Sodium perchlorate induces non-alcoholic fatty liver disease in developing stickleback

Highlights

• Perchlorate induces non-alcoholic fatty liver disease in developing stickleback.

• Perchlorate exposure increases lipid accumulation in stickleback liver.

• Stickleback liver appears to be more sensitive to perchlorate than thyroid tissue.

Abstract

Perchlorate is a pervasive, water-soluble contaminant that competitively inhibits the sodium/iodide symporter, reducing the available iodide for thyroid hormone synthesis. Insufficient iodide uptake can lead to hypothyroidism and metabolic syndromes. Because metabolism, obesity and non-alcoholic fatty liver disease (NAFLD) are tightly linked, we hypothesized that perchlorate would act as an obesogen and cause NAFLD via accumulation of lipids in liver of developing threespine stickleback (Gasterosteus aculeatus). We performed an upshift/downshift exposure regime (clean water to perchlorate treated water or perchlorate treated water to clean water) on stickleback embryos at two concentrations (30 mg/L and 100 mg/L) plus the control (0 mg/L) over the course of 305 days. Adult stickleback were euthanized, H&E stained and analyzed for liver morphology. Specifically, we counted the number of lipid droplets, and measured the area of each droplet and the total lipid area of a representative section of liver. We found that perchlorate treated fish had more and larger lipid droplets, and a larger percentage of lipid in their liver than control fish. These data indicate that perchlorate causes NAFLD and hepatic steatosis in stickleback at concentrations commonly found at contaminated sites. These data also indicate the potential of perchlorate to act as an obesogen. Future studies should investigate the obesogenic capacity of perchlorate by examining organ specific lipid accumulation and whether perchlorate induces these effects at concentrations commonly found in drinking water. Work is also needed to determine the mechanisms by which perchlorate induces lipid accumulation.

Introduction

Endocrine disrupting compounds (EDCs) are chemicals known to interfere with endocrine function of animals. Recent literature provides a growing list of EDCs that induce the accumulation of lipids and cause obesity in animals including humans, a link between chemicals and obesity known as the obesogen hypothesis (Baillie-Hamilton, 2002; Grün and Blumberg, 2009; Heindel and Schug, 2013; Janesick and Blumberg, 2016). Human rates of obesity have risen sharply leading to increases in many obesity-related illnesses and associated health care spending (Hebert et al., 2013). Although increased calorie consumption and reduced exercise may play important roles in the obesity pandemic, the obesogen hypothesis may explain the recent and dramatic increases in obesity as being due in part to exposure to certain EDCs (Heindel and Schug, 2014). Many EDCs are known to affect the regulation of sex hormones, the hypothalamic-pituitary-thyroid axis (HPT), and other endocrine axes (Grün and Blumberg, 2009; Heindel and Schug, 2014). Sex steroids in conjunction with key peptide hormones, such as growth hormone, inhibit lipid accumulation in tissues (Björntorp, 1997). Thus, disruption of this system, organs heavily involved in lipid mobilization and metabolism (liver), or endocrine axes that systemically regulate metabolism (e.g., HPT) have the potential to contribute to lipid accumulation and obesity (Lonardo et al., 2006).

Dysregulation of either androgens (Sato et al., 2003) or estrogens (Cooke and Naaz, 2004) can influence lipid accumulation and result in obesity. Estrogens can be obesogenic at certain developmental time points causing fattening and high circulating leptin levels (Ruhlen et al., 2008). Xenoestrogenic chemicals such as bisphenol-A can also cause obesity by upregulating adipocyte genes (Masuno et al., 2002). Disruption of the HPT axis can lead to depression of circulating thyroxine (T₄) and tri-iodothyronine (T₃) levels and thereby produce obese phenotypes due to downstream effects on lipid metabolism (Grün and Blumberg, 2009). For example, short-term exposure to polybrominated diphenyl ethers (PBDEs) lowers T₄ levels, and thus affects lipolysis in adipocytes (Zhou et al., 2001). Hypothyroidism with associated lower circulating T₄ levels affects overall metabolic rate and causes weight gain (Garber et al., 2012).

Commonly associated with obesity is nonalcoholic fatty liver disease (NAFLD (Braillon et al., 1985; Browning et al., 2004; Nasrallah et al., 1981)). NAFLD is associated with the accumulation of lipids in hepatic tissues at a level greater than 5–10% of the liver's weight without the presence or consumption of alcohol (Bruce and Byrne, 2009). This disease represents a spectrum of disorders that range from fatty liver to nonalcoholic steatohepatitis (NASH – accumulation of lipids coupled with hepatocyte injury and inflammation) which can further progress to cirrhosis and liver failure. This disease progression is known as the two-hit model where the liver first accumulates lipids (first hit) and then experiences inflammation and scarring (second hit) (Day and James, 1998). As of 2004, approximately one third of adults in the United States were diagnosed with some form of NAFLD (Browning et al., 2004), an occurrence rate approaching the obesity rate in the United States in 2015 of nearly 40% (Spengler and Loomba, 2015).

