Agricultural dust exposure and pneumoconiosis 2000). We report that young male agricul- tural workers have a higher prevalence of pneumoconiosis and small airway disease associated with mineral dust exposure than do nonagricultural workers living in the same environment. Methods Population. Left lungs from 112 Hispanic male autopsies were collected at the Fresno County coroner’s office from June 1994 to June 1995. Demographic information includ- ing age, residence duration in Fresno County, and occupational histories were obtained from the medical examiner and the coroner’s files. Smoking histories were available for a minor- ity of subjects; therefore, smoking status was classified by pathologic criteria. The study subjects ranged in age from 16 to 73 years and had died suddenly or unexpectedly. An autopsy was performed at the coroner’s office to determine the cause and manner of death, as dictated by state statute. The autopsies were performed within 12‒24 hr after death. The research team independently evaluated a sagittal slice of lung. The project was thor- oughly reviewed and approved by the Human Subjects Review Committee of the University of California, Davis. We did not have access to nor did we contact the next of kin. Tissue preparation. The left lung of each deceased individual was cannulated through the left mainstem bronchus and inflation- fixed with 2% glutaraldehyde at a hydrostatic pressure of 30 cm of water for 2 hr from a constant-pressure gravity apparatus. The lungs were cut in the sagittal plane to include the mainstem bronchus, hilar structures, and the medial aspect of both the upper and lower lobes of the left lung. If the left lung was not suitable because of trauma, the right lung was processed in an equivalent manner. Initial fixation and cutting was done by the coroner’s staff. The lung section was stored in fixative and shipped to the University of California, Davis. On arrival, each lung was photo- graphed from the cut sagittal surface as well as a medial view, and selected gross features were documented on a standard form; these included pleural pigmentation, fibrosis, and emphysema. Selected airways were microdis- sected (Pinkerton et al. 2000) and examined for mucous plugs or aspirated material within the lumen. Histopathologic evaluation. Samples of airways of varying size and airway generation, together with samples of pulmonary paren- chyma, were taken from the upper and lower lobes using a standardized schema (Figure 1). Each tissue block was embedded in paraffin (Fischer Scientific, Fair Lawn, NJ, USA), and 5-µm-thick sections were cut using a rotary microtome and stained with hematoxylin and eosin. Sirius red and elastic trichrome stains were used to confirm the presence of collagen and smooth muscle within tissue sections. We applied standard diagnostic cri- teria for the recognition of pneumoconio- sis (Craighead et al. 1982, 1988) including macules, defined as collections of dust-laden macrophages within the walls of respiratory bronchioles but without significant fibrosis; nodules, defined as fibrotic lesions up to 1 cm in size with round, irregular, or serpiginous borders; and interstitial fibrosis. The tracheobronchial lymph nodes were graded for fibrosis and dust. We evaluated pathologic changes consistent with chronic bronchitis and asthma using standard patho- logic criteria (Green and Churg 1998a, 1998b). Small airways disease—subdivided into mineral dust-associated small airways dis- ease (Churg 1992) and smoking-related small airways disease (Adesina et al. 1991; Cosio et al. 1980)—was diagnosed by accepted cri- teria. We assessed exposure to cigarette smoke in the recent past on the basis of accumula- tion of characteristic smokers’ macrophages within the respiratory bronchioles and adjacent alveoli (Niewoehner et al. 1974; Wright et al. 1992). Details of the diagnostic criteria for the lesions and the grading systems are provided in more detail in the Supplemental Material (available online at http://www.ehponline.org/ members/2009/0800144/suppl.pdf). Mineral dust analysis. We used tissue samples from areas adjacent to those sam- pled for histology for the minerologic assays (Figure 1). The tissues were digested in filtered sodium hypochlorite, and the residual dust was analyzed by two methods. CSi was assayed by X-ray diffraction (XRD) by DataChem Laboratories (Salt Lake City, UT, USA) using NIOSH (National Institute for Occupational Safety and Health) method 7500. We ana- lyzed separate aliquots by scanning electron microscopy (SEM) combined with X-ray spec- trometry (XRS) and image analysis (Stettler et al. 1991) to determine elemental composi- tion and particle size. For the SEM/XRS anal- yses, 1,000 or more inorganic particles were analyzed for each lung sample. Particles were placed into classes of minerals based on their elemental composition. Particles with > 90% silicon were classified as silica; particles with aluminum with or without other cations, that did not fit the criteria for silica, were classi- fied as aluminum silicates (AlSi) (Stettler et al. 1991). Greater detail of the minerologic meth- ods are given in the Supplemental Material (available online at http://www.ehponline.org/ members/2009/0800144/suppl.pdf). Structural remodeling of small airways. The following airways were evaluated: mem- branous bronchioles and first-generation respiratory bronchioles cut in cross-sec- tional and perpendicular planes to the lon- gitudinal profile of the airway, respectively. For respiratory bronchioles fibrosis, muscle hypertrophy, inflammation, intraluminal macrophages, opaque and birefringent par- ticles were recorded. For membranous bron- chioles, mucous cell hyperplasia was also recorded. Each feature was graded from 0 to 3, where 0 represented no evidence of that feature and 1 to 3 represented increas- ing grades of severity (Churg and Wright 1983). Variability of the readings and inter- observer reproducibility were established by having three readers (F.H.Y.G., K.E.P., and V.V.) each independently evaluate the same 30 histologic slides on two different occa- sions. More detail of the grading systems is given in the online Supplemental Material (available at http://www.ehponline.org/ members/2009/0800144/suppl.pdf). Statistical analysis. We analyzed 112 male Hispanic cases for this study. Nine subjects were of undefined occupational status and were not included in comparisons of farmworker to nonagricultural occupation. Assessment of smoking was defined by histologic evidence of ever having smoked. Diseases that met the required pathologic criteria for their diagnosis [see Supplemental Material (available at http:// www.ehponline.org/members/2009/0800144/ suppl.pdf)] were coded dichotomously. Analysis used standard statistical methods and the SAS software procedures (SAS Institute Inc., Cary, NC, USA). Continuous variables are described by mean ± SD, median, quartiles, or ranges. Categorical variables are described using frequencies and percentages. Group com- parisons for continuous variables were achieved using t-tests or analysis of variance for normally distributed data or the Kruskal–Wallis test for non-normally distributed data. For categorical data, we used chi-square analysis or logistic regression analysis to compare groups. We con- ducted bivariate analyses between independent variables and the outcome of interest. If the association between them generated a p-value < 0.05, the variable was considered for multi- variate analysis. Dust analysis (2.5 g) Histology J I E A K Apical F B G Airway morphometry Anterior C D H Figure 1. Sampling strategy for histopathologic and analytical lung tissue including conducting airways of varying sizes, central and peripheral lung paren- chyma, and hilar lymph nodes. Histology was taken from sites A–K, samples for dust analysis from sites E, F, G, and H. Environmental Health Perspectives • volume 117 | number 6 | June 2009 989