An in vitro-in silico workflow for predicting renal clearance of PFAS.

Per- and poly-fluoroalkyl substances (PFAS) have a wide range of elimination half-lives (days to years) in humans, thought to be in part due to variation in proximal tubule reabsorption. While human biomonitoring studies provide important data for some PFAS, renal clearance (CLrenal) predictions for hundreds of PFAS in commerce requires experimental studies with in vitro models and physiologically-based in vitro-to-in vivo extrapolation (IVIVE). Options for studying renal proximal tubule pharmacokinetics include cultures of renal proximal tubule epithelial cells (RPTECs) and/or microphysiological systems. This study aimed to compare CLrenal predictions for PFAS using in vitro models of varying complexity (96-well plates, static 24-well Transwells and a fluidic microphysiological model, all using human telomerase reverse transcriptase-immortalized and OAT1-overexpressing RPTECs combined with in silico physiologically-based IVIVE. Three PFAS were tested: one with a long half-life (PFOS) and two with shorter half-lives (PFHxA and PFBS). PFAS were added either individually (5 µM) or as a mixture (2 µM of each substance) for 48 h. Bayesian methods were used to fit concentrations measured in media and cells to a three-compartmental model to obtain the in vitro permeability rates, which were then used as inputs for a physiologically-based IVIVE model to estimate in vivo CLrenal. Our predictions for human CLrenal of PFAS were highly concordant with available values from in vivo human studies. The relative values of CLrenal between slow- and faster-clearance PFAS were most highly concordant between predictions from 2D culture and corresponding in vivo values. However, the predictions from the more complex model (with or without flow) exhibited greater concordance with absolute CLrenal. Overall, we conclude that a combined in vitro-in silico workflow can predict absolute CLrenal values, and effectively distinguish between PFAS with slow and faster clearance, thereby allowing prioritization of PFAS with a greater potential for bioaccumulation in humans.

Differences in the Rate of in Situ Mammary Gland Development and Other Developmental Endpoints in Three Strains of Female Rat Commonly Used in Mammary Carcinogenesis Studies: Implications for Timing of Carcinogen Exposure.

The potential of chemicals to alter susceptibility to mammary tumor formation is often assessed using a carcinogen-induced study design in various rat strains. The rate of mammary gland (MG) development must be considered so that the timing of carcinogen administration is impactful. In this study, in situ MG development was assessed in females of the Harlan Sprague-Dawley (Hsd:SD), Charles River Sprague-Dawley (Crl:SD), and Charles River Long-Evans (Crl:LE) rat strains at postnatal days 25, 33, and 45. Development was evaluated by physical assessment of growth parameters, developmental scoring, and quantitative morphometric analysis. Although body weight (BW) was consistently lower and day of vaginal opening (VO) occurred latest in female Hsd:SD rats, they exhibited accelerated pre- and peripubertal MG development compared to other strains. Glands of Crl:SD and Crl:LE rats exhibited significantly more terminal end buds (TEBs) and TEB/mm than Hsd:SD rats around the time of VO. These data suggest a considerable difference in the rate of MG development across commonly used strains, which is independent of BW and timing of VO. In mammary tumor induction studies employing these strains, administration of the carcinogen should be timed appropriately, based on strain, to specifically target the peak of TEB occurrence.

Application of Sholl analysis to quantify changes in growth and development in rat mammary gland whole mounts.

Studies that utilize the rodent mammary gland (MG) as an endpoint for assessing the developmental toxicity of chemical exposures typically employ either basic dimensional measurements or developmental scoring of morphological characteristics as a means to quantify MG development. There are numerous means by which to report these developmental changes, leading to inconsistent translation across laboratories. The Sholl analysis is a method historically used for quantifying neuronal dendritic patterns. The present study describes the use of the Sholl analysis to quantify MG branching characteristics. Using this method, we were able to detect significant differences in branching density in MG of peripubertal female Sprague Dawley rats that had been exposed to vehicle or a potent estrogen. These data suggest the Sholl analysis can be an effective tool for quantitatively measuring an important characteristic of MG development and for examining associations between MG growth and density and adverse effects in the breast.

Histopathologic changes in the uterus, cervix and vagina of immature CD-1 mice exposed to low doses of perfluorooctanoic acid (PFOA) in a uterotrophic assay.

The estrogenic and antiestrogenic potential of perfluorooctanoic acid (PFOA) was assessed using an immature mouse uterotrophic assay and by histologic evaluation of the uterus, cervix and vagina following treatment. Female offspring of CD-1 dams were weaned at 18days old and assigned to groups of equal weight, and received 0, 0.01, 0.1, or 1mg PFOA/kg BW/d by gavage with or without 17-ß estradiol (E(2), 500µg/kg/d) from PND 18-20 (n=8/treatment/block). At 24h after the third dose (PND 21), uteri were removed and weighed. Absolute and relative uterine weights were significantly increased in the 0.01mg/kg PFOA only group. Characteristic estrogenic changes were present in all E(2)-treated mice; however, they were minimally visible in the 0.01 PFOA only mice. These data suggest that at a low dose PFOA produces minimal histopathologic changes in the reproductive tract of immature female mice, and does not antagonize the histopathologic effects of E(2).