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INFLAMMATION AND IMMUNOPHARMACOLOGY
Department of Microbiology and Immunology, Medical College of Virginia Campus/Virginia Commonwealth University, Richmond, Virginia
Received July 30, 2003; accepted September 8, 2003.
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Abstract |
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). Previous toxicological studies in animals and culture caused both the Environmental Protection Agency and World Health Organization to classify GaAs as an immunotoxicant and potential carcinogen (Luster et al., 1992; World Health Organization, 1992). Exposure of mice to GaAs, either through intratracheal instillation or intraperitoneal administration, causes systemic inhibition of immune function. Immunosuppression is associated with defects in antigen processing by splenic macrophages, resulting in faulty activation of helper T cells (Lewis et al., 1998a,b) and dose-dependent reduction of the IgM humoral response (Sikorski et al., 1989, 1991a,b; Lewis et al., 1996). Impaired antigen processing is attributable to decreased proteolytic activity of cathepsins D, B, and L (Lewis et al., 1998b; Harrison and McCoy, 2001), correlating with decreased interleukin (IL)-2 production by helper T lymphocytes.
In contrast to systemic immune defects, GaAs has the opposite effect at the exposure site. A single intratracheal dose of GaAs or chronic inhalation in rodents results in pulmonary edema, fibrosis, and pneumocyte hyperplasia with accompanying accumulation of macrophages and neutrophils (Webb et al., 1986; National Toxicology Program, 2000; Tanaka et al., 2000). These symptoms are indicative of an inflammatory immune response. Following intraperitoneal exposure, both resident and thioglycollate-induced macrophages exhibit an enhanced ability to activate T lymphocytes in response to protein antigens (Hartmann and McCoy, 1996). This enhancement is mediated both by increased proteolytic activity of thiol cathepsins B and L (Lewis et al., 1998b) and increased surface expression of major histocompatibility complex class II and costimulatory molecules (Hartmann and McCoy, 1996; Caffrey-Nolan and McCoy, 1998). Due to up-regulation of these molecules, GaAs-exposed macrophages might exist in an activated state. In vitro GaAs exposure of macrophages augments cathepsin activity, but does not increase expression of class II molecules (Harrison and McCoy, 2003). Although GaAs directly affects peritoneal macrophages, the in vivo microenvironment also appears to contribute to a completely activated phenotype.
Currently, little is known about the mechanisms by which GaAs enhances macrophage function by GaAs-exposed peritoneal cells. Cytokines are one means by which cells of the immune system modulate their ability to mount responses to antigen. Importantly, cytokines are relevant to inflammatory responses akin to those induced by GaAs administration. The current study investigated the ability of GaAs to influence cytokine expression of a panel of cytokines at local (peritoneal) and distal (splenic) sites. During a time course, GaAs rapidly augmented steady-state mRNA levels of multiple inflammatory cytokines in leukocytes from the exposure site, whereas transcripts of three inhibitory cytokines were decreased. Increased protein quantity of four cytokines was present in the peritoneal fluid. Mainly, macrophages along with some B cells produced three inflammatory cytokine proteins. These findings indicate that GaAs alters cytokine expression at both the mRNA and protein levels, which could contribute to the chemical's adverse biological effects.
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Materials and Methods |
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Monoclonal and Polyclonal Antibodies. Purified rabbit anti-mouse IL-1
and rabbit anti-mouse IL-18 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Biotinylated monoclonal antibodies directed against Mac-1, B220 and Thy1.2, and phycoerythrin-conjugated anti-Mac-3 and anti-MCP-1 (anti-CCL2) were purchased from BD Biosciences PharMingen (San Diego, CA). Goat anti-rabbit IgG was purchased from Molecular Probes (Eugene, OR). Purified rabbit IgG, fluorescein-conjugated streptavidin, and normal mouse IgG were purchased from Zymed Laboratories (South San Francisco, CA). Culture supernatant containing monoclonal 2.4G2 antibody (anti-Fc
receptor) was a gift from Dr. Gregory Burton (Brigham Young University, Provo, UT).
