The fat of the land

Metabolic health disorders such as obesity and diabetes have been increasing dramatically both in the US and worldwide. Currently, about 40% of US adults are considered obese, and about 10% have been diagnosed with diabetes, contributing to more than $600 million in annual health care costs in the US alone.

Despite these worsening public health trends, interventions have resulted in only limited benefits, suggesting other potential contributory factors. This is supported by recent analyses suggesting that decreased activity, increased caloric intake, and genetics are insufficient to explain the rate and magnitude of these worsening trends. Mirrored in the worsening human health trends, researchers reported several years ago that animals from 24 populations (monkeys, laboratory and urban mice, cats, dogs, etc.) have also been experiencing increased weight gain within a similar timespan.

Research into other potential causative factors of this worsening health trend have suggested a role for environmental chemicals. The development of fat cells and adipose tissue is controlled in part by hormones, which are highly conserved across vertebrate animals. As such, toxicants that can activate or block these hormone pathways may be capable of influencing fat cell development or otherwise influencing energy homeostasis, circuity controlling food intake, energy expenditure, appetite and satiety, and other mechanisms that may contribute to impacted metabolic health.

There is a rich history of experimental evidence demonstrating how various environmental toxicants can induce metabolic disruption. Among these, antibiotics have been appreciated as a method of inducing weight gain in agricultural animals since the 1950’s, seemingly operating through effects on the gut microbiota and subsequent impacts on carbohydrate processing. Diethylstilbestrol (DES), a pharmaceutical intended to reduce incidence of miscarriage, premature labor, and abortions, which was prescribed to pregnant women from the 1940’s through 1970’s, has also been demonstrated to disrupt metabolic health. DES appears to stimulate fat cell development in a cell model through an estrogen receptor-mediated mechanism, and both gestational and perinatal exposure in animal models results in increased body weight, body fat, and altered lipid profiles; gestational exposure has even been correlated with increased risks of developing obesity in humans.

Hydraulic Fracturing/Unconventional Oil and Gas Operations

Unconventional oil and natural gas (UOG) operations have drastically changed oil and natural gas production, particularly within the US, over the last several decades. This technology combines horizontal drilling, now capable of drilling for miles through shale deposits thousands of feet below the surface, and hydraulic fracturing, a common form of stimulation involving the high-pressure injection of water, sand, and chemicals to fracture target geological layers and release previously inaccessible and unprofitable oil or natural gas. The UOG industry reports using more than 1,000 chemicals across the country, dependent on company and geology.

Wastewater from this process is recovered following injection and for the life of the well, and spills are reported at between 2-20% of active well sites, mainly from storage and transport of these fluids. As such, numerous federal and academic labs have reported localised contamination of surface water and groundwater near UOG operations, of hydraulic fracturing chemicals, heavy metals, dissolved gases, radionuclides, and other compounds.

Our laboratory has previously reported that UOG chemicals can act as endocrine disrupting chemicals (EDCs) using both cell and animal models. EDCs are chemicals able to disrupt normal hormone action and contribute to disease in both humans and animals. We’ve previously shown that 23 common hydraulic fracturing chemicals can inhibit and/or activate five important hormone systems: the estrogen, androgen, progesterone, thyroid, and glucocorticoid receptors.

We’ve also demonstrated increased endocrine activities in surface, ground, and drinking water samples near some UOG operations. Through a series of collaborative studies, we’ve also assessed potential health consequences following exposure to a mixture of the 23 common hydraulic fracturing chemicals. A mixture of these chemicals was provided via drinking water to pregnant laboratory mice from early pregnancy through birth. After birth, the health of the offspring were tracked into young adulthood. We observed increased body and organ weights in males and females, increased serum testosterone and decreased sperm counts in males, and disrupted hormone levels, ovarian follicle development, increased susceptibility to immune challenge, and the development of intraductal hyperplasia in the mammary glands of females.

This evidence of effects on birth and body weight through young adulthood led to further assessing these fluids for their potential metabolic health effects in our recent study.

Fracking chemicals as metabolic disruptors

For this study, we utilised a well-established model for fat cell development, the 3T3-L1 cell line. These cells are mouse fibroblasts, first described in the 1970’s, which upon exposure to chemicals that activate specific molecular pathways, differentiate into fat cells, accumulate lipids, and come to resemble what we would think of as a mature white fat cell with large central lipid droplet and displaced nucleus. Using this cell model, we can measure lipid accumulation as a marker for differentiation into mature fat cells (we can think of this as fat cell size, as cells increase in size to accommodate the large lipid droplets), and can also measure precursor fat cell proliferation by measuring DNA content (we can think of this as potential fat cell number).

