G 418 br Fig Wheat bracts and
Fig. 1. Wheat bracts and the siliceous prickles on their surfaces observed by scanning electron microscopy and light microscopy. A) Wheat seed and bracts including palea, lemma, and glume. B) Single prickle (pr) on the surface of the glume (Source: Hodson and Sangster, 1989). C) Abundant silicified prickles on the surface of palea, with silica concentrated on the prickle tips (Source: Hodson and Sangster, 1988). D) The bases of broken prickles on the lemma (Source: Hodson and Sangster, 1988). E) Single prickle with bulbous G 418 and the base of another broken prickle (arrow) from the ashed bracts (Source: Hodson and Sangster, 1989). F) Prickles and spherical particles contained in the fine wheat dust (Source: Martin, 1981).
higher concentrations of silica than wheat grains or roots (Sangster et al., 2001).
Future work to test whether silica fibers from wheat husks might be involved in the etiology of ESCC in north China should at least include the following studies:
1) Silicon content and silica fibers should be measured in esophageal tissues from high- and low-incidence regions;
2) Silicon content and silica fibers in wheat bracts (glume, lemma, and palea; see Fig. 1A) should be compared between regions of diﬀerent ESCC incidence;
3) Geographical distribution of silicon in blood, urine, and hair should be mapped in relation to ESCC risk;
4) The carcinogenicity of silica fibers from wheat husks can be directly tested in chicken or other animals.
The current study is an initial attempt to measure silicon and silica fibers in esophageal tissues from north China. We aim to examine whether phytoliths can be detected in esophageal tissues and whether silicon distribution varies between tumor tissue and distant normal tissue. Phytoliths (from Greek, “plant stone”) are amorphous silica particles deposited in cells of plants. As these phytoliths persist after the decay of the plant, they have been used as microfossils in archae-ological and palaeoenvironmental studies. Phytoliths in dental calculus have been used to study ancient plant foodstuﬀs (Fox et al., 1996). The preliminary findings here on phytolith analysis in esophageal tissues will guide the design of further systematic research into biogenic silica in relation to esophageal cancer in north China.
3. Materials and methods
3.1. ESCC tissue sample preparation
From the archives of the Pathology Department of Heping Hospital, Changzhi Medical College, we selected six pairs of formalin-fixed samples, tumor tissues and distant normal tissues, of six patients op-erated for ESCC who had no history of workplace exposure to silica dust. For the first three pairs of tissue samples, one 0.5 cm*0.5 cm*0.5 cm piece of each sample was pre-frozen at −20 °C and then lyophilized in a freeze-dryer at −50 °C for 48 h (Wu et al., 2012); subsequently, two pairs of these dried tissue samples were used for phytolith analysis and another pair for microanalysis with Trans-mission Electron Microscope (TEM). For the other three pairs of tissue samples, ultrathin sections were prepared for TEM analysis. Ethical approval for this study was obtained from the Institutional Review Board of Heping Hospital (2018-004).
3.2. Phytolith analysis
A wet ashing method modified after Lu et al. (2006) was used to extract phytoliths from the esophageal tissue samples. (i) Each dried esophagus tissue sample was cleaned with distilled water in a water bath to remove any surface contaminants. (ii) The cleaned sample was placed in 20 ml of saturated nitric acid for 12 h to completely oxidize the organic materials. (iii) The solution was then centrifuged at 2000 r.p.m. for 10 min, decanted and rinsed twice with distilled water, and rinsed with 95% ethanol until the supernatants were clear. (iv) Any residue detected in the solution was recovered and mounted onto mi-croscopic slides in Canada Balsam medium for photomicrography. (v) Light photomicrography at 400× magnification was used to identify and determine the morphology of the recovered phytoliths.
3.3. Transmission electron microscopy (TEM) analysis
The dried tissue samples, including the tumor tissue and distant normal tissue of one ESCC patient operated for ESCC, were mounted on 200 mesh Cu TEM grids with carbon film support after grinding with isopropyl alcohol and ultrasonicating for 25 min. For the three other pairs of formalin-fixed samples not treated by the freeze-drying process, blocks of 1 mm in diameter were fixed in glutaraldehyde in cacodylate buﬀer, postfixed in osmium tetroxide, and ultrathin sections were prepared and stained with lead citrate for examination under TEM. All the tissue samples on the TEM grids were analyzed with a JEM-2100 TEM operated at 200 kV (Li and Shao, 2009). Elements heavier than carbon (C) were semiquantitatively determined by an energy-dispersive X-ray spectrometer (EDS). For each segment of tissue found on the grid, at least 4 areas were chosen from the center and periphery of a sam-pling spot for elemental analysis. Approximately 20 low-magnification TEM images were taken for each ESCC tumor tissue sample and its corresponding distant normal tissue sample, respectively.