MEHP and plasma omega-6 polyunsaturated fatty acid levels in women with obesity participating in the NHANES 2003-2004 Study
Participants’ characteristics are presented in Table 1. While the majority of the plasma omega-6 fatty acid levels did not display a significant association with urinary MEHP, a positive association was observed between urinary MEHP levels and gamma-linolenic acid (0.436 ± SE: 0.182; p=0.019) as well as arachidonic acid (7.62 ± SE: 1.73; p=0.000) in women with obesity (Table 2). Furthermore, r2 adjusted model value explained 35% of the individual variation observed in arachidonic acid levels. Interestingly, it is MEHP which has the largest effect size (f2=0.212) in the linear model explaining variance in plasma arachidonic acid levels. However, while r2 adjusted model value explained 24% of the individual variation observed in gamma-linolenic acid levels with a major influence of ethnicity, MEHP still influenced the model with an effect size of f2=0.056.
Gamma-linolenic acid is a precursor of arachidonic acid. Moreover, increasing adult human dietary gamma-linolenic acid or arachidonic acid induces an increase in tissue and plasma arachidonic acid content (27,28). Plasma arachidonic acid is a major source of skeletal tissue arachidonic acid (29). Unmetabolized free arachidonic acid is converted into arachidonoyl-CoA by acyl-coenzyme A synthetases long-chain (ACSL), which play an important role in fatty acid metabolism and are believed to be involved in pathophysiological events(30–34). Based on previous work on rat fibroblasts where these enzymes were shown to play a role in arachidonic acid metabolism, we studied the effects of MEHP on exogenous long-chain fatty acid oxidation, mitochondrial respiration, and glycolysis in C2C12 myotubes (35).
Evaluation of MEHP cytotoxicity on C2C12 cells
Prior to studying the effects of increasing concentrations of MEHP on C2C12 myotubes, its cytotoxicity was studied. There was no significant difference in the number of apoptotic and necrotic cells with increasing concentrations of MEHP exposure on C2C12 myotubes, measured by condensed nuclei and PI positive cells (Figure 1). Furthermore, cell death always remained below 5% of the cell population.
The effects of MEHP on exogenous fatty acid oxidation and mitochondrial respiration in C2C12 myotubes
To characterize the metabolic effects of MEHP, 14C-palmitic acid oxidation as well as palmitate-induced respiration were studied in C2C12 myotubes. There was an overall decrease in total fatty acid oxidation following the exposure of myotubes to increasing concentrations of MEHP (Figure 2 a; p=0.035). Specifically, a decrease was observed between the control 0.1% DMSO (1.183 nmol/hr/mg ± 0.035) and MEHP exposed cells of 10 µM (1.033 nmol/hr/mg ± 0.048; p=0.011), 100 µM MEHP (1.076 nmol/hr/mg ± 0.012; p=0.048) and the highest concentration 300 µM MEHP (1.034 nmol/hr/mg ± 0.022; p=0.012). This reduction in fatty acid oxidation, was accompanied by a reduction in spare respiratory capacity (p=0.012) when studying the muscle cell effects of MEHP on oxygen consumption rate (OCR) (Figure 2 b and c). A decrease in spare respiratory capacity was observed between 0.1% DMSO (1.87 pmol/min/µg ± 0.121), 10 µM (1.85 pmol/min/µg ± 0.177) and the higher concentrations of MEHP, 100 µM (1.10 pmol/min/µg ± 0.279; vs 0.1% DMSO p=0.042; vs 10 µM DMSO p=0.047) and 300 µM (0.65 pmol/min/µg ± 0.281 vs 0.1% DMSO p=0.005; vs 10 µM DMSO p=0.005). While a trend towards a decrease in maximal respiration was observed between 10 µM (1.75 pmol/min/µg ± 0.382) and higher concentrations of 100 µM (0.62 pmol/min/µg ± 0.263) and 300 µM MEHP (0.60 pmol/min/µg ± 0.466), results were not significant. No difference in basal respiration rate was observed between C2C12 myotube exposure to different MEHP concentrations. Furthermore, metabolic effects were only observed during fatty acid-driven oxidation, as mitochondrial stress test demonstrated no difference with exposure to increasing concentrations of MEHP (Supplementary Figure 1).
The effects of MEHP on cellular glycolysis levels
Based on altered fatty acid metabolism in C2C12 myotubes exposed to MEHP, the effects of the toxicant on glucose utilization were studied, since we hypothesized that the latter may act as a compensatory pathway for ATP production. Specifically the extracellular acidification rate (ECAR) was studied in the Seahorse XF analyzer. ECAR measured during mitochondrial stress test, illustrated a trend towards an increase in basal glycolysis levels following increased exposure to MEHP (Supplementary Figure 1). Furthermore, ECAR measured by the glycolysis stress test in C2C12 myotubes, also illustrated a trending increase in basal glycolysis levels, specifically between 0.1% DMSO control (1.10 ± 0.097) and 300 µM MEHP (1.40 ± 0.135) (Figure 3).
The effects of MEHP on metabolism related proteins
After examining the effects of MEHP exposure on muscle fatty acid and glucose utilization/metabolism, we studied the toxicant’s effect on levels of acyl-CoA synthetase long chain protein 5 (ACSL5), a key protein in long-chain fatty acyl-CoA production from free fatty acids. Increasing the exposure of C2C12 myotubes to MEHP appeared to have no effect on ACSL5 (data not shown). However, there was a decrease in ACSL5 levels (p=0.032) with increasing MEHP in myoblasts (Supplementary Figure 2), specifically between 10 µM (Volume Intensity: 1.00 ± 0.075) and higher concentrations including 100 µM (Volume Intensity: 0.464 ± 0.137; p=0.007) and 300 µM MEHP (Volume Intensity: 0.467 ± 0.116; p=0.008) when adjusted for 0.1% DMSO control. These results reveal alteration in ACSL5 levels in myoblasts exposed to MEHP toxicant.