Characterizing Lung Inflammation in Agricultural Respiratory Exposures Between the Sexes

Agriculture workers, in Saskatchewan and worldwide, are exposed to numerous potential pollutants, including grain dust and pesticides. Although female workers make up approximately 25% of the agricultural working population, the majority of current research on the respiratory and inflammatory effects in agriculture has been conducted on male animal models and workers; very little has been studied of the female response. Females are able to mount a more robust and efficient early immune response leading to improved prognosis in surviving acute infections arising from a variety of pathogens (bacteria, viruses, trauma) compared to males. However, increased efficiency mean females are more predisposed to developing autoimmune diseases. Agriculture workers are commonly exposed to more than one pollutant at a time; how the interaction between glyphosate and lipopolysaccharide (a component of grain dust) will differentially affect the sexes is not known. Currently, there has been minimal work done to evaluate how a respiratory glyphosate exposure may differentially impact the sexes. The following study evaluates the differences in the inflammatory respiratory response between the sexes following a short-term agriculture respiratory exposure and is the first study to do so. It uses a mouse model. C57BL/6 mice were intranasally treated with glyphosate (1µg), lipopolysaccharide (LPS) (0.5µg), combined LPS + glyphosate (LPS: 0.5 µg + glyphosate: 1µg), or Hank’s Balanced Salt solution (HBSS) for 5 days. These studies were performed to characterize the inflammatory effects in mice following a short-term intranasal exposure to LPS plus glyphosate including 1) evaluating inflammatory effects of the combined exposure to glyphosate and LPS in female mice compared to exposure to each individual agent; 2) comparing the female response to the combined LPS plus glyphosate exposure vs. the male response; and 3) observing the structural lung changes of the combined exposure to glyphosate and LPS in female mice as measured using multiple image radiography. Female mice, exposed to LPS and glyphosate for 5 days showed higher levels of inflammatory mediators compared to control animals, or those treated with only LPS or glyphosate. Inflammatory mediators, such as proinflammatory cytokines, were elevated in the LPS plus glyphosate treated animals, indicating that after 5 days, the addition of the glyphosate impacts the ability of female mice to ameliorate the effects of LPS, compared to the animals treated only with LPS or glyphosate. Further, this study revealed that female mice display a different inflammatory respiratory response compared to male mice. Female mice demonstrated: less lung architecture damage across treatment groups; significantly lower levels of inflammatory markers; and lower levels of proinflammatory cytokine expression as compared to male mice. This is the first study to validate that a significant difference exists between the male and female immune response following a short-term agriculture respiratory exposure to LPS and glyphosate. Finally, comparisons of lung effects using multiple techniques (multiple image radiography, and histology) were utilized to evaluate a short-term common agriculture respiratory exposure in female mice. Histology revealed greater recruitment of cells into alveolar regions in the lungs of the mice and disruption to the bronchial epithelium from the combined LPS and glyphosate treated group as compared to other treatment groups. MIR images revealed mice exposed to LPS and both LPS plus glyphosate showed compromised lung tissue compared to other treatment groups. Taken together, these results reveal that female mice exposed to the combination of LPS and glyphosate displayed physiological and structural effects that were different from mice exposed to LPS or glyphosate alone. However, the inflammatory effects of the combined exposure were not as pronounced in the female mice compared to the male mice, highlighting the importance of using a structural evaluation technique such as multiple image radiography to reveal the impact to the lungs of such exposures. Overall, we observed that female mice, exposed to an agriculturally relevant concentration combining LPS plus glyphosate for 5 days, exhibited respiratory inflammatory effects significantly different compared to each single exposure. Additionally, we demonstrated that there is a significantly different respiratory inflammatory response between the females and males at 5 days of LPS plus glyphosate exposure. While the precise mechanisms remain to be elucidated, the differences may be due to the protective effects of estrogen. This study is the first research to characterize a short term respiratory inflammatory exposure to LPS plus glyphosate in female mice, to compare these results to those obtained from male mice, and to utilize multiple image radiography technology to do so. We were able to detect the differences between exposure groups using MIR and refined this technique during our study. The results suggest that MIR may become a paramount tool in future lung imaging experiments

Characterization of Lung Inflammation Induced by Exposure to Fipronil

Fipronil is an insecticide that acts at the gamma-aminobutyric acid receptor and glutamate-gated chloride channels in the central nervous systems of target organisms. The use of fipronil is increasing across the globe. Presently, very little data exist on the potential impact of exposure to fipronil on the lungs. We studied the same by exposing mice to fipronil intranasally (N=8) or orally (N=7) for 7 days followed by collection of blood, broncho-alveolar lavage (BAL) fluid and lung tissues. Control mice were given corn oil (N=15). The oral and intranasal exposure routes were chosen because these are the most common routes of exposure for humans and animals. Hematoxylin-eosin stained lung sections showed normal histology in the control lungs compared to the thickened alveolar septa, disruption of the airways epithelium and damage to vascular endothelium in the intranasal and the oral groups. Lung sections stained for von Willebrand factor showed that mice exposed to fipronil either orally or intranasally had increased staining in the endothelium and septal capillaries. Compared to the control mice, TLR4 expression in lungs from animals treated orally with fipronil was reduced while animals exposed intranasally had increased TLR4 staining in the airway epithelium. Similarly, TLR9 stained lungs showed that orally treated animals had reduced TLR9 reaction in the airway epithelial cells but intranasally exposed animals had intense TLR9 staining in the alveolar septa and airway epithelium. The slides were also scored blindly to gain a quantitative understanding of the staining; there were a significantly higher number of TLR4 positive stained cells in the intranasal fipronil group (P=0.010) but no significant differences between treatments for TLR9 positive stained cells (P=0.226). The U937 cell line was employed to compliment the in vivo work. Cells were exposed to fipronil in DMSO at concentrations of 0.29 µm to 5.72 µm per 1 ml for various times from 3, 9 and 24 hours. Viability was assessed and western blots on Toll-like receptors 4 and 9 were completed in addition to immunofluorescence. Cell death was determined with trypan blue method. A significant increase in cell death was observed when the cell line was exposed to higher concentrations of fipronil (P<0.0001). Western blots on TLR4 and 9 revealed no significant differences (TLR4 {P=0.49}, TLR9 {P = 0.94}) between cells exposed to fipronil and those exposed to the control (DMSO). The data taken together show that fipronil causes cell dealth in vitro, and induces lung inflammation following oral or intranasal exposure but has different effects on the expression of TLR4 and TLR9 in vivo. Because of the central roles of TLR4 and TLR9 in lung inflammation, fipronil-induced changes in the expression of these receptors would alter the pulmonary response to bacterial infections in the host exposed to fipronil. Further studies are needed to examine the mechanisms through which fipronil regulates expression of immune receptors and also the pulmonary response of fipronil-exposed animals to subsequent microbial infections