Shotgun ecotoxicoproteomics of Daphnia pulex: biochemical effects of the anticancer drug tamoxifen.

Among pollutants released into the environment by human activities, residues of pharmaceuticals are an increasing matter of concern because of their potential impact on ecosystems. The aim of this study was to analyze differences of protein expression resulting from acute (2 days) and middle-term (7 days) exposure of aquatic microcrustacean Daphnia pulex to the anticancer drug tamoxifen. Using a liquid chromatography-mass spectrometry shotgun approach, about 4000 proteins could be identified, providing the largest proteomics data set of D. pulex published up to now. Considering both time points and tested concentrations, 189 proteins showed a significant fold change. The identity of regulated proteins suggested a decrease in translation, an increase in protein degradation and changes in carbohydrate and lipid metabolism as the major effects of the drug. Besides these impacted processes, which reflect a general stress response of the organism, some other regulated proteins play a role in Daphnia reproduction. These latter results are in accordance with our previous observations of the impact of tamoxifen on D. pulex reproduction and illustrate the potential of ecotoxicoproteomics to unravel links between xenobiotic effects at the biochemical and organismal levels. Data are available via ProteomeXchange with identifier PXD001257.

Inhalation of Microplastics-A Toxicological Complexity.

Humans are chronically exposed to airborne microplastics (MPs) by inhalation. Various types of polymer particles have been detected in lung samples, which could pose a threat to human health. Inhalation toxicological studies are crucial for assessing the effects of airborne MPs and for exposure-reduction measures. This communication paper addresses important health concerns related to MPs, taking into consideration three levels of complexity, i.e., the particles themselves, the additives present in the plastics, and the exogenous substances adsorbed onto them. This approach aims to obtain a comprehensive toxicological profile of deposited MPs in the lungs, encompassing local and systemic effects. The physicochemical characteristics of MPs may play a pivotal role in lung toxicity. Although evidence suggests toxic effects of MPs in animal and cell models, no established causal link with pulmonary or systemic diseases in humans has been established. The transfer of MPs and associated chemicals from the lungs into the bloodstream and/or pulmonary circulation remains to be confirmed in humans. Understanding the toxicity of MPs requires a multidisciplinary investigation using a One Health approach.

Human skin absorption of three phthalates.

Population studies reveal widespread exposure to phthalates. Understanding their absorption, distribution, metabolism, and excretion is vital to reduce exposure. However, data on skin absorption remain limited. We thus aim to characterize the skin permeation of three phthalates in a mixture, neat or in emulsion; di(2-ethylhexyl) phthalate (d4-DEHP), dibutyl phthalate (d4-DBP), and diethyl phthalate (d4-DEP), by comparing in vitro human skin (800 µm) permeation (24 hours) results using flow-through diffusion cells with urine results obtained from volunteers exposed to the same mixture applied to a forearm (40 cm2). Metabolites were analyzed in receptor fluids and urine. Phthalates crossed the skin barrier and metabolized into monoesters before elimination. Increased permeation was observed for phthalates in emulsion compared to neat substances, with polyethylene glycol (PEG) in the receptor fluid enhancing emulsion permeation, but not affecting neat substances. In vitro results mirrored in vivo findings: DEP showed rapid permeation (J: ∼2 ug/cm2/h) and urinary excretion peaking at six hours post-application, whereas DBP exhibited slower kinetics (J: ∼0.1 ug/cm2/h), with a urinary peak at 15-17 hours post-application. DEHP had minimal permeation (J: ∼0.0002 ug/cm2/h) with no observable urinary peak. These findings underscore the importance of comprehending phthalate skin absorption for effective exposure mitigation strategies.

Novel Strategy to Assess the Neurotoxicity of Organic Solvents Such as Glycol Ethers: Protocol for Combining In Vitro and In Silico Methods With Human-Controlled Exposure Experiments.

