Observed Effect Level Research Articles

Biological Response after 14-Day Cannabidiol and Propylene Glycol Inhalation in Sprague-Dawley Rats.

Objective: Cannabidiol (CBD), a phytocannabinoid of increasing interest for its purported therapeutic effects, is primarily consumed via ingestion and inhalation. While the toxicology of orally administered CBD has been reported, little is known about the effects of CBD inhalation. Doses selected for the present analysis allowed for evaluation of dose-response at concentrations >100-fold higher than typical human consumption levels. Materials and Methods: CBD (98.89% pure) was formulated in propylene glycol (PG) and aerosolized by nebulization to evaluate biological response after nose-only inhalation. Sprague Dawley rats (n = 35 males, 30 females) were exposed to 1.0 and 1.3 mg/L nominal concentrations of CBD and PG, respectively, for 12-180 min. Resulting average daily presented dose ranges were 8.9-138.5 mg/kg CBD and 11.3-176.0 mg/kg PG. Aerosols of 1.4 µm median diameter were achieved. Biological response indicators included clinical signs, clinical chemistry, hematology, body/organ weights, and pulmonary/systemic histopathology. Results: Inflammatory and necrotic responses were observed in the nose at the highest doses of CBD. Limited findings in the larynx and lung were mainly observed at higher doses. There were no histological findings in extrapulmonary organs. Dosimetry modeling differentiated the no observable adverse effect level between the nasal region and lungs to be 2.8 and 10.6 mg/kg CBD, respectively. Conclusions: Dose-depending findings of histological changes in the respiratory tract are observed at high doses. At lower doses consistent with typical over-the-counter vape products there appears to be substantial safety margin in the present study (93- and 353-fold lower for nose and lung, respectively).

Preclinical Safety Evaluation of Etripamil Nasal Spray in Cynomolgus Macaques (Macaca fascicularis) to Assess for Safety in Patients With Paroxysmal Supraventricular Tachycardia.

Etripamil is a calcium channel blocker currently in Phase 3 trials for the treatment of paroxysmal supraventricular tachycardia (PSVT). Systemic and local toxicity following once-weekly intranasal administration of etripamil was evaluated in cynomolgus macaques to support clinical development. Groups of animals (N = 8, 4 males and 4 females) were administered etripamil into the left nostril weekly at dose levels of 0 (vehicle), 1.9, 3.8, or 5.7mg/kg/dose for 26 doses. Persistence, reversibility, and progression of findings were examined following a 28-day recovery period. Clinical signs were transient and were related to the intranasal administration (e.g., nasal discharge, sneezing, etc.) of etripamil. There were no macroscopic or systemic microscopic findings at any dose. Etripamil-related adaptive and reactive local changes affecting the nasal cavity, larynx, and nasopharynx were observed at ≥1.9mg/kg/dose. Minimal to severe dose-dependent nasal epithelial damage was observed, mainly affecting respiratory and transitional epithelium. Following the 28-day recovery period, microscopic changes were confined to the left nasal cavity and nasopharynx. These changes were significantly lower in incidence and severity, with noticeable reversal of the adaptive and reactive changes, indicating partial to complete recovery of the epithelial lining. Based on the lack of systemic toxicity and the minimal and transient nasal changes, the systemic, no observable adverse effect level (NOAEL) of etripamil in monkeys was the high dose, 5.7mg/kg/dose. The NOAEL for local toxicity was 1.9mg/kg/dose. Collectively, these data support further study of etripamil in human trials as a potential treatment for PSVT.

TOXICOLOGICAL EVALUATION OF OLEANOLIC ACID (PENTACYCLIC TRITERPENOID) EXTRACTED FROM LANTANA CAMARA ROOTS FOLLOWING ORAL EXPOSURE IN WISTAR RATS