Chemical Exposure. Mice were injected intraperitoneally with 200 mg/kg body weight GaAs crystals with a 1.5-µm mean diameter (Research Triangle Institute, Research Triangle Park, NC) suspended in saline containing 0.05% Tween 80 (Sigma-Aldrich Chemical Co., St. Louis, MO). This dose induces systemic immunosuppression without general toxicity (Sikorski et al., 1989; Lewis et al., 1996). Injections were performed intraperitoneally because this route produces results similar to intratracheal instillation without concomitant pulmonary damage (Hartmann and McCoy, 1996). Control mice were injected with saline containing 0.05% Tween 80 (vehicle) or 200 mg/kg body weight latex beads with a 1.0-µm mean diameter (Sigma-Aldrich) suspended in the vehicle. Cell suspensions were prepared from four mice per exposure group, and sera were pooled from two mice per group.
Preparation of Cell and Fluid Samples. At 6, 18, and 48 h after exposure blood was collected by cardiac puncture and sera were prepared and stored at –20°C until used. For RNA isolation, resident peritoneal leukocytes (RPL) were harvested by lavage and splenic cell suspensions were prepared as described (Lewis et al., 1998b). Cells were suspended in Trizol reagent according to the manufacturer's directions (Invitrogen, Carlsbad, CA). RNA yield was determined by absorbance at 260 nm, and purity and quality were confirmed by 260 nm/280 nm and 260 nm/230 nm absorbance ratios. For enzyme-linked immunosorbent assay (ELISA), cell-free peritoneal fluid (PLF) was collected by lavage with 1 ml Hanks' balanced salt solution and stored at –20°C until used.
Immunofluorescence Staining and Flow Cytometric Analysis. Cells were stained as described (Hartmann and McCoy, 1996). Briefly, RPL were incubated with normal mouse IgG and/or monoclonal 2.4G2 antibody to block Fc receptors. For cell surface staining, cells were incubated with lineage-specific monoclonal antibodies or irrelevant control antibody for 30 min at 4°C and washed. When using biotinylated antibodies, cells were then incubated with fluorescein-labeled streptavidin and washed. Intracellular cytokine staining was conducted using the Cytofix/Cytoperm system (BD Biosciences PharMingen) following the manufacturer's protocol. Briefly, cells were incubated with GolgiPlug (brefeldin A) and stained with lineage-specific antibodies as above. Cells were fixed and permeabilized in Cytofix/Cytoperm buffer, stained with anticytokine or irrelevant control antibody for 30 min at 4°C, and then washed. Fluorescence intensity was measured by a FACScan (BD Biosciences, San Jose, CA). Each experiment analyzed at least 10,000 cells, gated on forward and side scatter to exclude debris. Data analysis was conducted using Cell Quest Pro software (BD Biosciences).
Ribonuclease Protection Assay (RPA). Total RNA was prepared as described above and analyzed using the RiboQuant MultiProbe Ribonuclease Protection Assay System (BD Biosciences PharMingen). The MultiProbe template sets, including mCK2b, mCK3b, and mCK5b, assessed a panel of 25 different cytokines as well as two housekeeping genes, L32 and GAPDH. Briefly, 2-µg total RNA was incubated overnight with radiolabeled probes, subjected to digestion by Ribonuclease A + T1, and protected probes were resolved on an 8 M urea-5% acrylamide sequencing gel. Dried gels were exposed overnight to Kodak X-OMAT blue film (Eastman Kodak, Rochester, NY), or exposed in a Molecular Dynamics PhosphorImager cassette (Amersham Corp., Sunnyvale, CA). Cassette exposures were quantitated on a PhosphorImager 445Si, and data analysis and densitometry were achieved using ImageQuant 5.1 (Amersham Corp.). Band intensities for cytokines were calculated as a proportion of the band density of GAPDH to normalize data among lanes and experiments.
