Systemic toxicity induced by topical application of perfluoroheptanoic acid (PFHpA), perfluorohexanoic acid (PFHxA), and perfluoropentanoic acid (PFPeA) in a murine model

Data Collection Methods

1. 1. Animal Exposures
      • Female B₆C₃F₁ mice (7-8 weeks at start of study)
      • Perfluoroheptaoinc acid (PFHpA), perfluorohexanoic aicd (PFHxA), and perfluoropentanoic acid (PFPeA) (25 µl/ear; 1.25-5%) or acetone on dorsal surface of both ears for up to 28 days
   2. Tissue Collection
      • Kidney and thymus were collected, weighted, and then discarded.
      • Liver was weighed, caudate lobe was collected in RNA later and then homogenized on a TissueLyser II in Buffer RLT for RNA extraction.  The remainder of the liver was collected in 10% formalin for histopathology analysis.
      • Spleen was weighed, 1/2 was collected in 4 mL RPMI and processed into single cell suspensions by mechanical disruption of tissues between frosted microscope slides.  The remainder of the spleen was collected in 10% formalin for histopathology analysis.
      • Ear pinnas (1 per mouse; split into ventral and dorsal halves) was collected in RPMI and either and processed into single cell suspensions prepared by incubating with a 0.25 mg/ml Liberase-TL Research grade (Roche) enzymatic digestion for 90 min at 37°C in RMPI with 100 µg/ml DNase I.  The second ear pinna (1/2) collected in 10% formalin for histopathology analysis and 1/2 was collected in RNA later and then homogenized on a TissueLyser II in Buffer RLT for RNA extraction.
      • Right and left auricular draining lymph nodes (dLNs) collected in 2 mL sterile phosphate-buffer saline (PBS) (pH 7.4) and single cell suspensions (2 nodes/animal) were prepared by mechanical disruption of tissues between frosted microscope slides
   3. Serum chemistries
      • Blood samples were collected via cardiac puncture and separated by centrifugation.  Selected serum chemistries were evaluated using Catalyst DX Chemistry Analyzer.
   4. Analytical PFAS detection
      • Serum and urine samples were collected and analyzed for individual PFAS by Vista Analytical Laboratories as described in (Shoemaker et al. 2008).
   5. Gene expression
      • Real-time PCR (Applied Biosystems 7500 RT-PCR System).
   6. Immune Cell Subsets
      • Flow cytometry using BD LSRII Flow Cytometer and data was analyzed using FlowJo software.
   7. Histology
      • Tissue samples were stained via hematoxylin and eosin (H&E) and evaluated by a veterinary pathologist.