Per- and poly-fluoroalkyl substances (PFAS) are widely found in the environment because of their extensive use and persistence. Although several PFAS are well studied, most lack toxicity data to inform human health hazard and risk assessment. This study focussed on four model PFAS: perfluorooctanoic acid (PFOA; 8 carbon), perfluorobutane sulfonate (PFBS; 4 carbon), perfluorooctane sulfonate (PFOS; 8 carbon), and perfluorodecane sulfonate (PFDS; 10 carbon). Human primary liver cell spheroids (pooled from 10 donors) were exposed to 10 concentrations of each PFAS and analyzed at four time-points. The approach aimed to: (1) identify gene expression changes mediated by the PFAS; (2) identify similarities in biological responses; (3) compare PFAS potency through benchmark concentration analysis; and (4) derive bioactivity exposure ratios (ratio of the concentration at which biological responses occur, relative to daily human exposure). All PFAS induced transcriptional changes in cholesterol biosynthesis and lipid metabolism pathways, and predicted PPARα activation. PFOS exhibited the most transcriptional activity and had a highly similar gene expression profile to PFDS. PFBS induced the least transcriptional changes and the highest benchmark concentration (i.e., was the least potent). The data indicate that these PFAS may have common molecular targets and toxicities, but that PFOS and PFDS are the most similar. The transcriptomic bioactivity exposure ratios derived here for PFOA and PFOS were comparable to those derived using rodent apical endpoints in risk assessments. These data provide a baseline level of toxicity for comparison with other known PFAS using this testing strategy.

Per- and polyfluoroalkyl substances (PFAS) are some of the most prominent organic contaminants in human blood. Although the toxicological implications of human exposure to perfluorooctane sulfonate (PFOS) and perfluorooctanoate (PFOA) are well established, data on lesser-understood PFAS are limited. New approach methodologies (NAMs) that apply bioinformatic tools to high-throughput data are being increasingly considered to inform risk assessment for data-poor chemicals. The aim of this study was to compare the potencies (i.e., benchmark concentrations: BMCs) of PFAS in primary human liver microtissues (3D spheroids) using high-throughput transcriptional profiling. Gene expression changes were measured using TempO-seq, a templated, multiplexed RNA-sequencing platform. Spheroids were exposed for 1 or 10 days to increasing concentrations of 23 PFAS in three subgroups: carboxylates (PFCAs), sulfonates (PFSAs), and fluorotelomers and sulfonamides. PFCAs and PFSAs exhibited trends toward increased transcriptional potency with carbon chain-length. Specifically, longer-chain compounds (7 to 10 carbons) were more likely to induce changes in gene expression and have lower transcriptional BMCs. The combined high-throughput transcriptomic and bioinformatic analyses support the capability of NAMs to efficiently assess the effects of PFAS in liver microtissues. The data enable potency ranking of PFAS for human liver cell spheroid cytotoxicity and transcriptional changes, and assessment of in vitro transcriptomic points of departure. These data improve our understanding of the possible health effects of PFAS and will be used to inform read-across for human health risk assessment.

Human populations throughout the world are exposed daily to low levels of environmental contaminants. The consequences of potential interactions of these compounds to human endocrine, reproductive, and immune function remain unknown. The current study examines the effects of subchronic oral exposure to a complex mixture of ubiquitous persistent environmental contaminants that have been quantified in human reproductive tissues. The dosing solution used in this study contained organochlorines (2,3,7,8-tetrachlorodibenzo-p-dioxin [TCDD], polychlorinated biphenyls [PCBs],p,p'-dichlorodiphenoxydichloroethylene [p,p'-DDE],p,p-dichlorodiphenoxytrichloroethane [p,p'-DDT], dieldrin, endosulfan, methoxychlor, hexachlorobenzene, and other chlorinated benzenes, hexachlorocyclohexane, mirex and heptachlor) as well as metals (lead and cadmium). Each chemical was included in the mixture at the minimum risk level (MRL) or tolerable daily intake (TDI) as determined by the U.S. EPA or ATSDR or, for TCDD, at the no observable effect level (NOEL) used to calculate the TDI. Sexually mature male rats were exposed to this complex mixture at 1, 10, 100, and 1000 times the estimated safe levels daily for 70 days. On day 71, all animals were sacrificed and a variety of physiological systems assessed for toxic effects. Evidence of hepatotoxicity was seen in the significant enlargement of the liver in the 1000x group, reduced serum LDH activity (100x), and increased serum cholesterol and protein levels (both 1000x). Hepatic EROD activities were elevated in animals exposed to10x and above. The mixture caused decreased proliferation of splenic T cells at the highest dose and had a biphasic effect on natural killer cell lytic activity with an initial increase in activity at 1x followed by a decrease to below control levels in response to 1000x. No treatment-related effects were seen on bone marrow micronuclei, daily sperm production, serum LH, FSH, or prolactin levels or weights of most organs of the reproductive tract. The weights of the whole epididymis and of the caput epididymis were significantly decreased at 10x and higher doses, although no effect was seen on cauda epididymal weight. The sperm content of the cauda epididymis was increased at the 1x level but not significantly different from control at higher dose levels. A slight, but significant, increase in the relative numbers of spermatids was seen in the animals from the 1000x group with a trend towards reduced proportion of diploid cells at the same dose. Only minor, nondose related changes were seen in parameters related to condensation of chromatin, as determined by flow cytometry, in epididymal sperm. We conclude that the mixture induced effects on the liver and kidney and on general metabolism at high doses but caused only minor effects on immune function, reproductive hormone levels, or general indices of reproductive function measures. These data suggest that additive or synergistic effects of exposure to contaminants resulting in residue levels representative of contemporar...

