Investigating the cytotoxicity of per- and polyfluoroalkyl substances in HepG2 cells: A structure-activity relationship approach.

Per- and polyfluoroalkyl substances (PFAS) are a family of man-made chemicals with currently over 4'700 compounds identified. While toxicological data are available for some of the legacy PFAS, such as PFOA and PFOS, a knowledge gap remains concerning both emerging and legacy PFAS' toxicity due to the diversity of the PFAS. Therefore, a better understanding of the PFAS structure-activity relationship may prove helpful. The present study investigated a potential structure-activity relationship between PFAS and hepatotoxicity. As such, the effects of thirteen PFAS with varying carbon chain-length and functional head-groups (in a concentration range of 0-800 µM) on the cell viability of HepG2 cells and intracellular reactive oxygen species formation have been tested using the MTT and DCFH assay, respectively. The exposure times were either 3 or 24 h. In addition, intracellular PFAS levels were determined in HepG2 after 24 h exposure. The present study demonstrated that the cytotoxicity of PFAS is dependent on their chain-length as cell viability decreased with increasing chain-length at both exposure times. Calculated Relative Potency Factors (RPF), based on the TC50 values, were used for a tentative ranking of PFAS regarding their hepatotoxicity: PFNA ˃ PFDA ˃ PFOS ≥ PFOA ˃ PFHxS ˃ PFBS ˃˃ PFHpA = PFHxA = PFBA = PFPrA = 6:2 FTOH = 4:2 = FTOH = 3:1 FTOH. Similar results were observed regarding intracellular reactive oxygen species generation at both exposure times, with a tentative ranking of: PFNA ˃ PFOS ˃ PFOA ≥ PFDA ˃ PFHxS ˃ PFBS ˃ PFBA ˃ PFHpA ≥ PFHxA ˃ PFPrA ˃ 6:2 FTOH = 4:2 FTOH = 3:1 FTOH. Moreover, a concentration-dependent reactive oxygen species generation has been observed for all PFSAs and PFCAs, but not for the FTOHs. In conclusion, the carbon chain-length and functional head-group of a PFAS determine their in vitro toxicity for the two toxicological endpoints assessed in the present study. Moreover, no effects were observed for the tested FTOHs. As such, the present study established a potential structure-activity relationship that opens the possibility of developing a predictive model to help with the risk assessment of PFAS in the future.

The impact of legacy and novel perfluoroalkyl substances on human cytochrome P450: An in vitro study on the inhibitory potential and underlying mechanisms.

Per- and polyfluoroalkyl substances (PFASs) are a group of synthetic compounds with a wide range of industrial applications. PFOA and PFOS have been the most extensively studied and have been associated with hepatotoxicity. Recently, the interaction with cytochrome P450 (CYP) has been proposed as a potential key molecular event leading to PFAS-induced hepatotoxicity. In the present study, we aimed to determine a structure-activity relationship between thirteen PFASs and their inhibitory potential on the activities of four CYPs (CYP2E1, CYP2D6, CYP3A4 and CYP2C19). The influence of PFASs (5-3200 µM) on CYP enzyme activities was measured using the Vivid® P450 metabolism assays. Using the same assays, Michaelis-Menten saturation curves were determined to explore the type of PFAS-induced CYP inhibition. Most PFASs were capable of inhibiting activity of the tested CYPs, as shown by their IC50 values. CYP2E1 is particularly inhibited by 3:1 FTOH, PFOA, and PFOS, whereas CYP2D6 is inhibited by PFHxS, PFHpA, PFOA, PFOS, PFNA, and PFDA. Additionally, CYP3A4 is most strongly inhibited by PFHxS, PFOA, PFOS, PFNA, and PFDA. Finally, CYP2C19 is inhibited by PFBS, PFHxS, PFHpA, PFOA, PFOS, PFNA, and PFDA. Interestingly, PFHxA and PFHxS induced an increase in CYP2E1 activity, whereas 4:2 FTOH strongly induced CYP2D6 activity. The mechanism of inhibition of CYPs by PFASs differed per CYP isoenzyme. CYP3A4 was competitively inhibited by PFBS, PFHxS, PFOS, PFNA and PFDA and non-competitively by PFOA. Additionally, CYP2C19 was competitively inhibited by PFHxA, PFOS and PFNA, whereas PFBS and PFHxS induced a mixed inhibition. Inhibition of CYP2C19 by PFHpA was atypical with an increased Vmax and a decreased Km. Finally, PFHxS competitively inhibited CYP2D6, whereas PFBS, PFOA, PFOS, PFDA and PFNA induced an atypical inhibition. Our results show that CYP inhibition by PFASs appears to be structure-dependent as well as CYP dependent. Inhibition of CYP2D6, CYP2C19 and CYP3A4 increased with increasing chain-lengths between six and nine carbons. The PFTOHs were only able to inhibit CYP2E1 and did not affect any of the other CYPS. Some PFASs remarkably induced the enzyme activity of CYPs. These results indicate that in addition to PFOA and PFOS, multiple novel PFASs may alter drug metabolism by the interference with CYPs.

