Erionite exposure in North Dakota and Turkish villages with mesothelioma

Erionite Exposure Occurs in the United States.

To test the hypothesis that erionite exposure might occur in the United States due to increased urban development, we conducted pilot studies in different areas in the United States known to contain geological deposits of erionite (15). In 2006, we learned that erionite-containing geologic formations in the North Killdeer Mountains in Dunn County, ND were being used since the 1980s (16) to produce gravel. Thus, we focused our studies in Dunn County and our research team determined that over the past two to three decades, more than 300 miles of roads, including 32 miles of school bus routes, parking lots, playgrounds, and baseball fields were paved with erionite-containing gravel (Fig. 1). We tested whether the use of erionite on these roads caused human exposure to this potent carcinogen. Air sampling was performed during activities that disturb gravel such as driving, raking, and sweeping, with air monitoring done in the breathing zone of the individuals performing the activities. We found that when erionite-containing gravel is disturbed, erionite fibers become airborne and can enter the personal breathing zone (Table 1).

Air Concentrations of Erionite in ND and Turkish MM Villages.

Air concentrations of erionite associated with an increased risk of mesothelioma have not been established. Therefore, our team conducted an in-depth survey of the mineral fiber concentrations in the air of five villages in Cappadocia, Turkey, where some of us had previously observed an excess of MM caused by exposure to erionite (1–3, 14, 17, 18). These data allowed us to establish air concentrations of erionite that are associated with an increased risk of MM (Table 2). Next, the results of exposure-point measurements in ND, locations which are shown in Fig. S2 A and B, were compared with similar measurements performed in the Turkish MM villages. Results for erionite concentrations in personal breathing zones during outdoor activities involving raking of gravel in a community parking lot, ball field, and gravel pits in ND were lower than exposures measured during sweeping of roads in Turkish MM villages (Table 2). Erionite concentrations in stationary outdoor samples (defined in legends of Tables 1 and 2) taken along the road near a school bus stop in ND [0.11 structures/cubic centimeter (s/cc) total transmission electron microscopy (TEM), 0.01 s/cc phase contrast microscopy equivalent (PCME)] exceeded all stationary roadside samples collected along the main town roads in the Turkish MM villages (range: <0.00–0.01 s/cc total TEM, all <0.00 s/cc PCME) (Tables 1 and 2). As shown in Tables 1 and 2, results of indoor air samples in personal breathing zones collected in a road maintenance garage and a social services office in ND during sweeping and housekeeping activities (0.18 s/cc total TEM, 0.06 s/cc PCME) were comparable to those in Boyali and Karlik, villages that have MM mortality of 6.25 and 7.41%, respectively (17, 18). Elevated airborne erionite concentrations were also found within school buses (0.10 s/cc total TEM, 0.01 s/cc PCME), inside cars (0.27 s/cc total TEM, 0.02 s/cc PCME), and during bicycle riding (0.59 s/cc total TEM, 0.05 s/cc PCME) over erionite-containing gravel roads in ND. Peak concentrations of 2.74 s/cc total TEM (0.2 s/cc PCME) were measured inside cars and school buses during these driving scenarios (Table 1). Asbestos was also found in total TEM indoor samples in ND and in four of the Turkish villages (except for Tuzkoy, all indoor PCME samples were nondetectable; Table 2). Outdoor asbestos concentrations were observed most notably during activity-based air sampling in the Turkish villages and were not detected in ND (Table 2).

Physical Characteristics and Chemical Composition of ND and Turkish Erionite.

Length and diameter of mineral fibers have been linked to carcinogenesis and only fibers with a diameter of about 0.3 μm or less reach the pleura (19). Scanning electron microscopy (SEM) and TEM analyses revealed that the average length of ND and Turkish erionite fibers was 2.20 and 3.57 μm, respectively. The average width of fibers was 0.31 μm for both locations (Table S1). Compositions of erionite from Old Sarihidir in Cappadocia, Turkey and from ND were compared using an electron microprobe with procedures to minimize loss of cations during analysis. Repeated scans for Sr and Ba showed them to be below the detection limit of 200 ppmw for erionite from both locations. The analysis included K, Na, Ca, Mg, and Fe and framework Al and Si. Accuracy of analysis was confirmed by the “balance error” calculation in which the sum of the nonframework cation charge should equal the charge loss in the framework due to Al⁺³ substitution for Si⁺⁴. As shown in Fig. 2B, resulting atomic Si/(Si+Al) lie between 0.78 and 0.80, at the high end of erionite published values (20), but with ND values slightly lower than the Turkish ones. Atomic Na/(Na⁺ Ca) values discriminate between ND (<0.10) and Turkish erionite (∼0.5). On the conventional K-Mg-(Ca+Na) ternary diagram there appears to be a small difference (Fig. 2C). In summary, our data show that the physical and chemical characteristics of Turkish and ND erionite are very similar.

Biological Activity of ND and Turkish Erionite.

To determine whether the minor differences in the physical properties or chemical composition between erionite from ND and Cappadocia could alter its capacity to induce transformation of human mesothelial cells (HM), we compared the biological activity of erionite from both sources in vitro and in vivo. HM were exposed to erionite in the presence of macrophages in a coculture system that mimics the process of macrophage recruitment and activation to sites of fiber deposition (21). After 8 wk in culture, high numbers of tridimensional foci developed in HM treated with erionite from ND (78.67 ± 3.480), Oregon (52.00 ± 5.686), and Turkey (24.33 ± 3.480) (Fig. 3 A and B). No foci developed in HM cultures with or without macrophages, unexposed or treated with glass beads as controls.