Cell Culture. RPL and splenocytes were cultured overnight at 106 cells/ml in Dulbecco's modified Eagle's medium fortified with 10% heat-inactivated fetal bovine serum (BioSource International, Camarillo, CA), 100 U/ml penicillin/streptomycin (Invitrogen), 2 mM L-glutamine (Invitrogen), and 0.1 mM 2-mercaptoethanol (Sigma-Aldrich). Cell-free culture supernatants were harvested and stored at –20°C until used.
Cytokine ELISA. Murine tumor necrosis factor (TNF)-
and IL-1 were measured using the Quantikine kit following the manufacturer's recommended protocol (R&D Systems, Minneapolis, MN). IL-18 was measured using a commercially available kit (MBL, Nagoya, Japan), as was CCL2 (Pierce Endogen, Rockford, IL) according to the manufacturers' directions. Samples were assayed in triplicate. Absorbance was measured on a SpectraMax 250 plate reader running SOFTmax Pro 3.1.2 software (Molecular Devices Corp., Sunnyvale, CA). Cytokine quantities in samples were calculated from standard curves of recombinant cytokines.
Statistical Analysis. Parametric analysis of variance was performed using Student's two-tailed t test for unmatched pairs in all experiments. GaAs-exposed samples were compared with the vehicle control, and to the latex bead control when applicable. A value of p < 0.05 was considered significant.
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Results |
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; Caffrey-Nolan and McCoy, 1998). However, these observations occurred after a 5-day exposure, allowing for the possibility of early modulation of cell populations. To determine whether early changes in cellular composition are initiated by GaAs exposure mice were administered GaAs intraperitoneally, and at 18 h RPL were characterized by flow cytometric analysis for lineage-specific surface molecules including Mac-1, Mac-3, B220, and Thy1.2. As shown in Fig. 1, alteration of the peritoneal population was evident following GaAs exposure compared with vehicle control. RPL from control mice consisted mainly of Mac-1hi/Mac-3+ and B220+/Mac-3– cells with a negligible fraction (<5%) of Thy1.2+ cells. In contrast, RPL from GaAs-exposed mice were skewed toward a single Mac-1mod/Mac-3mod phenotype consistent with monocytes with relatively few (15%) B220+ cells. The proportion of Thy1.2+ cells appeared to be slightly reduced by GaAs. Thus, peritoneal subpopulations changed early after GaAs exposure.
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Altered Cytokine mRNA Expression after GaAs Exposure. A rapid shift in the cellular profile of RPL may be due to monocyte recruitment to the exposure site, perhaps mediated by proinflammatory cytokines. Multiprobe RPA was used to determine the presence and relative concentration of proinflammatory cytokine mRNA transcripts following GaAs exposure. Latex bead exposure was included as an additional control to determine the effect of nonspecific phagocytosis of particles on cytokine production. Steady-state levels of 25 cytokine transcripts were determined during a time period up to 48 h after exposure. Of these, 21 transcripts in RPL showed altered expression at one or more time points, while the others were unaffected by GaAs exposure during the time course. RPAs were also performed on samples of splenic leukocytes. For all cytokines tested, mRNA expression levels in splenocytes were not significantly different among the exposure groups during the 48-h period (data not shown). Thus, significant changes in cytokine transcript expression after GaAs exposure were limited to RPL.
As shown in a representative gel in Fig. 2, numerous cytokine transcripts were detected following 18-h GaAs exposure, including CCL5, CCL4, CCL3, CXCL1, and CCL2. In contrast, neither vehicle control nor latex bead-exposed RPL appeared to express high levels of these cytokines. Figure 3 depicts a 48-h time course for mRNA expression of three cytokines. During this period, chemokine CXCL1 transcript levels were rapidly elevated at 6 h, and gradually returned to basal level by 48 h (Fig. 3A). A similar expression pattern was observed for the monocyte chemotactic protein CCL2 (Fig. 3B). Vehicle administration caused a small increase in the 6-h expression of these transcripts compared with later time points. Latex bead exposure caused only a slight, short-lived increase in the expression of CXCL1 at 6 h (p < 0.05), the magnitude of which was significantly less than that of GaAs exposure (p < 0.01). Monocyte migration inhibitory factor (MIF) mRNA in RPL also displayed a rapid increase (Fig. 3C); however, its expression was maintained above control throughout the 48-h time course.