Objective: The aim of the present study was to perform an acute toxicity study to obtain information on the possible adverse effects from a single oral administration of Oleanolic acid in Wistar rats as the onset of toxicity, and to determine the range of exposure (to the LD50 cut-off criteria). Following a sub-chronic 90-days Repeated toxicity study by oral route to determine any potential indication of its dose response relationship and determine no observed effect level (NOEL)/ no-observed adverse effect level (NOAEL)/ low observed effect level (LOEL)/ low observed adverse effect level (LOAEL). Methods: A dose level of 2000mg/kg body weight was employed as step one for single acute study. Based on the survival pattern of the previously dosed animals after 48 hours, same dose 2000 mg/kg body weight) was repeated for Step 2 as a confirmatory test. For the 90-day toxicity study, the highest dose was determined as 1000mg/kg Body weight, and the middle and lower doses were 500 and 250mg/kg Body weight respectively. The rat group was held for a 14-day recovery period after the last dose administration, to observe for any persistence or reversal of toxic effects. Results: Results of the acute toxicity study showed no mortality on the dose level of 2000mg/kg body weight with no significant clinical and body weight changes. During the 90-day Repeated dose oral sub-chronic toxicity study, no rats died. There were no significant clinical changes related to the test item in terms of functional evaluation, body weight, food and water consumption, ophthalmological tests, urine analysis, necropsy findings, or organ weight, Hematology, and biochemistry at the highest dose level of 1000 mg/kg bwt. Conclusion: It is concluded that LD50 Cut-off Value of “Oleanolic acid” in from acute oral toxicity study in Wistar Rats is 2000 mg/kg b.wt. In addition, 90 days study have showed no significant changes with respect to any hematological or blood chemical analyses in 1000, 500 and 250 mg/kg bwt groups. Based on histopathological findings, clinical signs and other parameters, it may be concluded that upon repeated once oral administration for consecutive 90 days, the Oleanolic acid (Pentacyclic Triterpenoid) extracted from Lantana camara roots at the dose level of 1000 mg/kg body weight have caused no adverse effect in both sexes of Wistar Rats.

Ecological impact of pharmaceutical pollutants and options of river health improvements - A risk analysis-based approach

Pharmaceuticals are one of the emerging pollutants (EPs) in river waters across the world. Due to their toxic effects on aquatic organisms, they have drawn the global attention of the scientific community concerned with river ecosystems. This paper reviews the existing occurrence data for various pharmaceutical pollutants (PPs) reported in river waters in some part of the world and their ecological impacts. Using algae, macroinvertebrates (MI), and fish as biotic indicator groups in water to reflect river health conditions, an attempt has been made to assess the ecological risk due to the presence of PPs in the water environment.After ascertaining the predicted no-effect concentration (PNEC) of PPs for selected groups of aquatic organisms, the risk quotient (RQ) is estimated based on their measured environmental concentration (MEC). When MEC > PNEC and RQ > 1 for any of the biotic indicator, ecologically it is ‘high risk’ condition. The determination of PNEC uses a minimum assessment factor (AF) of 10 due to uncertainty in data over the no observed effect level (NOEL) or lowest observed effect level (LOEL). Accordingly, MEC 10 times higher than PNEC, (RQ = 10) represents a threshold risk concentration (RCT) beyond which adverse effects may start showing observable manifestations. In the present study, a new classification system of ‘high risk’ conditions for RQ = 1–10 has been proposed, starting from ‘moderately high’ to ‘severely high’. For RQ > 10, the ecological condition of the river is considered ‘impaired’.For river health assessment, in the present study, the whole range of physico-chemical characteristics of river water quality has been divided into three groups based on their ease of measurement and frequency of monitoring. Dissolved oxygen related parameters (DORPs), nutrients (NTs), and EPs. PPs represent EPs in this study. A framework for calculating separate indicator group score (IGS) and the overall river health index (RHI) has been developed to predict indicator group condition (IGC) and river health condition (RHC), respectively. Color-coded hexagonal pictorial forms representing IGC and RHC provide a direct visible perception of the existing aquatic environment and a scientific basis for prioritization of corrective measures in terms of treatment technology selection for river health improvements.The analyses indicate that many rivers across the world are under ‘high risk’ conditions due to PPs having MEC > PNEC and RQ > 1. Up to RCT, (where RQ = 10), the ‘high risk’ condition varies from ‘moderately high’ to ‘severely high’. In many instances, RQ is found much more higher than 10, indicating that the ecological condition of river may be considered as ‘impaired’. Algae is the most frequently affected group of biotic indicators, followed by MI and fish.A review of treatment methods for selection of appropriate technology to reduce the pollution load, especially PPs from the wastewater streams has been summarized. It appears that constructed wetlands (CWs) are at present the most suitable nature-based solutions, particularly for the developing economies of the world, to reduce the concentrations of PPs within limits to minimize the ecological impacts of pharmaceutical compounds on biotic indicators and restore the river health condition. Some suggestive design guidelines for the CWs have also been presented to initiate the process.