High mobility group box-1 (HMGB1) is localized in the nucleus of most cell types and it is released in the cytoplasm and the extracellular space during programmed cell necrosis, and it is actively secreted in the extracellular space by activated macrophages and by HM exposed to asbestos (21). Release of HMGB1 induces tumor necrosis factor-α (TNF-α) production and secretion by HM and macrophages (22), a process that has been linked to mineral fiber carcinogenesis (21). We tested whether, similar to asbestos, exposure to erionite could lead to the release of HMGB1 (21) and secretion of TNF-α (22). As shown in Fig. 3C, HMGB1 and TNF-α were specifically detected in the conditioned medium of HM and macrophages exposed to either US or Turkish erionite. To compare the hallmarks of MM pathogenesis in vivo (i.e., chronic inflammation in response to mineral fiber deposition that, over time, leads to MM development) (21) groups of three BALB/c mice were injected with 1 mg of ND or Turkish erionite and the organs were analyzed for signs of inflammation after 2 wk. In separate groups of three BALB/c mice, injection of 1 mg of asbestos was used as a positive control and vehicle injection was used as negative control. Inflammatory cell infiltration and mesothelial hyperplasia were detected in the peritoneum of mice injected with any of the mineral fibers (Fig. 3D). Immunohistochemistry showed that most cells surrounding tissue-embedded fibers were positive for the macrophage marker, F4/80. Some macrophages contained fibers or fiber fragments in their cytoplasm. Mesothelial cells were localized in the same areas, as verified by immunostaining for cytokeratin. HMGB1 staining was localized exclusively in both the nuclei and cytoplasm of reactive macrophages surrounding the fibers and in the nearby extracellular space, consistent with HMGB1 release. In vehicle-injected mice, neither chronic inflammation nor cytoplasmic or extracellular staining of HMGB1 was detected. These data revealed no significant difference in biological activities of erionite from ND or Turkey.

Collection of Air Samples.

Currently, there are no applicable or relevant and appropriate requirements or erionite-specific health benchmarks available to establish “safe” levels of human exposure to erionite fibers. Given the current lack of guidance regarding the nature and concentrations of erionite fiber exposure associated with adverse health effects in human populations and similarities with asbestos in physical characteristics, toxicology, and health effects, we used current approaches and benchmarks for asbestos exposures as a starting point for assessment of erionite-contaminated sites. Therefore, air samples were collected using a modified International Standards Organization (ISO) method 10312 “Ambient Air–Determination of Asbestos Fibers–Direct-Transfer Transmission Electron Microscopy Method” or 13794 (indirect method) at a nominal flow rate at or below 10 L per minute (l/min) using 0.8-μm pore size mixed cellulose ester (MCE) filters. Activity-based sampling (defined in the legend of Table 2) was performed to collect personal breathing zone air samples in a variety of settings both indoors and outdoors in Turkey and ND.

Fiber Counting, Identification, and Analysis of Erionite Composition.

Fiber counting, identification, and analysis of erionite composition were performed according to standard procedures (SI Methods). Compositions of Turkish and ND erionite were determined using an electron microprobe according to standard procedures (SI Methods).

Coculture and Foci Formation.

Primary HM were cultured and characterized as described in ref. 22 and used between passages 2 and 3. THP-1 human monocytes (American Type Culture Collection, ATCC) were differentiated into macrophages as described in ref. 38. Primary HM were seeded in 6-well plates until 80% confluence. Macrophages were cocultured in an insert chamber placed on top of the HM. The bottom of the insert chamber has 0.4-μm pores, allowing cytokines and growth factors produced by macrophages to reach the lower chamber where HM are cultured. Erionite (5 μg/cm²) from North Dakota, Oregon, or Karain, Turkey were added into the culture and glass beads (3–10 μm; Polysciences) were used at the same concentration as control. Erionite fibers or glass beads were added once at the beginning of the experiment to both HM cells and macrophages. The cells were kept in culture up to 2 mo. The HM media, together with freshly differentiated macrophages were replaced two times a week. Data were compared between treatment groups by a two-tailed Student's t test.

Western Blotting.

Western blotting was performed using standard procedures (21, 22) (SI Methods).

Animal Experiments.

All procedures were performed in accordance with institutional guidelines and approved by the University of Hawaii Institutional Animal Care and Use Committee. Groups of three 21-d old BALB/c mice were injected intraperitoneally with 1-mg single injection of the following fibers: crocidolite asbestos (ASB), North Dakota erionite (ND), or Turkey erionite (TUR). Control group was injected with PBS. After 2 wk, animals were killed and the tissues and organs were evaluated histologically by hematoxylin-eosin (H&E) staining.

Immunohistochemistry.

Immunohistochemistry was performed according to standard procedures (21, 22) (SI Methods).

Statistical Analysis.

Data were analyzed using the two-sided Student's t test and considered statistically significant when the P value was <0.05. Bar graphs represent the mean, and error bars represent 95% confidence intervals. Statistical analyses were performed using GraphPad Prism version 4.0.