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.

Estimating biotransformation rate constants of organic chemicals from modeled and measured elimination rates.

In this study, biotransformation rate constants are estimated for a large set of organic compounds. Biotransformation (km) is considered part of the total elimination, further consisting of physico-chemical elimination to water (kw), depuration by feces (kf) and growth dilution (gamma). Existing models are used to estimate kw and kf, and gamma. The difference between measured elimination rate constants and the sum of predicted elimination rate constants for water, feces and growth indicates the ration of biotransformation in the total elimination. In all examined animal classes, polycyclic aromatic hydrocarbons seem to be metabolized at an intermediate rate. Because of the relative low hydrophobicity of some of the studied compounds, their physico-chemical elimination rate constant is relatively high, and the relative contribution of metabolism to total elimination of these compounds is therefore relatively low. Fish seem to be capable of metabolizing chlorodibenzo-p-dioxins and -furans, DDT, chloroanilines and phenol.

The power of size. 1. Rate constants and equilibrium ratios for accumulation of organic substances related to octanol-water partition ratio and species weight.

Most of the thousands of substances and species that risk assessment has to deal with are not investigated empirically because of financial, practical, and ethical constraints. To facilitate extrapolation, we have developed a model for accumulation kinetics of organic substances as a function of the octanol-water partition ratio (Kow) of the chemical and the weight, lipid content, and trophic level of the species. The ecological parameters were obtained from a previous review on allometric regressions. The chemical parameters, that is, resistances that substances encounter in water and lipid layers of organisms, were calibrated on 1,939 rate constants for absorption from water for assimilation from food and for elimination. Their ratio was validated on 37 laboratory bioconcentration and biomagnification regressions and on 2,700 field bioaccumulation data. The rate constant for absorption increased with the hydrophobicity of the substances with a Kow up to about 1,000 and then leveled off, decreasing with the weight of the species. About 39% of the variation was explained by the model, while deviations of more than a factor of 5 were noted for labile, large, and less hydrophobic molecules as well as for algae, mollusks, and arthropods. The efficiency for assimilation of contaminants from food was determined mainly by the food digestibility and thus by the trophic level of the species. A distinction was made between substances that are stable, that is, with a minimum elimination only, and those that are labile, that is, with an excess elimination probably largely due to biotransformation. The rate constant for minimum elimination decreased with the hydrophobicity of the substance and the weight of the species. About 70% of the variation was explained by the model, while deviations of more than a factor of 5 were noted for algae, terrestrial plants, and benthic animals. Labile substances were eliminated faster than isolipophilic stable compounds, but differences in laboratory elimination and accumulation were small compared with those in field accumulation. Excess elimination by vertebrates was faster than by invertebrates. Differences between terrestrial and aquatic species were attributed to water turnover rates, whereas differences between trophic levels were due to the food digestibility. Food web accumulation, expressed as organism-organic solids and organism-food concentrations ratios could be largely explained by ecological variables only. The model is believed to facilitate various types of scientific interpretation as well as environmental risk assessment.

Natural and synthetic organic compounds in the environment-a symposium report.

In March 2000, an international two-day symposium was organized in Noordwijkerhout, The Netherlands, on 'Natural and synthetic organic compounds in the environment'. The emphasis of the symposium was on the following classes of compounds: polycyclic aromatic hydrocarbons, xeno-estrogens, phyto-estrogens, and veterinary drugs. Sources, environmental distribution, uptake, biotransformation and toxic effects from the molecular to the population level were discussed. Other important aspects were the development of biomarkers, analytical methods, bioassays, molecular modelling and other research tools. Finally, the implications of the findings for government policies were discussed. In this paper, a summary is given of the most important facts and views presented at the symposium.

Environmental risk limits for two phthalates, with special emphasis on endocrine disruptive properties.