Total cucurbitacins from Herpetospermum pedunculosum pericarp do better than Hu-lu-su-pian (HLSP) in its safety and hepatoprotective efficacy.

The pericarp of Herpetospermum pedunculosum (HPP) has traditionally been used for treating jaundice and hepatitis. However, the specific hepatoprotective components and their safety/efficacy profiles remain unclear. This study aimed to characterize the total cucurbitacins (TCs) extracted from HPP and evaluate their hepatoprotective potential. As a reference, Hu-lu-su-pian (HLSP), a known hepatoprotective drug containing cucurbitacins, was used for comparison of chemical composition, effects, and safety. Molecular networking based on UHPLC-MS/MS identified cucurbitacin B, isocucurbitacin B, and cucurbitacin E as the major components in TCs, comprising 70.3%, 26.1%, and 3.6% as determined by RP-HPLC, respectively. TCs treatment significantly reversed CCl4-induced metabolic changes associated with liver damage in a dose-dependent manner, impacting pathways including energy metabolism, oxidative stress and phenylalanine metabolism, and showed superior efficacy to HLSP. Safety evaluation also showed that TCs were safe, with higher LD50 and no observable adverse effect level (NOAEL) values than HLSP. The median lethal dose (LD50) and NOAEL values of TCs were 36.21 and 15mg/kg body weight (BW), respectively, while the LD50 of HLSP was 14mg/kg BW. In summary, TCs extracted from HPP demonstrated promising potential as a natural hepatoprotective agent, warranting further investigation into synergistic effects of individual cucurbitacin components.

Pre-clinical safety and toxicity assessment of Limosilactobacillus fermentum NCDC 400 in murine model

Comprehensive safety assessment of potential probiotic strains is crucial in the selection of risk-free strains for clinical translation. This study aimed to evaluate the biosafety of Limosilactobacillus fermentum NCDC 400, a potential probiotic strain, using oral toxicity tests in a Swiss albino mouse model. Mice were orally gavaged with low (108 CFU/mouse/day) and high (1010 CFU/mouse/day) doses of NCDC 400 for 14 (acute), 28 (subacute), and 90 (subchronic) days to assess behavioral, hematological, biochemical, immunological, and histological effects. The administration of NCDC 400 did not result in any observable adverse effects on general health parameters, including body weight, feed and water intake, and organ indices. Hematological and biochemical parameters, such as glucose, serum enzymes, urea, creatinine, serum minerals, total serum proteins, and lipid profile, remained largely unaffected by the test strain. Notably, NCDC 400 administration led to a significant reduction in harmful intestinal enzymes and improvement in gut health indices, as indicated by fecal pH, lactate, ammonia, and short-chain fatty acids. There were no instances of bacterial translocation of NCDC 400 to blood or extra-intestinal organs. Immune homeostasis was not adversely affected by repeated exposure to NCDC 400 in all three oral toxicity studies. Histopathological examination revealed no strain-related changes in various tissues. Based on these findings, a dose of 1010 CFU/mouse/day was considered as the No Observable Effect Level (NOEL) in healthy mice. In conclusion, this study demonstrates the safe and non-toxic behavior of L. fermentum NCDC 400. The results support and ensure the safety and suitability for clinical trials and eventual translation into clinical practice as potential probiotic.

Oil droplet fouling on lesser sandeel (Ammodytes marinus) eggshells does not enhance the crude oil induced developmental toxicity

The oil industry's expansion and increased operational activity at older installations, along with their demolition, contribute to rising cumulative pollution and a heightened risk of accidental oil spills. The lesser sandeel (Ammodytes marinus) is a keystone prey species in the North Sea and coastal systems. Their eggs adhere to the seabed substrate making them particularly vulnerable to oil exposure during embryonic development. We evaluated the sensitivity of sandeel embryos to crude oil in a laboratory by exposing them to dispersed oil at concentrations of 0, 15, 50, and 150 µg/L oil between 2 and 16 days post-fertilization. We assessed water and tissue concentrations of THC and tPAH, cyp1a expression, lipid distribution in the eyes, head and trunk, and morphological and functional deformities. Oil droplets accumulated on the eggshell in all oil treatment groups, to which the embryo responded by a dose-dependent rise in cyp1a expression. The oil exposure led to only minor sublethal deformities in the upper jaw and otic vesicle. The findings suggest that lesser sandeel embryos are resilient to crude oil exposure. The lowest observed effect level documented in this study was 36 µg THC/L and 3 µg tPAH/L. The inclusion of these species-specific data in risk assessment models will enhance the precision of risk evaluations for the North Atlantic ecosystems.

