Chemosphere xxx (2013) xxx–xxx

1. 2013.02.28 Mechanistic toxicity study of perfluorooctanoic acid in zebrafish suggests mitochondrial dysfunction to play a key role in PFOA toxicity Kim Hwan-Young Chemosphere xxx (2013) xxx–xxx

2. Abstract • - The aquatic environment is an important site for perfluorooctanoic acid (PFOA) deposit. Nevertheless, the exact mode of action and its resulting toxicological effects in aquatic organisms remain largely unknown. • To gain a better understanding of the mode of action of teleost PFOA toxicity, transcriptomics, proteomics, biochemical parameters and reproduction were integrated in this study. • Male and female zebrafish were exposed to nominal concentrations of 0.1, 0.5 and 1 mg L-1PFOA for 4 and 28 d resulting in PFOA accumulation which was higher in males than in females. These gender-related differences were likely caused by different elimination rates due to distinct hormone levels and differences in transport activity by solute carriers. --The general mode of action of PFOA was described as an increase of the mitochondrial membrane permeability followed by an impairment of aerobic ATP production. Depletion of liver glycogen stores together with altered expression levels of transcripts involved in carbohydrate metabolism, with emphasis on anaerobic metabolism, was probably a means of compensating for this decreased aerobic efficiency. • The mitochondrial dysfunction further resulted in effects on oxidative stress and apoptosis at the gene transcript and protein level. As a consequence, evidence for the replacement of the affected cells and organelles to sustain tissue homeostasis was found at the transcript level, resulting in an even greater glycogen depletion. Despite this increase in metabolic expenditure, no effects on reproduction were found indicating that the fish seemed to cope with exposure to the tested concentrations of PFOA during the exposure period of 1 month.

3. Highlights • The mode of action of PFOA was assessed using an integrative • approach in zebrafish. • PFOA affected mRNA and protein expression, liver energy • stores but not reproduction. • Results indicated altered membrane permeability and • mitochondrial dysfunction. • PFOA also decreased the mitochondrial electron transport • system activity. • Glycogen stores were depleted to compensate for inefficient • aerobic respiration.

4. Perfluorooctanoic acid (PFOA) Perfluorooctanoic acid (PFOA) is one of the most used perfluorinated compounds (PFCs). Substances of this chemical class consist of a hydrophilic functional group and a hydrophobic alkyl chain in which all hydrogens are replaced by fluorine. PFOA has been detected in industrial waste, stain resistant carpets, carpet cleaning liquids, house dust, microwave popcorn bags, water, food, some cookware. PFOA is a carcinogen, liver toxicant, a developmental toxicant, an immune system toxicant, and also exerts hormonal effects including alteration of thyroid hormone levels. Animal studies show developmental toxicity from reduced birth size, physical developmental delays, endocrine disruption, and neonatal mortality.

5. Materials and methods • Exposure experiments • fish were exposed to nominal concentrations of 0.1, 0.5 and 1 mg L1 PFOA (purity 96%, Sigma–Aldrich, Bornem, Belgium) for 4 and for 28 d • Determination of PFOA concentrations • Transcriptomics • - RNA extraction: Trizol-RNA extraction procedure (Gibco BRL, Life Technologies) • - Construction of labeled cRNA : Quick Amp labeling kit (Agilent Technologies) • - Microarray hybridisation • - Microarray scanning and bioinformatics analysis : Genepix Personal 4100A • confocalscanner (Axon instruments) • 4. Proteomics • - Protein extraction and labeling • - Two-dimensional difference gel electrophoresis (2D DiGE) • - Gel scanning and bioinformatics analysis • 5. Energy budget • 6. Assessment of reproduction • 7. Mitochondrial electron transport chain

6. Results Table 1 Whole body concentration and bioconcentration rates (bioconcentration factor/day) of PFOA in male and female zebrafish after exposure for 4 and 28 d to different PFOA concentrations (n = 6).

7. Table 2 List of the significantly altered gene transcripts and protein levels on which the discussion is based.

11. Fig. 1. Representation of the most affected biological processes in males and females exposed to 1 mg L1 expressed as the percentage of the total number of differential gene transcripts within each gender.

12. Fig. 2. The expression profiles of the unique identified differentially expressed proteins with their accession number.

13. Fig. 3. (A) The mean glycogen content in whole bodies (B) The mitochondrial electron transport activity

14. Fig. 4. Proposed working mechanism of PFOA in zebrafishliver

15. Summary • Gender-related differences in PFOA accumulation, with males accumulating more PFOA compared to females, is suggested to be caused by different elimination rates due to distinct hormone levels and differences in transport activity by solute carriers. • The general mode of action of PFOA was described as an increase of the mitochondrial permeability with evidence of effects at the MPT-pore at the transcript level. These changes affect the oxidative phosphorylation and can result in oxidative stress and cell death. • As a consequence, affected cells and organelles had to be replaced to sustain tissue homeostasis which was demonstrated by the increased cell cycle and DNA replication at the transcript level. • Tissue repair is an energy draining process. To cope with the higher energy demand due to these adverse effects and to compensate for the incapacitated aerobic respiration, the glycogen stores were used as an energy source.