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Expression of cytokine transcripts of IL-1 family members indicated that each was specifically up-regulated following GaAs exposure (Fig. 4). As before, transcripts accumulated rapidly after exposure and returned to control levels within 48 h. IL-1 displayed the largest increase in relative expression of all cytokines tested, with a maximal level nearly three times that of the housekeeping gene GAPDH. IL-1 transcript expression after vehicle or latex bead exposure was virtually undetectable. In addition, mRNA transcripts for the proinflammatory cytokines, TNF- and IL-18, displayed expression time courses similar to those observed for the above cytokines (Fig. 5).
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Members of the tumor growth factor (TGF)- family of cytokines are generally considered to have anti-inflammatory activity. In marked contrast to the preceding cytokines, transcripts for TGF-2 and TGF-3 were reduced to below detection level by GaAs exposure (Fig. 6, A and B). Reduction was observed at 6 h, and was complete by 18 h. Although a slight induction of TGF-1 was apparent in vehicle- and latex bead-exposed cells, its expression remained low in GaAs-exposed RPL throughout the time course (Fig. 6C).
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Altered levels of several additional cytokine mRNAs were detected within RPL following GaAs exposure (Table 1). CCL3 and IL-6 mRNA were significantly up-regulated at the 6-h time point, while TNF- mRNA was slightly reduced. By 18 h, CCL3 and IL-6 transcripts remained elevated (Table 1), whereas TNF- returned to basal level (data not shown). Furthermore, up-regulation of six more cytokines occurred at 18 h (Table 1), and included the chemokines CCL4, CCL1, and CCL5 and the cytokines interferon (IFN)-, IL-6, IL-12 p40, IL-10, and IL-12 p35. Again, the mRNA expression of these nine cytokines was normal at 48 h (data not shown).
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Induction of Cytokine Secretion by GaAs. To determine whether increases in cytokine transcripts were reflected at the protein level, four cytokines were selected for further analysis at the 18-h time point. These were selected to represent both proinflammatory cytokine and chemokine families. Following exposure, sera and peritoneal lavage fluids were tested for the presence of IL-1, IL-18, CCL2, and TNF- protein by ELISA. Additionally, harvested RPL and splenocytes were cultured in medium without a stimulus overnight, and culture supernatants were assayed. As shown in Fig. 7A, IL-1 protein was elevated in PLF from GaAs-exposed mice, while no significant change was detected in sera or culture supernatants. IL-18, like IL-1, was significantly increased only in PLF from GaAs-exposed mice (Fig. 7B). The monocyte chemokine CCL2 was elevated in both peritoneal fluid and sera from GaAs-exposed mice (Fig. 7C). Secretion of TNF- was elevated in PLF, but not in serum (Fig. 7D). No differences in cytokine protein levels were detected in culture supernatants generated from RPL or splenocytes. Overall, the increased level of inflammatory cytokine mRNA within the peritoneum was mirrored by similar increases in secreted protein.
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Identification of Cells Producing Cytokines. To determine which cells in the peritoneum were producing inflammatory cytokines, intracellular immunofluorescence staining of RPL for CCL2, IL-1, and IL-18 was performed. A representative experiment is shown in Fig. 8. Following GaAs exposure, intracellular protein expression of cytokines was detected. Approximately 11% of RPL were CCL2+ with the overwhelming majority (>95%) costaining for Mac-1. IL-1 was expressed in approximately 16% of RPL, of which most (58%) were Mac-1+, and the remainder positively stained for B220. IL-18 was expressed in approximately 6.5% of RPL. Again, most (66%) were Mac-1+, and the remainder were B220+. No intracellular cytokine staining was detected in Thy1.2+ cells. Hence, the main cell type producing the three cytokines examined was Mac-1+.