Arsenic Impairs Wound Healing Processes in Dermal Fibroblasts and Mice.

Inorganic arsenic (NaAsO2) is a naturally occurring metalloid found in water resources globally and in the United States at concentrations exceeding the U.S. Environmental Protection Agency Maximum Contamination Level of 10 ppb. While exposure to arsenic has been linked to cancer, cardiovascular disease, and skin lesions, the impact of arsenic exposure on wound healing is not fully understood. Cultured dermal fibroblasts exposed to NaAsO2 displayed reduced migration (scratch closure), proliferation, and viability with a lowest observable effect level (LOEL) of 10 µM NaAsO2 following 24 h exposure. An enrichment of Matrix Metalloproteinase 1 (MMP1) transcripts was observed at a LOEL of 1 µM NaAsO2 and 24 h exposure. In vivo, C57BL/6 mice were exposed to 10 µM NaAsO2 in their drinking water for eight weeks, then subjected to two full thickness dorsal wounds. Wounds were evaluated for closure after 6 days. Female mice displayed a significant reduction in wound closure and higher erythema levels, while males showed no effects. Gene expression analysis from skin excised from the wound site revealed significant enrichment in Arsenic 3-Methyltransferase (As3mt) and Estrogen Receptor 2 (Esr2) mRNA in the skin of female mice. These results indicate that arsenic at environmentally relevant concentrations may negatively impact wound healing processes in a sex-specific manner.

Updated Chronic Copper Bioavailability Models for Invertebrates and Algae.

Chronic copper (Cu) bioavailability models have been successfully implemented in European risk assessment frameworks and compliance evaluations. However, they were developed almost two decades ago, which calls for an update. In the study, we present updated chronic Cu bioavailability models for invertebrates and algae. They consider recent ecotoxicity data sets and use the more recent speciation model Windermere Humic Aqueous Model (WHAM) VII and an optimized model structure (i.e., a generalized bioavailability model [gBAM]). Contrary to the classic biotic ligand model, a gBAM models the effect of pH on Cu2+ toxicity via a log-linear relationship parametrized through the pH slope SpH . The recalibrated SpH parameters are -0.208 for invertebrates (Daphnia magna, two clones) and -0.975 for algae (Raphidocelis subcapitata and Chlorella vulgaris). The updated models predict 80% to 100% of the observed effect levels for eight different species within a factor of 2. The only exception was one of the two data sets considering subchronic 7-day mortality to Hyalella azteca: the prediction performance of the updated invertebrate model at pH ≥ 8.3 was poor because the effect of pH on Cu2+ toxicity appeared to be dependent on the pH itself (with a steeper pH slope compared with the updated invertebrate model at pH ≥ 8.1). The prediction performance of the updated Cu bioavailability models was similar to or better than that of the models used for regulatory application in Europe until now, with one exception (i.e., H. azteca). Together with the recently published fish bioavailability model, the models developed in the present study constitute a complete, updated, and consistent bioavailability model set. Overall, the updated chronic Cu bioavailability model set is robust and can be used in regulatory applications. The updated bioavailability model set is currently used under the European Union Registration, Evaluation, Authorisation, and Restriction of Chemicals framework regulation to guide the safe use of Cu. Environ Toxicol Chem 2024;43:450-467. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Chemometric modeling of the lowest observed effect level (LOEL) and no observed effect level (NOEL) for rat toxicity

Humans and other living species of the ecosystem are constantly exposed to a wide range of chemicals of natural as well as synthetic origin.

Pancreatic Antioxidative Defense and Heat Shock Proteins Prevent Islet of Langerhans Cell Death After Chronic Oral Exposure to Cadmium LOAEL Dose.