Migration of formaldehyde from 'biobased' bamboo/melamine cups: A Dutch retail survey.

Bamboo/melamine cups had become popular, due to their ecofriendly and sustainable image. Some cups are marketed as biobased and biodegradable. These cups are made from melamine-formaldehyde resin to which bamboo fibers or powder are added. They may release formaldehyde and melamine into the beverage. In 2019 the Netherlands Food and Consumer Product Safety Authority conducted a market study. 46 cups were sampled in duplicate and tested for formaldehyde migration. 88% of the cups complied with the migration limit of 15 mg/kg. Some cups showed very high migration values, over 200 mg/kg. There is a high exposure of formaldehyde from other sources (food, environment). When applying a realistic worst-case scenario of a daily intake of 0.4 L of beverage from the cups, the health based guidance value is exceeded at a migration of 19 mg/kg and higher. For some of the cups that had a high migration of formaldehyde, health risks may occur. For food-utensils for babies and young children, the exposure is higher due to their lower body weight. The SML does not provide sufficient protection for babies and young children. Bamboo is not authorized in the EU as additive for plastic food contact materials. Therefore the Netherlands, Belgium, Luxembourg and other EU member states have actively banned these products from the market. Finally, bamboo/melamine tableware would suggest having a 'green' image and would contribute to the goals of the circular economy to use biobased raw materials, but by using it in combination with melamine-formaldehyde resin may affect food safety.

Monitoring and risk assessment of hazardous chemicals in toy-slime and putty in the Netherlands.

In 2019, the Dutch Food and Consumer Product Safety Authority performed a market surveillance for toy-slime (23 samples) and putty (16 samples). For 35% of the toy-slimes and 13% of the putties, the migration of boron exceeded the European legal limit of 300 and 1200 mg/kg respectively. In 36% of the toy samples, methylisothiazolinone (MI) and chloromethylisothiazolinone (CMI) were detected in levels up to 25 and 38 mg/kg, respectively, much higher than the European legal limit for aqueous toys intended for children younger than three. 59% of the toys contained other preservatives such as 2-phenoxyethanol, p-hydroxybenzoic acid and parabens. In 2 toy-slimes and 2 putties N-nitrosodiethanolamine (NDELA) was found in amounts up to 2.3 mg/kg. A risk assessment was performed for boron and NDELA. The estimated exposure to boron did not exceed the health based guidance value. The estimated exposure to NDELA from 2 toy-slimes may pose a health risk. For 2 putties the estimated exposure to NDELA was somewhat lower, but health risks could not be excluded. The presence of isothiazolinones may lead to skin sensitisation. It is recommended to extend the legal limit for NDELA, MI and CMI in finger-paint and labelling requirements to other aqueous toys.