Inhalation of polycarbonate emissions generated during 3D printing processes affects neuroendocrine function in male rats

Data Collection Methods

Animals.  Male Sprague Dawley rats ([H1a: (SD) CVF, n = 6 rats/group (N=60); 6-7 weeks of age, 200 – 250 g) were obtained from Hilltop Lab Animals, Inc., Scottdale, PA.  All animals were free of viral pathogens, parasites, myoplasm’s, Heliobacter and cilia-associated respiratory (CAR) bacillus.

Exposure  After 1 week of acclimation to the facility, animals were exposed to filtered-air (controls) or emissions generated by 3D printing.  More specifically, 3 desktop 3D-printers (LuluBot Mini, Fargo Additive Manufacturing Equipment 3D LLC, Fargo, ND) were placed in an airtight stainless-steel chamber. Black polycarbonate filament was fed into each printer and the printers were operated continuously during the 4 h exposure period. The emissions were blown into an exposure chamber where animals were housed in individual chambers (12 animals/exposure). Analysis of volatile organic compounds by GC/MS. Emissions were collected using the canister method (NIOSH Manual of Analytic Methods (NMAM 3900))

and were analyzed by GC/MS over a 4-h collection period. Particulate was also collected onto glass fiber filters and bisphenols were measured by HPLC (BVNA labs; Novi, MI)

Tissue collection:  Animals were exposed for 4 h/day 4 days/week.  Groups of animals (6 air control and 6 treated) were euthanized by injection of pentobarbital (100 mg/kg i.p.) 24 h after 1, 4, 8, 15 or 30 days of exposure.  Brains were collected and half the brain along with the pituitaries were frozen on dry ice and stored at -80ºC until sectioned by histology and immunohistochemistry.  The other half was dissected and individual regions of interest were stored at  -80Cº until assayed. Plasma was separated from whole blood and stored at -80Cº until assayed.  Testes were fixed in 10% formalin, paraffin embedded and sectioned for histological and immunohistochemical analyses.

Tissue preparation: Histology.  The left hemisphere of the brain was sectioned (20 µm) for histological or immunohistochemical analyses, one slide from each animal and each region was processed. The right testis was also dissected from each animal and placed in 10% buffered formalin and sectioned (10µm) for immunohistochemistry and histology.

Microscopy.  Fluorescent-labeled slides were examined using an Olympus microscope (new microscope) photomicrographs were taken at a magnification of 20x using DP73 camera and CellSense version 510 (Fisher Scientific, Indianapolis IN).  Densitometry was performed on photomicrographs using ImageJ.  To perform densitometry, a threshold was set to identify regions that were immuno-stained.  The area to be quantified was outlined and the total area of the outlined structure was measured along with area of the immunolabeled cells that were at or above the set threshold.  Depending on the region, 3-5 sections were analyzed.  The average immune-stained area was calculated and used for analyses.

ELISAs.  ELISAs for estradiol, prolactin, thyroid stimulating hormone (TSH) and follicle stimulating hormone (FSH) were performed using plasma samples collected from animals after 1, 4, 8, 15 or 30 days of exposure.  ELISAs were performed using methods supplied by the manufacturers.

Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR).  qRT-PCR was performed to determine if exposure to 3D printing fumes resulted in changes in transcript levels in the olfactory bulb and hypothalamus described in (Krajnak et al., 2006).