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Discussion |
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family members) were diminished to below detection level. Changes in mRNA expression in response to latex beads had a markedly reduced magnitude and duration compared with GaAs, and in many cases were not different from the vehicle control, indicating that the effects of particulate GaAs were specific to the nature of the particle and not simply due to phagocytosis. Generally, cytokine mRNA steady-state levels rapidly increased, and then gradually returned to basal levels by 48 h. In contrast, suppression TGF- transcript expression remained complete at 48 h, which could also promote inflammation. Our findings are the first evidence that in vivo GaAs exposure affects cytokine mRNA expression.
For altered steady-state mRNA cytokine levels to initiate an inflammatory response, the gene products must be efficiently translated and secreted at the exposure site. We found that four transcripts, which were examined, were efficiently translated in vivo, yielding increased cytokine protein levels within the peritoneal fluid of GaAs-exposed mice relative to vehicle control. In contrast, splenic cells did not significantly differ in proinflammatory cytokine expression after GaAs exposure, neither at the mRNA nor protein level. Only the chemokine CCL2, whose function is dependent upon dissemination for formation of a chemotactic gradient, was detected in sera. Cytokine expression, as determined by intracellular cytokine staining, was predominantly restricted to Mac-1+ cells, although some immunofluorescence staining was detected in B220+ cells. Other investigators have reported that B lymphocytes are capable of secreting proinflammatory cytokines, including IL-1, IL-18, and TNF- (Bonnefoy et al., 1989; Nakanishi et al., 2001; Ferreira et al., 2003). Hence, GaAs may induce direct activation of these cells in vivo. Alternatively, cytokine production by peritoneal B cells might be attributed to the secondary activation of these cells by monocyte-derived IL-1. Secreted cytokines were mainly detected only within the peritoneal lavage fluid, indicating that the inflammatory response is localized, not systemic. Therefore, we conclude that direct exposure of peritoneal macrophages to GaAs results in production of inflammatory cytokines at the exposure site.
The kinetics pattern of proinflammatory cytokine mRNA expression may indicate that the GaAs-mediated inflammatory reaction has a limited duration. However, GaAs particles persist at the exposure site for extended periods up to 28 days (Webb et al., 1986; Burns et al., 1991). This persistence may trigger a second wave of cytokine production, possibly leading to chronic inflammation. Furthermore, secretion of one cytokine typically causes the subsequent induction of others, which might also contribute to a second round of cytokine production. Thus, analysis of later time points may identify additional cytokines that could influence leukocyte recruitment and activation, and inflammation.
The IL-1 family, including IL-1, IL-1, and the IL-1 receptor antagonist (IL-1Ra), is implicated in inflammatory reactions in many experimental systems. The massive up-regulation of IL-1 mRNA (>60-fold) and protein (8-fold), which we observed following GaAs exposure, indicates that this cytokine may be a primary driver of the inflammatory response to GaAs. Furthermore, transcripts of five other cytokines that regulate cell migration were expressed at >3-fold higher than the vehicle control (CCL2, CCL3, CCL5, CXCL1, and MIF). The relatively modest increases in the expression of the other proinflammatory cytokines, such as TNF- and IL-6, suggest these cytokines may play a more limited role. Signaling through the Type I IL-1 receptor activates monocytes and macrophages (Fenton et al., 1987, 1988) and results in the generation of inflammatory lipid mediators, such as prostaglandins or leukotrienes (Gronich et al., 1994). Such a mechanism is consistent with our previous findings of an activated state of GaAs-exposed macrophages (Hartmann and McCoy, 1996; Caffrey-Nolan and McCoy, 1998).
Monocytes are strongly attracted to CCL2 present in inflamed tissues (Kuziel et al., 1997; Siebert et al., 2000). CCL2 was found in sera from GaAs-exposed mice, where it potentially produces a concentration gradient for directed migration of monocytes into peritoneum. After exiting the blood, monocytes mature into macrophages. Macrophages can enhance and sustain inflammation via cytokine secretion (Warren et al., 1988; Bogdan and Nathan, 1993), or differentiate into dendritic cells that migrate to secondary lymphoid organs (Kabel et al., 1989; Zhou and Tedder, 1996). We hypothesize that recruited monocytes, which are evident within the peritoneum at 18 h exposure, phagocytose GaAs particles, become activated, and acquire the macrophage phenotype that is observed 5 days after exposure.