BACKGROUND: Chemicals are not required to be tested systematically for their neurotoxic potency, although they may contribute to the development of several neurological diseases. The absence of systematic testing may be partially explained by the current Organisation for Economic Co-operation and Development (OECD) Test Guidelines, which rely on animal experiments that are expensive, laborious, and ethically debatable. Therefore, it is important to understand the risks to exposed workers and the general population exposed to domestic products. In this study, we propose a strategy to test the neurotoxicity of solvents using the commonly used glycol ethers as a case study. OBJECTIVE: This study aims to provide a strategy that can be used by regulatory agencies and industries to rank solvents according to their neurotoxicity and demonstrate the use of toxicokinetic modeling to predict air concentrations of solvents that are below the no observed adverse effect concentrations (NOAECs) for human neurotoxicity determined in in vitro assays. METHODS: The proposed strategy focuses on a complex 3D in vitro brain model (BrainSpheres) derived from human-induced pluripotent stem cells (hiPSCs). This model is accompanied by in vivo, in vitro, and in silico models for the blood-brain barrier (BBB) and in vitro models for liver metabolism. The data are integrated into a toxicokinetic model. Internal concentrations predicted using this toxicokinetic model are compared with the results from in vivo human-controlled exposure experiments for model validation. The toxicokinetic model is then used in reverse dosimetry to predict air concentrations, leading to brain concentrations lower than the NOAECs determined in the hiPSC-derived 3D brain model. These predictions will contribute to the protection of exposed workers and the general population with domestic exposures. RESULTS: The Swiss Centre for Applied Human Toxicology funded the project, commencing in January 2021. The Human Ethics Committee approval was obtained on November 16, 2022. Zebrafish experiments and in vitro methods started in February 2021, whereas recruitment of human volunteers started in 2022 after the COVID-19 pandemic-related restrictions were lifted. We anticipate that we will be able to provide a neurotoxicity testing strategy by 2026 and predicted air concentrations for 6 commonly used propylene glycol ethers based on toxicokinetic models incorporating liver metabolism, BBB leakage parameters, and brain toxicity. CONCLUSIONS: This study will be of great interest to regulatory agencies and chemical industries needing and seeking novel solutions to develop human chemical risk assessments. It will contribute to protecting human health from the deleterious effects of environmental chemicals. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/50300.

Hematological variations in healthy participants exposed 2 h to propylene glycol ethers under controlled conditions.

Glycol ethers are solvents used in a plethora of occupational and household products exposing the users to potential toxic effects. Several glycol ethers derived from ethylene glycol induce hematological toxicity, such as anemia in workers. The exposure effects on blood cells of glycol ethers derived from propylene glycol are unknown in humans. The aim of our study was to evaluate blood parameters indicative of red blood cell (RBC) hemolysis and oxidative stress in participants exposed to propylene glycol (propylene glycol monobutyl ether (PGBE) and propylene glycol monomethyl ether (PGME)), two extensively used propylene glycol derivatives worldwide. Seventeen participants were exposed 2 h in a control inhalation exposure chamber to low PGME (35 ppm) and PGBE (15 ppm) air concentrations. Blood was regularly collected before, during (15, 30, 60, and 120 min), and 60 min after exposure for RBC and oxidative stress analyses. Urine was also collected for clinical effects related to hemolysis. Under the study conditions, our results showed that the blood parameters such as RBCs, hemoglobin concentration, and white blood cells tended to increase in response to PGME and PGBE exposures. These results raise questions about the possible effects in people regularly exposed to higher concentrations, such as workers.

Repeated Human Exposure to Semivolatile Organic Compounds by Inhalation: Novel Protocol for a Nonrandomized Study.