Environmental risk limits (ERLs) are derived for di-n-butyl phthalate (DBP) and di(2-ethylhexyl) phthalate (DEHP). The ERLs are derived using data on (eco)toxicology and environmental chemistry. Endpoints used are survival, growth, and reproduction. The resulting ERLs in water are 10 and 0.19 microg/L for DBP and DEHP, respectively; in fresh soil and sediment with 10% organic matter the derived ERLs are respectively 0.7 and 1 mg/kg fresh wt. In The Netherlands, measured concentrations of DBP are seldom above the ERLs, while reported concentrations for DEHP are 3 to 20 times higher than the ERL. As phthalates as a group are commonly mentioned as chemicals with possible endocrine disruptive effects, in vivo and in vitro tests for a series of phthalates with endpoints related to endocrine disruption are reviewed. In vitro and in vivo tests give a similar distinction between phthalates that can or cannot act as endocrine disrupters. The significance of these tests for the derivation of ERLs is discussed. It is concluded that the ERLs derived will give sufficient protection against endocrine disruptive effects. There is no need to include additional data for DBP and DEHP, related to endpoints other than survival, growth, or reproduction, in the derivation of ERLs.

Extraction and isolation of linear alkylbenzenesulfonate and its sulfophenylcarboxylic acid metabolites from fish samples.

Linear alkylbenzenesulfonate (LAS) is the most widely used synthetic surfactant. In fish, assessment of the environmental risk and investigation of the biotransformation behavior of LAS require compound-specific methods for extraction and isolation of LAS and its biotransformation products, sulfophenylcarboxylic acids (SPC). Matrix solid-phase dispersion (MSPD) extraction with subsequent ion-pair liquid-liquid (IP-LL) partitioning of the extract was a time-efficient sample preparation method for analysis of LAS. The recovery of parent LAS from spiked fish exceeded 70%, and the limit of quantitation ranged around 0.2 mg.kg-1 corresponding to 0.6 mumol.kg-1. In a simultaneous determination of LAS and SPC in fish, the analytes were MSPD extracted in different fractions. The target compounds were separated from the sample matrix by protein precipitation and subsequent isolation of (a) SPC by graphitized carbon black solid-phase extraction of the supernatant and (b) parent LAS by IP-LL partitioning of the pellet obtained after protein precipitation. The recoveries of the model compounds C12-2-LAS and C4-3-SPC were 84 +/- 6 and 65 +/- 11%, respectively. The use of C3-3-SPC as an internal standard corrected for the loss of the biotransformation product during sample workup. The suitability of both methods was demonstrated by analyzing fish containing LAS and SPC incurred during aqueous exposure.

Lipid content and time-to-death-dependent lethal body burdens of naphthalene and 1,2,4-trichlorobenzene in fathead minnow (Pimephales promelas).

For naphthalene (NAPH) and 1,2,4-trichlorobenzene (TCBz), lethal body burdens (LBB) of 8 +/- 3.1 mmol/kg wet wt (ww) and 14 +/- 4.5 mmol/kg (ww), respectively, were determined in fathead minnow (Pimephales promelas). LBBs of both compounds were found to increase with increasing lipid content and time-to-death of fish within the same aquarium. The correlation with time-to-death suggests that besides lipid content at least one other factor causes intraspecies variation in LBBs. When intraspecies variation is excluded by comparing mean LBBs from different aquaria and exposure regimes, LBBs still vary with time-to-death. In contrast to the situation within one aquarium, between different aquaria and exposure regimes often a decrease in LBB with time-to-death is found. The present study and others indicate that LBBs can vary with time-to-death. Therefore, a time-independent LBB cannot be assumed.

An energy budget model for the biodegradation and cometabolism of organic substances.

Expression of bacterial cellular processes (maintenance, heat loss, growth, cometabolism and substrate degradation) into energy units yielded surprisingly realistic figures on the bacterial energy balance in spite of the severe approximations and assumptions that had to be made. For studies without cometabolism, 47-83% of the calculated amount of available energy was consumed by growth; for maintenance + heat loss this percentage was 20-35%. When involved, cometabolism consumed 7-13% of the total energy budget. Overall, 67-118% of the calculated amount of energy generated was spent on these energy-consuming processes. This shows that the model is internally consistent. Relationships between growth and cometabolism may offer predictions of persistence of cometabolizable chemicals under different conditions. The energy budget model as presented provides a starting point for the development of such relationships. In addition, the method is used to explain threshold concentrations, below which normally degradable compounds are not degraded.

