Genotoxicity assessment of per- and polyfluoroalkyl substances mixtures in human liver cells (HepG2).

Per- and polyfluoroalkyl substances (PFAS) are ubiquitous, toxic, and persistent environmental chemicals of concern that have been widely detected in all environmental matrices including human biological fluids. Although humans are exposed to complex mixtures of PFAS, it remains uncertain whether the co-exposure to PFAS mixtures could induce genotoxic damage in humans. Hence, this study evaluated the combined genotoxicity of PFAS mixtures in a human cell line system. To assess the possible genotoxic damage caused by human exposure to PFAS and their mixtures, we investigated their potential to induce cytotoxicity (cell viability) and genotoxicity (DNA damage) in a human liver cell line (HepG2). The selected PFAS include perfluorononanoic acid (PFNA), perfluorooctane sulfonic acid (PFOS), perfluorodecanoic acid (PFDA), perfluorooctanoic acid (PFOA), and perfluorohexane sulfonic acid (PFHxS). The interaction toxicities of these PFAS in binary mixtures were also determined using the additive index approach. The results revealed that exposure to PFNA, PFOS, PFDA, PFOA, and PFHxS singly and in binary mixtures induced a concentration-dependent decrease in cell viability. The additive index values indicated that the binary mixtures of PFOS + PFNA, PFOS + PFDA, and PFOS + PFOA displayed synergistic interaction, whereas the binary mixtures of PFOS + PFHxS, PFOA + PFNA, PFOA + PFDA, and PFOA + PFHxS behaved additively. Using the alkaline Comet assay, the potential of PFAS and their mixtures to induce DNA damage was evaluated based on a 1:1 ratio of the concentration of respective compounds required to produce a 1/10th of effective concentrations causing 50 % inhibition in cell viability (EC50). The results revealed that exposure to PFNA, PFOS, PFDA, PFOA, and PFHxS singly and in binary mixtures (PFOS + PFNA, PFOS + PFDA, PFOS + PFOA, PFOS + PFHxS, PFOA + PFNA, PFOA + PFDA, and PFOA + PFHxS) caused a moderate increase in cellular DNA damage, but no dose-response relationship was observed. Overall, this study indicates that the tested PFAS causes a concentration-dependent decrease in cell viability and only a modest increase in cellular DNA damage under these conditions.

Combined effects of mixed per- and polyfluoroalkyl substances on the Nrf2-ARE pathway in ARE reporter-HepG2 cells.

Although the Nrf2-ARE pathway plays a critical role in cellular protection against toxicity and oxidative stress from environmental chemical stressors, the association between exposure to per- and polyfluoroalkyl substances (PFAS) mixtures and the changes of Nrf2-ARE pathway remains largely unexplored. This study evaluated the potential of PFAS to induce the Nrf2-ARE pathway as individual compounds and as binary, ternary, and multicomponent mixtures in the ARE reporter-HepG2 cells and compared the mixture toxicity data to the predictions by concentration addition (CA) model. The toxicological interactions between PFAS mixture components were also determined by the model deviation ratio (MDR) between the CA predicted and mixture toxicity values. The induction of the Nrf2-ARE pathway was quantified using the luciferase system, and the endpoint assessed was the concentration that induced an induction ratio (IR) of 1.5 (ECIR1.5). The results showed that exposures to both individual and mixed PFAS induced the Nrf2-ARE pathway in ARE reporter-HepG2 cells. Based on the MDRs, the combinations with PFOS showed synergistic interactive effects, while the combinations with PFOA showed additive effects. These results indicate that the CA model underestimated the mixture toxicity of PFAS with PFOS co-exposures and may have health risk assessment implications.

Toxicity assessment of historical aqueous film-forming foams (AFFFs) using cell-based assays.

Aqueous film-forming foam (AFFF) has historically contained high concentrations of long-chain per-and polyfluoroalkyl substances (PFAS), which have been linked with adverse health outcomes. However, the toxicity of historical AFFFs remains largely unknown, presenting uncertainties in their risk assessment. This study assessed the toxicity of historical AFFFs by exposing human liver cells (HepG2) to various dilutions of 3M Light Water AFFF or Ansulite AFFF (0.001%, 0.002%, 0.005%, 0.009%, 0.019%, 0.038%, 0.075%, 0.15%, and 0.3%) for 24 h. The effects of the two AFFF formulations on the cell viability, intracellular reactive oxygen species (ROS) production, Nrf2-ARE activity, and DNA damage were assessed by CellTiter 96® Aqueous One Solution Cell Proliferation Assay (MTS kit), dichlorofluorescein diacetate assay, luciferase assay, and alkaline Comet assay, respectively. The results revealed that the two brands of AFFFs tested were toxic to HepG2 cells at dilutions lower than the recommended 3% application formulation. Specifically, exposure to 3M Light Water AFFF or Ansulite AFFF induced a dilution-dependent decrease in cell viability, increased intracellular ROS production, and increased Nrf2-ARE activity. However, except for the highest concentration (lowest dilution) of 3M Light Water AFFF tested (0.038%.), both 3M Light Water AFFF and Ansulite AFFF did not significantly induce cellular DNA damage. Overall, 3M Light Water AFFF was more toxic than Ansulite AFFF. The findings from this study provided valuable in vitro toxicity data that may better inform the health risk assessment of these historical AFFFs.

