxide. Green curves are residuals and indicate good fits for all three samples. (g,h) Optical microscopy, µ-XRF analysis of intravascular structures in B. canadensis. (i,j) µ-XRD and iron µ-XANES chemical analysis of intravascular structure (location 1) in B. canadensis identifies these features as crystalline goethite with an additional fraction of biogenic iron oxyhydroxide, which is amorphous (i.e. poorly diffracting). (k–n) Investigation of intravascular structures in T. rex revealed similar findings (i.e. location 13 was identified as a combination of goethite and biogenic Fe oxyhydroxide). Whole pattern µ-XRD insets of (i,m) exhibit thin, continuous rings for both B. canadensis and T. rex, indicating that the goethite in both samples is nanocrystalline. Labels associated with peaks (e.g. 200, 101) represent h, k, l Miller indices of diffracting planes. XRD was used to identify the nature of the crystalline iron oxyhydroxides and XANES spectroscopy was used to probe the poorly crystalline or amorphous (i.e. poorly diffracting) phase(s) using least-square linear combination fitting and a set of iron standards. (Online version in colour.)

Figure 3.

Overlay and fluorescence microscopy images of dinosaur ‘osteocytes’ and vessels after exposure to polyclonal antibodies. (a–h) Cells exposed to antibodies against actin protein; (i–p) vessels exposed to antibodies raised against elastin protein, a component of vessel walls. (a,b) Brachylophosaurus canadensis and (e,f) T. rex osteocytes show minimal reactivity to antibodies before treatment with PIH; treatment with the iron chelator PIH result in increase in binding to actin antibodies in both B. canadensis (c,d) and T. rex (g,h) isolated osteocytes. Similar increase in antibody binding is visible in dinosaur vessels. (i,j) isolated vessel from B. canadensis exposed to elastin antibodies without PIH; binding is greatly increased after chelation (k,l). A similar pattern is seen for T. rex vessels before (m,n) and after chelation (o,p). All data collection parameters are identical for each condition. Scale bar is 50 µm for each image. (Online version in colour.)

Figure 4.

Ostrich vessels isolated from extant bone. (a,b) Vessels incubated in HB in deoxygenated condition; (c,d) vessels incubated in HB, but exposed to oxygen. (e,f) Deoxygenated vessels incubated in distilled water; (g,h) vessels incubated in distilled water and oxygenated. Black arrows show endothelial layer is much thicker and better preserved in HB-soaked vessels. Other details are seen in HB vessels, for example possible endothelial nuclei on vessel surfaces (white arrowhead). White asterisks indicate red blood cells within vessel of only HB-oxygenated condition; black asterisks show microbial invasion. Scale bar as indicated. (Online version in colour.)