Comparative in vitro toxicity assessment of perfluorinated carboxylic acids Cecon T. Mahapatra a *, Nur P. Damayanti b , Samuel C. Guffey a , Jennifer S. Serafin a , Joseph Irudayaraj b and Maria S. Sepúlveda a ABSTRACT: Perfluoroalkyl and polyfluoroalkyl substances (PFASs) are synthetic fluorinated compounds that are highly bioaccumulative and persistent organic pollutants. Perfluorooctanoic acid (PFOA), an eight-carbon chain perfluorinated carboxylic acid, was used heavily for the production of fluoropolymers, but concerns have led to its replacement by shorter carbon chain homologues such as perfluorohexanoic acid (PFHxA) and perfluorobutanoic acid (PFBA). However, limited toxicity data exist for these substitutes. We evaluated the toxicity of PFOA, PFHxA and PFBA on a zebrafish liver cell line and investigated the effects of exposure on cell metabolism. Gross toxicity after 96 h of exposure was highest for PFOA and PFO – , while PFHxA and PFBA exhibited lower toxicity. Although the structural similarity of these compounds to fatty acids suggests the possibility of interference with the transport and metabolism of lipids, we could not detect any differential expression of peroxisome proliferator-activated receptor (ppar-α,-β and -γ), fabp3 and crot genes after 96 h exposure to up to 10 ppm of the test compounds. However, we observed localized lipid droplet accumulation only in PFBA-exposed cells. To study the effects of these compounds on cell metabolism, we conducted fluorescence lifetime imaging microscopy using naturally fluorescent biomarkers, NADH and FAD. The fluorescence lifetimes of NADH and FAD and the bound/free ratio of each of these coenzymes decreased in a dose- and carbon length-dependent manner, suggesting disruption of cell metabolism. In sum, our study revealed that PFASs with shorter carbon chains are less toxic than PFOA, and that exposure to sublethal dosage of PFOA, PFHxA or PFBA affects cell metabolism. Copyright © 2016 John Wiley & Sons, Ltd. Keywords: Perfluoroalkyl substances; Perfluorohexanoic acid; Perfluorobutanoic acid; Zebrafish; Liver; Fluorescence Lifetime Imaging Microscopy Introduction Perfluoroalkyl and polyfluoroalkyl substances (PFASs) are synthetic chemicals widely used in numerous industrial and consumer appli- cations. They are very stable compounds due to their strong carbon–fluorine bonds (Prevedouros et al., 2006) and thus are highly persistent organic pollutants (Buck et al., 2011; Giesy & Kannan, 2002). The eight-carbon perfluorooctanoic acid (PFOA) is the best-studied member of a subclass of PFASs called perfluorinated carboxylic acids. PFOA is one of the most com- monly detected PFASs in the environment and it exhibits a high bioaccumulation potential (Kennedy et al., 2004; Lau et al., 2007; Maloney & Waxman, 1999). The existing literature on the effects of exposure to PFASs of eight or more carbon atoms in length suggests that vertebrate an- imals primarily suffer disturbances to the liver and pancreas (Biegel et al., 2001; Kennedy et al., 2004; Kudo & Kawashima, 2003). PFOA exhibits structural similarity to fatty acids and has been shown to affect lipid metabolism by acting through the transcription factor peroxisome proliferator-activated receptor (PPAR)-α (Maloney & Waxman, 1999; Rosen et al., 2007; Takacs & Abbott, 2007; Wang et al., 2013). PFOA can induce hepatomegaly and hepatocellular adenomas in rats (Lau et al., 2007) and it was shown variously to cause weight loss, increase total cholesterol and perturb cellular metabolism, including lipid metabolism, glucose metabolism, oxi- dative phosphorylation and mitochondrial dysfunction in mice, zebrafish and human systems (Abbott et al., 2012; Hagenaars et al ., 2013; Nelson et al., 2010; Peng et al., 2013; Yan et al., 2015). Existing literature shows that the toxicity of PFASs is related to the carbon chain length (Kleszczyński et al., 2007; Ulhaq et al., 2013; Wang et al., 2014), which can directly impact bioaccumula- tion and elimination rates of these compounds (Kudo et al ., 2006). In rats, a correlation was found between urinary excretion and carbon chain length of PFASs (Ohmori et al., 2003). The shorter carbon chain compounds were excreted rapidly from the body and thus resulted in lesser accumulation in the serum and liver of mice (Kudo et al., 2001). In vitro toxicity tests using mammalian cell lines showed a positive correlation between the toxicity of the PFASs and the carbon chain length (Mulkiewicz et al., 2007). Owing to their adverse effects and high environmental persis- tence, longer chain PFASs such as PFOA are being phased out and replaced with shorter carbon chain compounds such as six-carbon perfluorohexanoic acid (PFHxA) and four-carbon perfluorobutanoic acid (PFBA) (Ye et al., 2009). Although many studies have been conducted on the toxicity of PFOA, few data are available on the toxicity effects of shorter chain PFASs. PFHxA seems to be more rapidly eliminated than PFOA in mammalian systems and thus is less bioaccumulative (Chengelis et al., 2009; Gannon et al., 2011; Russell et al., 2013). In humans, the half-life is *Correspondence to: Cecon T. Mahapatra, Purdue University, Department of For- estry and Natural Resources, West Lafayette, IN 47907, USA. E-mail: cmahapat@purdue.edu a Purdue University, Department of Forestry and Natural Resources, West Lafayette, IN, 47907, USA b Bindley Bioscience Center, Purdue University, West Lafayette, IN, USA J. Appl. Toxicol. 2016 Copyright © 2016 John Wiley & Sons, Ltd. Research article Received: 16 May 2016, Revised: 25 October 2016, Accepted: 25 October 2016 Published online in Wiley Online Library (wileyonlinelibrary.com) DOI 10.1002/jat.3418