Cadmium, a hazardous environmental contaminant, is associated with metabolic disease development. The dose with the lowest observable adverse effect level (LOAEL) has not been studied, focusing on its effect on the pancreas. We aimed to evaluate the pancreatic redox balance and heat shock protein (HSP) expression in islets of Langerhans of male Wistar rats chronically exposed to Cd LOAEL doses, linked to their survival. Male Wistar rats were separated into control and cadmium groups (drinking water with 32.5 ppm CdCl2). At 2, 3, and 4 months, glucose, insulin, and cadmium were measured in serum; cadmium and insulin were quantified in isolated islets of Langerhans; and redox balance was analyzed in the pancreas. Immunoreactivity analysis of p-HSF1, HSP70, HSP90, caspase 3 and 9, and cell survival was performed. The results showed that cadmium exposure causes a serum increase and accumulation of the metal in the pancreas and islets of Langerhans, hyperglycemia, and hyperinsulinemia, associated with high insulin production. Cd-exposed groups presented high levels of reactive oxygen species and lipid peroxidation. An augment in MT and GSH concentrations with the increased enzymatic activity of the glutathione system, catalase, and superoxide dismutase maintained a favorable redox environment. Additionally, islets of Langerhans showed a high immunoreactivity of HSPs and minimal immunoreactivity to caspase associated with a high survival rate of Langerhans islet cells. In conclusion, antioxidative and HSP pancreatic defense avoids cell death associated with Cd accumulation in chronic conditions; however, this could provoke oversynthesis and insulin release, which is a sign of insulin resistance.

Toxicological Assessment of Higher Olefins in OECD TG 422 Repeated Dose and Reproductive /Developmental Toxicity Screening Tests in Han Wistar Rats.

Higher olefins (HO) are used primarily as intermediates in the production of other chemicals, such as polymers, fatty acids, plasticizer alcohols, surfactants, lubricants, amine oxides, and detergent alcohols. The potential toxicity of five HO (i.e., 1-Octene, Nonene, Decene, Hexadecene, and 1-Octadecene) with carbon ranging from C8 to C18 was examined in a combined repeated dose and reproduction/developmental toxicity screening study (OECD TG 422). These five HO were administered to Han Wistar rats by gavage at 0 (controls), 100, 300, and 1000mg/kg bw/day. As a group of substances, adaptive changes in the liver (liver weight increase without pathological evidence), as well as increased kidney weight in male rats, were observed in HO with carbon numbers from C8 to C10. The overall systemic no observed adverse effect level (NOAEL) for all HO was determined at 1000mg/kg bw/day. In the reproductive/developmental toxicity assessment, offspring viability, size, and weights were reduced in litters from females treated with Nonene at 1000mg/kg bw/day. The overall no observed effects level (NOEL) for reproductive toxicity was considered to be 300mg/kg bw/day for Nonene and 1000mg/kg bw/day for the other four HO, respectively. These data significantly enrich the database on the toxicity of linear and branched HO, allowing comparison with similar data published on a range of linear and branched HO. Comparisons between structural class and study outcome provide further supportive data in order to validate the read-across hypothesis as part of an overall holistic testing strategy.

3D Airway Epithelial-Fibroblast Biomimetic Microfluidic Platform to Unravel Engineered Nanoparticle-Induced Acute Stress Responses as Exposome Determinants.

Insights into how biological systems respond to high- and low-dose acute environmental stressors are a fundamental aspect of exposome research. However, studying the impact of low-level environmental exposure in conventional in vitro settings is challenging. This study employed a three-dimensional (3D) biomimetic microfluidic lung-on-chip (μLOC) platform and RNA-sequencing to examine the effects of two model anthropogenic engineered nanoparticles (NPs): zinc oxide nanoparticles (Nano-ZnO) and copier center nanoparticles (Nano-CCP). The airway epithelium exposed to these NPs exhibited dose-dependent increases in cytotoxicity and barrier dysregulation (dominance of the external exposome). Interestingly, even nontoxic and low-level exposure (10 μg/mL) of the epithelium compartment to Nano-ZnO triggered chemotaxis of lung fibroblasts toward the epithelium. An increase in α smooth muscle actin (α-SMA) expression and contractile activity was also observed in these cells, indicating a bystander-like adaptive response (dominance of internal exposome). Further bioinformatics and network analysis showed that a low-dose Nano-ZnO significantly induced a robust transcriptomic response and upregulated several hub genes associated with the development of lung fibrosis. We propose that Nano-ZnO, even at a no observable effect level (NOEL) dose according to conventional standards, can function as a potent nanostressor to disrupt airway epithelium homeostasis. This leads to a cascade of profibrotic events in a cross-tissue compartment fashion. Our findings offer new insights into the early acute events of respiratory harm associated with environmental NPs exposure, paving the way for better exposomic understanding of this emerging class of anthropogenic nanopollutants.