BACKGROUND: Semivolatile organic compounds (SVOCs) comprise several different chemical families used mainly as additives in many everyday products. SVOCs can be released into the air as aerosols and deposit on particulate matter during use by dispersion, evaporation, or abrasion. Phthalates are SVOCs of growing concern due to their endocrine-disrupting effects. Human data on the absorption, distribution, metabolism, and excretion (ADME) of these compounds upon inhalation are almost nonexistent. OBJECTIVE: The goal of this study is to develop a method for repeated inhalation exposures to SVOCs to characterize their ADME in humans. METHODS: We will use diethylhexyl phthalate (DEHP), a major indoor air pollutant, as a model SVOC in this novel protocol. The Swiss official Commission on Ethics in Human Research, Canton de Vaud, approved the study on October 14, 2020 (project-ID 2020-01095). Participants (n=10) will be repeatedly exposed (2 short daily exposures over 4 days) to isotope-labeled DEHP (DEHP-d4) to distinguish administered exposures from background exposures. DEHP-d4 aerosols will be generated with a small, portable, aerosol-generating device. Participants will inhale DEHP-d4-containing aerosols themselves with this device at home. Air concentrations of the airborne phthalates will be less than or equal to their occupational exposure limit (OEL). DEHP-d4 and its metabolites will be quantified in urine and blood before, during, and after exposure. RESULTS: Our developed device can generate DEHP-d4 aerosols with diameters of 2.5 µm or smaller and a mean DEHP-d4 mass of 1.4 (SD 0.2) µg per puff (n=6). As of May 2023, we have enrolled 5 participants. CONCLUSIONS: The portable device can be used to generate phthalate aerosols for repeated exposure in human studies. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/51020.

Blood absorption toxicokinetics of glycol ethers after inhalation: A human controlled study.

Glycol ethers are organic solvents present in countless products for professional and domestic use. The main toxicological concerns are hematotoxicity, respiratory and reproductive toxicity. The general population can be exposed when using products containing one or several glycol ethers that evaporate or if sprayed, generate aerosols that can be inhaled. The rate at which glycol ethers enters blood following inhalation exposure are unknown in humans, and chemical risk assessors only rely on animal and in vitro toxicity studies. Propylene glycol monomethyl ether (PGME) and propylene glycol monobutyl ether (PGBE) are two examples of glycol ethers used worldwide. Our study aimed to provide human toxicokinetic data after inhalation exposure of low PGME and PGBE concentrations tested alone or in mixture. Healthy participants (n = 28) were exposed to 35 ppm (131 mg/m3) of PGME and 15 ppm (i.e., 83 mg/m3) of PGBE for 2 or 6 h. Blood was regularly collected during the exposure sessions. PGME and PGBE were immediately bioavailable in blood during exposure, and the mean absorption rates were up to 13 µg/L/min and 2.45 µg/L/min, respectively. Maximum mean blood concentration (Cmax) was 2.91 mg/L and 0.41 mg/L for PGME and PGBE. The cumulative internal doses over time (area under the curve, AUC) were 11 mg∗h/L and 1.81 mg∗h/L for PGME and PGBE. PGME and PGBE total blood uptake could possibly be higher in physically active individuals, such as workers. We recommend that glycol ethers present on the market undergo toxicological testing with the internal doses we found in our toxicokinetic study.

Influence of collection and storage materials on glycol ether concentrations in urine and blood.

Glycol ethers, such as propylene glycol monomethyl ether (PGME) and propylene glycol monobuthyl ether (PGBE) are solvents found in many professional and domestic products. In biomonitoring studies, the type of materials used to collect, store, and transport these samples can greatly influence the analytical results because materials can adsorb the analyte. Plastic tubes generally have a hydrophobic internal surface that can reduce the concentration of certain chemicals and result in an underestimation of workers' exposures. The aim of this study was to assess whether the storage of PGME and PGBE spiked blood and urine samples led to different PGME and PGBE concentrations in vials made of glass and common plastics (polypropylene (PP), polyethylene (PE) or polystyrene (PS)). Glycol ether concentrations were quantified with headspace gas chromatography equipped with a flame ionization detector. Our results show stable urinary PGME and PGBE concentrations in PP, while up to 15% variations in urinary PGME for PE and PS. For PGME and PGBE in blood, we observed no statistically significant losses in glass, while losses were recorded for all types of plastic tested (PS, PP and PE). We conclude that biomonitoring samples should be collected in glass for blood and PP for urine.