Obesity and NAFLD are associated through genetics, lifestyle and the endocrine system (Anstee and Day, 2013; Lonardo et al., 2006). For example, thyroid function (mediation of lipid metabolism) and NAFLD are linked in humans and animal models (Lonardo et al., 2006). Overfeeding geese for foie gras is associated with both fatty liver and hypothyroidism (Janan et al., 2000). Correlative studies in humans have also found that patients with NASH were more likely to also have hypothyroidism compared to patients without any form of liver disease (Liangpunsakul and Chalasani, 2003). Sex hormones also affect lipid accumulation in the liver. Women under treatment with the estrogen receptor blocker tamoxifen have a higher risk of developing NASH (Bruno et al., 2005). Male aromatase knockout mice also develop hepatic steatosis which can be ameliorated with the addition of exogenous estrogen (Hewitt et al., 2004). These effects can also span multiple generations through epigenetic mechanisms. For example, pregnant mice exposed to tributyltin, a compound that disrupts sex hormone synthesis through inhibition of aromatase, produce offspring with fatty livers and this phenotype persists for another two generations in unexposed individuals (Chamorro-García et al., 2013).

Perchlorate (ClO₄⁻) is a pervasive, water-soluble contaminant to which virtually all U.S. residents and residents of many other industrialized countries are exposed via ingestion of contaminated water and foods (EPA, 2005; FDA, 2004). As of 2009, perchlorate contamination had been detected in 45 U.S. states (GAO, 2010). It has been classified by the U.S. Environmental Protection Agency as a chemical of concern, occurring in 213 out of 285 common foods and drinks in the U.S. (Murray et al., 2008). A Centers for Disease Control national survey discovered perchlorate in urine samples of all 2820 people tested, with the highest levels found in children. The Centers for Disease Control found that a third of American women with low iodine levels experienced reduced thyroid hormone production at perchlorate exposure levels below the Environmental Protection Agency's 2005 “safe” threshold (Blount et al., 2007).

Perchlorate is ionically similar to iodide and competes with iodide at the sodium/iodide symporter thereby inhibiting the translocation of iodide across the basolateral membrane of thyroid follicular cells (Carr et al., 2006; Wolff, 1998). Insufficient uptake of iodide leads to hypothyroidism and to negative downstream effects on growth, development, metamorphosis and metabolism (Braverman and Cooper, 2012). Perchlorate exposure alters the development of the vertebrate thyroid leading to depleted colloid, thyrocyte hypertrophy and increased angiogenesis (Furin et al., 2015b; Patiño et al., 2003; Petersen et al., 2015), and is often associated with reduced levels of circulating T₄ and T₃ (Crane et al., 2005). Reduced levels of thyroid hormones can lead to obese phenotypes, and our research group discovered that perchlorate exposure induces lipid droplet formation around thyroid tissue in the model fish species the threespine stickleback (Gasterosteus aculeatus, hereafter “stickleback”); therefore, we determined that perchlorate is a candidate chemical obesogen (Gardell et al., 2017).

Perchlorate exposure falls within the paradigm of developmental origins of health and disease (DOHaD). Within this paradigm, early exposures to stressors may manifest later in life as specific phenotypes or diseases (Gillman, 2005). Thyroid disrupting compounds fit this paradigm (McDonald, 2002) and because perchlorate inhibits thyroid production and affects the developmental trajectories of numerous traits (Furin et al., 2015a, b; Petersen et al., 2016), it is likely that any obesogenic effects could be the result of early developmental exposures.

Perchlorate exposure also affects gonadal development (Furin et al., 2015b) and androgen levels in larval stickleback (Petersen et al., 2015). Perchlorate has been shown to alter sex ratios in zebrafish (Danio rerio) (Mukhi et al., 2007) and to masculinize stickleback (Bernhardt and von Hippel, 2008; Bernhardt et al., 2006), which may be due to perchlorate causing a reduction in primary germ cell number early in development (Petersen et al., 2016). Because perchlorate affects sex steroid levels and gonadal development, it could cause obese phenotypes through dysregulation of sex steroids or altered gonadal physiology in addition to or in concert with thyroid-mediated effects (Duarte-Guterman et al., 2014).

In this study, we investigate the effects of perchlorate exposure during development on adult liver phenotypes in stickleback. Liver is a key organ in lipid metabolism and therefore may play an important role in mediating the effects of obesogenic chemicals. Because other chemicals and disruptions to the HPT and the hypothalamic-pituitary-gonadal axis produce obese phenotypes at varying times during development (McDonald, 2002), we tested the hypothesis that perchlorate exposure over a variety of developmental time courses causes accumulation of lipids in the liver, leading to phenotypes similar to NAFLD. We assessed whether perchlorate exposed stickleback have more and larger lipid droplets and a greater proportion of lipid in the liver and other morphologies associated with NAFLD (Ludwig et al., 1980).