The role of organochlorine (OC) exposure in the etiology of breast cancer remains controversial. Thus, our objective was to determine whether the most abundant and toxic OCs found in human milk could, when ingested during the neonatal period, modulate the development of mammary tumors in the rat. We prepared a mixture composed of p,p'-dichlorodiphenyltrichloroethane (DDT), its major metabolite, p,p'-dichlorodiphenyldichloroethene (DDE), and 19 polychlorinated biphenyls (PCB) based on their concentrations found in the milk of Canadian women. Neonate rats at 1, 5, 10, 15, and 20 days of age were gavaged with this mixture, at 10, 100, and 1,000 times the amount that a human baby would consume. An additional group received 2.5 microg 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)/kg body weight (bw) by gavage at 18 days of age, instead of the mixture. On day 21, all treatment groups, except for a control group and a 1,000-mix group, received a single intraperitoneal injection of methylnitrosourea (MNU, 30 mg/kg bw), the initiator of the carcinogenic process. The average number of rats per treatment group was 33. Rats were sacrificed when their tumors reached 1 cm in size, or at 308 days of age. We prepared mammary tumors and mammary gland whole mounts for histologic analysis. There were no significant effects when only the malignant or only the benign tumors were considered. After all benign and malignant lesions were pooled, the number of mammary tumors differed among all MNU-treated groups (p = 0.02) with more lesions developing in the MNU-1,000[times] (median = 4.5; p = 0.05) and MNU-TCDD (median = 5.5; p = 0.07) compared to the MNU-0 rats (median = 2). Compared to the MNU-0 group, the percentage of rats that developed palpable tumors (benign plus malignant) was slightly higher (p = 0.06) in the MNU-TCDD group, but not in the MNU-1,000[times] group. The percentage of palpable tumors that were malignant was higher (p = 0.02) in the MNU-100[times] group (15/16, 94%) than in the MNU-0 group (10/18, 56%). The highest dose of the mixture delayed (p = 0.03) the development of tumors, but this was not observed with the MNU-TCDD treatment. These results suggest that neonatal exposure to high doses of organochlorines could favor the development of MNU-induced mammary lesions, but also delays the development of palpable tumors in the rat.

Background: Oxidative stress and inflammation are considered to be important pathways leading to particulate matter (PM)-associated disease. In this exploratory study, we examined the effects of metals and oxidative potential (OP) in urban PM on biomarkers of systemic inflammation, oxidative stress and neural function. Methods: Fifty-three healthy non-smoking volunteers (mean age 28 years, twenty-eight females) were exposed to coarse (2.5–10 μm, mean 213 μg/m3), fine (0.15–2.5 μm, 238 μg/m3), and/or ultrafine concentrated ambient PM (<0.3 μm, 136 μg/m3). Exposures lasted 130 minutes, separated by ≥2 weeks. Metal concentrations and OP (measured by ascorbate and glutathione depletion in synthetic airway fluid) in PM were analyzed. Blood and urine samples were collected pre-exposure, and 1-hour and 21-hour post exposure for assessment of biomarkers. We used mixed-regression models to analyze associations adjusting for PM size and mass concentration. Results: Results for metals were expressed as change (%) from daily pre-exposure biomarker levels after exposure to a metal at a level equivalent to the mean concentration. Exposure to various metals (silver, aluminum, barium, copper, iron, potassium, lithium, nickel, tin, and/or vanadium) was significantly associated with increased levels of various blood or urinary biomarkers. For example, the blood inflammatory marker vascular endothelia growth factor (VEGF) increased 5.3% (95% confidence interval: 0.3%, 10.2%) 1-hr post exposure to nickel; the traumatic brain injury marker ubiquitin C-terminal hydrolase L1 (UCHL1) increased 11% (1.2%, 21%) and 14% (0.3%, 29%) 1-hr and 21-hr post exposure to barium, respectively; and the systemic stress marker cortisol increased 1.5% (0%, 2.9%) and 1.5% (0.5%, 2.8%) 1-hr and 21-hr post exposure to silver, respectively. Urinary DNA oxidation marker 8-hydroxy-deoxy-guanosine increased 14% (6.4%, 21%) 1-hr post exposure to copper; urinary neural marker vanillylmandelic acid increased 29% (3%, 54%) 1-hr post exposure to aluminum; and urinary cortisol increased 88% (0.9%, 176%) 1-hr post exposure to vanadium. Results for OP were expressed as change (%) from daily pre-exposure biomarker levels after exposure to ascorbate-related OP at a level equivalent to the mean concentration, or for exposure to glutathione-related OP at a level above the limit of detection. Exposure to ascorbate- or glutathione-related OP was significantly associated with increased inflammatory and neural biomarkers including interleukin-6, VEGF, UCHL1, and S100 calcium-binding protein B in blood, and malondialdehyde and 8-hydroxy-deoxy-guanosine in urine. For example, UCHL1 increased 9.4% (1.8%, 17%) in blood 21-hr post exposure to ascorbate-related OP, while urinary malondialdehyde increased 19% (3.6%, 35%) and 8-hydroxy-deoxy-guanosine increased 24% (2.9%, 48%) 21-hr post exposure to ascorbate- and glutathione-related OP, respectively. Conclusion: Our results from this exploratory study suggest that metal constituents and OP in ambient PM may influence biomark...