Multigenerational effects of the anticancer drug tamoxifen and its metabolite 4-hydroxy-tamoxifen on Daphnia pulex.

Tamoxifen and its metabolite 4-hydroxy-tamoxifen (4OHTam) are two potent molecules that have anticancer properties on breast cancers. Their medical use is expected to increase with the increasing global cancer rate. After consumption, patients excrete tamoxifen and the 4OHTam metabolite into wastewaters, and tamoxifen has been already detected in wastewaters and natural waters. The concentrations of 4OHTam in waters have never been reported. A single study reported 4OHTam effects on the microcrustacean Daphnia pulex. The effects of tamoxifen and 4OHTam over more than two generations are unknown in aquatic invertebrates. The main goal of this study was to assess the long-term sensitivity of the microcrustacean D. pulex over four generations, based on size, reproduction, viability and the intrinsic rate of natural increase (r). Additional experiments were carried out to observe whether the effects of tamoxifen and 4OHTam were reversible in the next generation after descendants were withdrawn from chemical stress (i.e., recovery experiment), and whether the lowest test concentration of each chemical induced toxic effects when both concentrations were combined (i.e., mixture experiments). Our results showed that tamoxifen and 4OHTam induced the adverse effects at environmentally relevant concentrations. Tamoxifen and 4OHTam impaired size, viability, reproduction and the r in four generations of treated D. pulex, but these effects were not clearly magnified over generations. Tamoxifen was more potent than 4OHTam on D. pulex. When used in a mixture, the combination of tamoxifen and 4OHTam induced effects in offspring, whereas no effects were observed when these chemicals were tested individually. In the recovery experiment, the reproduction and size were reduced in offspring withdrawn from chemical exposures. Our results suggested that tamoxifen and its metabolite may be a relevant pharmaceutical to consider in risk assessment.

The anticancer drug metabolites endoxifen and 4-hydroxy-tamoxifen induce toxic effects on Daphnia pulex in a two-generation study.

Although pharmaceutical metabolites are found in the aquatic environment, their toxicity on living organisms is poorly studied in general. Endoxifen and 4-hydroxy-tamoxifen (4OHTam) are two metabolites of the widely used anticancer drug tamoxifen for the prevention and treatment of breast cancers. Both metabolites have a high pharmacological potency in vertebrates, attributing prodrug characteristics to tamoxifen. Tamoxifen and its metabolites are body-excreted by patients, and the parent compound is found in sewage treatment plan effluents and natural waters. The toxicity of these potent metabolites on non-target aquatic species is unknown, which forces environmental risk assessors to predict their toxicity on aquatic species using knowledge on the parent compounds. Therefore, the aim of this study was to assess the sensitivity of two generations of the freshwater microcrustacean Daphnia pulex towards 4OHTam and endoxifen. Two chronic tests of 4OHTam and endoxifen were run in parallel and several endpoints were assessed. The results show that the metabolites 4OHTam and endoxifen induced reproductive and survival effects. For both metabolites, the sensitivity of D. pulex increased in the second generation. The intrinsic rate of natural increase (r) decreased with increasing 4OHTam and endoxifen concentrations. The No-Observed Effect Concentrations (NOECs) calculated for the reproduction of the second generation exposed to 4OHTam and endoxifen were <1.8 and 4.3 µg/L, respectively, whereas the NOECs that were calculated for the intrinsic rate of natural increase were <1.8 and 0.4 µg/L, respectively. Our study raises questions about prodrug and active metabolites in environmental toxicology assessments of pharmaceuticals. Our findings also emphasize the importance of performing long-term experiments and considering multi-endpoints instead of the standard reproduction outcome.