This cell line has been well-characterised, and the pathways driving differentiation of these fat cell precursors into mature fat cells are well-understood and equivalent to the pathways underlying these processes in humans.

Due to this translational understanding, we can also use commonly-applied receptor reporter gene assays to query specific pathways that might be promoting the observed effects on fat cell development. In brief, these assays utilize transfected DNA plasmids (hormone receptor of interest and a modified hormone response element). The hormone response element, specific to your hormone receptor of interest, is inserted upstream of the luciferase gene in an expression vector. The idea behind this system is that when a chemical binds the receptor of interest and this subsequently binds the response element, it produces luciferase protein. You can then measure this by adding luciferin and quantifying the luminescence produced by the reaction (the same way that fireflies produce light) and then comparing this response to the response exhibited by known concentrations of a positive control hormone (such as testosterone for the androgen receptor).

Our goal for this study was to assess the potential for UOG wastewater, wastewater-impacted surface water from drilling regions, and common hydraulic fracturing chemicals to promote fat cell development across a range of concentrations. Specifically, we selected: a laboratory-created mixture of 23 common UOG chemicals (23-mix), which we previously demonstrated to promote increased birth and body weights in rodents exposed during gestation, two UOG wastewater samples collected from Colorado, and eight UOG-impacted surface water samples (four from Colorado, four from West Virginia, and one blank control sample in each).

We observed that the 23-mix was able to promote significant fat cell development (approximately 60% of the maximal response of our positive control, rosiglitazone, an antidiabetic drug known to induce weight gain in humans), as well as stimulate fat cell precursor proliferation by 80%. The environmental samples also produced significant effects at low concentrations; the UOG wastewater sample promoted approximately 80% lipid accumulation relative to the positive control and up to 60% proliferation, while the UOG-impacted water samples promoted lesser effects as would be expected from diluted samples. Notably, the environmental samples promoted significant effects at very low concentrations, such as 100-fold dilutions of the UOG wastewater sample and 25-fold dilutions of some UOG-impacted surface water samples.

We also further assessed the underlying pathway(s) that might be promoting these effects. The peroxisome proliferator activated receptor gamma (PPARg) is often considered the master regulator of fat cell development, the primary mechanism through which rosiglitazone, our positive control, exerts its effects. Activation of this receptor promotes robust differentiation of and lipid accumulation by these committed fat cell precursors. While this is a critical pathway to consider, many other hormone receptor pathways also contribute to this process, including activation of the glucocorticoid receptor (GR), or inhibition of the thyroid or androgen receptors (TR and AR, respectively).

We found that some samples, including the UOG wastewater sample and several UOG-impacted surface water samples, activated PPARg at concentrations that also promoted fat cell development, highlighting this as the likely underlying mechanism. For other samples, such as the 23-mix and other surface water samples, this was not the case, and other pathways may be responsible for the observed effects on fat cells. These samples exhibited inhibition of AR and/or TR at concentrations that promoted effects on fat cells, suggesting that these other, less studied mechanisms, may be responsible for some of the effects observed.

Implications and future directions

We know that exposure during pregnancy to a mixture of 23 common hydraulic fracturing chemicals, at concentrations suspected to be environmentally relevant, resulted in increased birth and body weights in the resulting mouse pups.

We also have some evidence that weight might be impacted for humans living in UOG regions. Several studies in the Marcellus Shale region have reported an increased risk of low birth weight and small for gestational age births among mothers living nearest UOG operations, while a study in Colorado reported an association of UOG well density and increased risk of high birth weight babies. While these studies seemingly report disparate effects on birth weight, both low and high birth weights are associated with greater risk of obesity later in life.

Together, these factors suggest that UOG operations may have the potential to influence the metabolic health of residents living nearby. However, much more research is needed to characterise exposure levels to specific UOG chemicals, identify the main pathways of exposure, and assess which are the causative chemicals capable of inducing these effects on fat cell development.

The paper is: Unconventional oil and gas chemicals and wastewater-impacted water samples promote adipogenesis via PPARg-dependent and independent mechanisms in 3T3-L1 cells. Kassotis et al., Science of the Total Environment, 2018, DOI: 10.1016/j.scitotenv.2018.05.030

Author Dr Christopher Kassotis is a Postdoctoral Research Scholar in the Nicholas School of the Environment at Duke University, studying endocrine toxicology; specifically, the potential for environmental contaminants to disrupt hormone action and contribute to chronic adverse health effects in humans.