Assessing the human health risks of per- and polyfluoroalkyl substances: A need for greater focus on their interactions as mixtures.

Humans are exposed to complex mixtures of per- and polyfluoroalkyl substances (PFAS). However, human health risk assessment of PFAS currently relies on animal toxicity data derived from individual substance exposure, which may not adequately predict the risk from combined exposure due to possible interactions that can influence the overall risk. Long-chain perfluoroalkyl acids (PFAAs), particularly perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) are recognised as global emerging contaminants of concern due to their ubiquitous distribution in all environmental media, wildlife, and humans, persistency, bioaccumulative-, toxic-, and human health-risk potentials. This article reviews the current understanding of the human health risks associated with PFAS exposure focusing on more recent toxicological and epidemiological studies from 2010 to 2020. The existing information on PFAA mixtures was also reviewed in an attempt to highlight the need for greater focus on their potential interactions as mixtures within the class of these chemicals. A growing number of toxicological studies have indicated several adverse health outcomes of PFAA exposure, including developmental and reproductive toxicity, neurotoxicity, hepatotoxicity, genotoxicity, immunotoxicity, thyroid disruption, and carcinogenicity. Epidemiological findings further support some of these adverse human health outcomes. However, the mechanisms underlying these adverse effects are not well defined. A few in vitro studies focusing on PFAA mixtures revealed that these compounds may act additively or interact synergistically/antagonistically depending on the species, dose level, dose ratio, and mixture components. Hence, the combined effects or potential interactions of PFAS mixtures should be considered and integrated into toxicity assessment to obtain a realistic and more refined human health risk assessment.

Evaluation of the individual and combined toxicity of perfluoroalkyl substances to human liver cells using biomarkers of oxidative stress.

Although human exposure is to mixtures of per- and polyfluoroalkyl substances (PFAS), their combined effects and underlying mechanisms remain largely unknown. In this study, the combined effects of PFAS was investigated by treating human liver cells (HepG2) with various concentrations of perfluorooctanoic acid (PFOA), perfluorooctane sulfonate (PFOS), perfluorodecanoic acid (PFDA), perfluorononanoic acid (PFNA), and perfluorohexanoic acid (PFHxS) individually or in binary combinations (PFOS + PFOA, PFOS + PFDA, PFOS + PFNA, PFOS + PFHxS, PFOA + PFDA, PFOA + PFNA, and PFOA + PFHxS) for 24 h using an orthogonal design. The individual and binary combination effects of PFAS on the cytotoxicity, intracellular reactive oxygen species (ROS) production, and glutathione (GSH) levels were determined by MTS assay, dichlorofluorescein diacetate assay, and GSH-Glo™ Glutathione assay, respectively. The results showed that exposure to PFOA, PFOS, PFDA, PFNA, and PFHxS individually and in binary combinations caused concentration-dependent cytotoxicity to HepG2 cells. Also, intracellular ROS production was not significantly induced in both the individual and co-treatment groups, indicating that ROS production may not be likely influencing the combined cytotoxicity of PFAS to HepG2 cells. However, the depletion of the intracellular glutathione levels was correlated with cytotoxicity. Moreover, the factorial analysis results showed no significant interactive effects between PFOS + PFOA, PFOS + PFDA, PFOS + PFNA, PFOS + PFHxS, PFOA + PFDA, PFOA + PFNA, and PFOA + PFHxS. Taken together, the results showed that both individual and combined PFAS could induce concentration-dependent cytotoxicity and depletion of GSH levels, but could not induce significant increases in ROS production at the concentration range tested. Overall, these results provided valuable toxicological data on the combined effects of mixed PFAS that may help to better assess their human health risk.

Combined effects and toxicological interactions of perfluoroalkyl and polyfluoroalkyl substances mixtures in human liver cells (HepG2).

The combined effects and toxicological interactions of perfluoroalkyl and polyfluoroalkyl substances (PFAS) mixtures remain largely unknown even though they occur as complex mixtures in the environment. This study investigated the toxicity of individual and combined PFAS to human liver cell line (HepG2). The Combination Index (CI)-isobologram equation method was used to determine the toxicological interactions of PFAS in binary, ternary and multi-component mixtures. The results indicated that the cytotoxicity of individual PFAS to HepG2 cells increased with increasing carbon chain lengths when separated into non-sulfonated and sulfonated groups. The respective cytotoxicity of PFAS is in the order of PFDA > PFNA > PFOA > PFHpA for perfluoroalkyl carboxylic acids and in the order of PFOS > PFHxS for perfluoroalkane sulfonic acids. The toxicological interaction of PFOS and PFOA with other PFAS clearly showed a different pattern of combined toxicity in HepG2 Cells. The binary, ternary, and multi-component combinations of PFOS with PFOA, PFNA, PFDA, PFHxS, and PFHpA displayed synergistic interactions for almost all inhibitory effect levels tested, whereas, either synergistic or antagonistic effect was observed in mixtures with PFOA. Overall, the pattern of interactions of PFAS mixtures is predominated by synergism, especially at low to medium effect levels; the exceptions to this were the antagonistic interactions found in mixture with PFOA, PFHxS, and PFHpA. These cytotoxicity results may have an implication on the health risk assessment of PFAS mixtures.