Exposure of macrophages to GaAs in culture stimulates enhanced expression of cathepsin L, a thiol protease, and invariant chain, a molecular chaperone (Harrison et al., 2003). However, in vitro exposure does not induce the complete activated macrophage phenotype that occurs after in vivo exposure. In striking contrast to in vivo chemical exposure, in vitro exposure does not stimulate secretion of IL-1, TNF-, or IL-12 (Harrison et al., 2003). The differential impact of in vivo and in vitro exposures may be due to differences in the myeloid cells examined. Our in vitro model utilized macrophage cell lines and thioglycollate-elicited macrophages. An alternative is that other leukocytes and/or another organ system may influence cytokine production, monocyte migration, and myeloid cell activation during in vivo GaAs exposure.
Other chemotactic factors were up-regulated within the peritoneum following GaAs exposure, including CXCL1 and CCL5, which are chemotactic for neutrophils and T lymphocytes, respectively. However, we did not detect an increased proportion of either cell type 18 h after exposure. Perhaps, neutrophils entered the peritoneum at even earlier time points. In other inflammatory models, neutrophil influx occurs as early as 4 h after treatment (Schramm et al., 2002). Alternatively, neutrophil chemotaxis to CXC chemokines is dependent upon TNF- (Schramm et al., 2000). Although increased TNF- levels were present in peritoneal fluid from GaAs-exposed mice, the amount may be insufficient to support neutrophil diapedesis. Although substantial up-regulation of T lymphocyte chemokine CCL5 occurred, we found relatively minor induction of T cell-related cytokines (IL-18, IFN-, IL-12), suggesting that the role of T cells is minimal early after chemical exposure. Effector T cells are often found at sites of chronic inflammation, days or weeks after initiation. Hence, T lymphocytes may possibly infiltrate the peritoneum later. Ultimately, the role of T cells in the initiation and maintenance of GaAs-induced inflammation requires further study.
In conclusion, GaAs exposure quickly elevated mRNA expression of multiple proinflammatory cytokines, and production appeared to be mainly local, not systemic. Monocytes were recruited to the site of GaAs-mediated inflammation under the direction of CCL2, and may be activated primarily by IL-1. Recruited monocytes may become further stimulated by other cytokines. Although health risks to industrial workers exposed to GaAs remains unclear, animal studies have established that chemical exposure causes detrimental biological effects. Intratracheal instillation and inhalation of GaAs results in pulmonary inflammation and edema in rodents (Webb et al., 1986; National Toxicology Program, 2000; Tanaka et al., 2000). Tissue damage is associated with accumulation of neutrophils and macrophages. Hence, the ability of GaAs to up-regulate proinflammatory cytokines may extend to the lungs, which could contribute to pulmonary damage. Identification of the genes whose expression is altered by GaAs exposure will lead to a better comprehension of the inflammatory reaction induced by the chemical.
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Acknowledgements |
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Footnotes |
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ABBREVIATIONS: GaAs, gallium arsenide; IL, interleukin; RPL, resident peritoneal leukocytes; ELISA, enzyme-linked immunosorbent assay; PLF, peritoneal lavage fluid; RPA, ribonuclease protection assay; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; TNF, tumor necrosis factor; MIF, migration inhibitory factor; TGF, transforming growth factor; IFN, interferon.
Address correspondence to: Dr. Kathleen McCoy, Dept. of Microbiology and Immunology, Box 980678, Virginia Commonwealth University, Richmond, VA 23298-0678. E-mail: kmccoy{at}hsc.vcu.edu
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), latex bead- (
), and vehicle-exposed (
) mRNA levels are expressed proportional to the expression of the housekeeping gene GAPDH. Expression levels were assayed in triplicate, and values are mean ± S.D. Significance between GaAs- and vehicle-exposed mRNA level was determined using Student's t test. **, p < 0.01; ***, p < 0.001.