Use of lethal body burdens to indicate species differences in susceptibility to narcotic toxicants.

Lethal body burdens (LBB) for 1,2- and 1,4-dihalogenated benzenes (F, Cl or Br) are determined in rainbow trout of two age-classes. LBBs range from 0.3 to 2.4 mmol/kg. There are no significant differences between the two tested age-classes of rainbow trout. The rainbow trout data are compared to LBBs for 1,2- and 1,4-difluorobenzene in fathead minnow which range from 2.7 to 3.0 mmol/kg, and to LBBs of dichloro- and dibromobenzenes in guppy and fathead minnow [Sijm et al. 1993] which range from 2.7 to 8.0 mmol/kg. Rainbow trout are more susceptible to dihalogenated benzenes than fathead minnow. The LBB can be used as an instrument to examine the intrinsic toxicity of a chemical to a species, and to indicate the susceptibility of a species. Possible reasons for differences in susceptibility among species are discussed.

Allometry in the uptake of hydrophobic chemicals determined in vivo and in isolated perfused gills.

Uptake rate constants of different classes of hydrophobic organic chemicals have been determined in isolated perfused gills of rainbow trout (Oncorhynchus mykiss) as an alternative for studies in vivo. The uptake rate constants have been compared to those determined in guppy, Poecilia reticulata, in vivo. The organic chemicals which have been used are anthracene, hexabromobenzene, octachloronaphthalene, octachlorodibenzo-p-dioxin, phenol, polychlorinated anisoles, polychlorinated benzenes, polychlorinated biphenyls, and tetrachloroveratrole. Uptake rate constants in guppy are higher than those in rainbow trout gills, and show relatively high variation in both gills and guppy. When uptake rate constants in each study are normalized for that of pentachlorobenzene (pCBz), variation is significantly reduced both in perfused gills and in guppy. All allometric relationship is derived between weight and uptake rate constant. Uptake rate constants determined in one fish can thus be used for prediction of those in other fishes. When a reference chemical, such as pCBz, is included, the gill perfusion experiments can be highly suitable to determine uptake rate constants of organic chemicals, which can be extrapolated to fish of different sizes.

Surfactant bioconcentration--a critical review.

Bioconcentration data for surfactants have been collected and critically reviewed. Twenty-two references report whole body bioconcentration data. Most of these data are inappropriate to quantitatively describe the bioconcentration of surfactants because the most frequently used analytical method, LSC without prior chromatographic separation of radiolabelled compounds, does not allow to distinguish between parent compound and metabolites. Hence, the measured concentrations very likely are overestimates of the concentration of the parent surfactant. In order to compare data we defined a comparability criterion. Data which fulfil this criterion consistently overestimate the true extent of bioconcentration. Fifty-four out of 100 whole body concentration ratios (CRs) were selected employing the above criterion, with 33, 11, and 10 Crs reported for anionic, cationic and nonionic surfactants, respectively. Further findings are: 1. Selected CRs range between 2.4 for octyltrimethylammonium chloride and 1960 for tallowtrimethylammonium chloride. In general, CRs increased with increasing alkyl chain length. 2. Surfactants of all classes are readily taken up across the gills. Hexadecylpyridinium and dialkyl dimethyl ammonium surfactants appear to be taken up rather slow. 3. Nonionic and anionic surfactants were demonstrated to be biotransformed. Tissue specific data demonstrated that elimination via the gall bladder is an important excretion route. 4. Environmental variables appeared to influence bioconcentration of ionic surfactants.

The influence of biochemical species differences on acute fish toxicity of organic chemicals.

1. Reported LC50s of hundreds of chemicals and data of many enzymes in many fishes have been reviewed in order to find the hypothesized influence of biotransformation on acute toxicity. 2. Biotransformation of carbaryl was correlated and biotransformation of endrin was inversely correlated with acute toxicity. 3. For most compounds no correlation was found which indicates that either biotransformation is not important in acute toxicity or enzyme activities are not representative parameters to quantify biotransformation reactions.