Safety and Toxicology Study of Hu7691, a Novel AKT Inhibitor, following Oral Administration in Rats.

Hu7691 represents a novel Pan-Akt kinase inhibitor, demonstrating excellent selectivity towards non-AGC kinase families and pronounced inhibitory effects on the proliferation of multiple tumor cell lines. However, there is currently a notable absence of in vivo toxicological research evidence concerning Hu7691. This study represents the first investigation into the 14-day repeated-dose toxicity of Hu7691 in male and female Sprague Dawley (SD) rats. Male rats were administered daily doses of 12.5, 50, 100, and 150 mg/kg/day, while female rats received doses of 12.5, 25, 50, and 75 mg/kg/day for 14 consecutive days. Hematological assessments, organ weights, and histopathological examinations revealed corresponding alterations, suggesting potential target organs for toxicity including the spleen, thymus, and gastrointestinal tract. It is worth noting that the test substance may also impact the liver, kidneys, heart, and ovaries. The No Observed Effect Level (NOAEL) was determined to be no greater than 12.5 mg/kg/day. Based on the observed gender-related toxicity differences in preliminary trials, it is recommended that the high dose reference dose for male animals in formal experiments should not be less than 100 mg/kg/day, while for female animals, it should be less than 50 mg/kg/day.

Development of a non-target strategy for evaluation of potential biological effects of inhalable aerosols generated during purposeful room conditioning using an in vitro inhalation model

Objectives: An integrated in vitro inhalation approach was outlined to estimate potential adverse acute inhalation effects of aerosols from commercial nebulizer applications used for purposeful room conditioning such as disinfection, scenting or others. Aerosol characterization, exposure estimation and evaluation of acute biological effects by in vitro inhalation were included to generate dose-response data, allowing for determination of in vitro lowest observable adverse effect levels (LOAELs). Correlation of these to estimates of human lung deposition was included for quantitative in vitro to in vivo extrapolation approach (QIVIVE) for acute effects during human exposure.Methods: To test the proposed approach, a case study was undertaken using two realistic test materials. An acute in vitro inhalation setup with air-liquid interface A549-cells in an optimized exposure situation (P.R.I.T.® ExpoCube®) was used to expose cells and analysis of relevant biological effects (viability, mitochondrial membrane potential, stress, IL-8 release) was carried out. Results: The observed dose-responsive effects in a sub-toxic dose-range could be attributed to the main component of one test material and its presence in the aerosol phase of the nebulized material. QIVIVE resulted in a factor of at least 256 between the in vitro LOAEL and the estimated acute human lung exposure for this test material.Conclusions: The case-study shows the value of the non-target in vitro inhalation testing approach especially in case of a lack of knowledge on complex product composition. It is expected that approaches like this will be of high value for product safety and environmental health in the future.

Determination of no observable effect level of nanoplastics on intestinal flora

The risk of human exposure to nanoplastics (NPs) is increasing due to the incomplete decomposition and accumulation of plastic debris in the environment. Dietary intake is the most common way for human body to ingest NPs. NPs cannot degrade in vivo and will accumulate in multiple tissues, which result in various tissue damages. Although numerous studies have focused on evaluating the influences of exposure to NPs, the exposure amounts investigated were much higher than the actual exposure level in human daily life. Therefore, it is necessary to evaluate the hazards of exposure to low-dose NPs. Intestinal flora is a generally recognized sensitive indicator for NPs exposure, so determining a safe threshold to intestinal flora can represent the maximal no-effective dose for the human body of NPs exposure. Based on these information, mice were exposed to NPs at concentrations of 0, 0.001, 0.005, 0.01 and 0.1 mg kg−1·BW, respectively. The composition changes of the intestinal flora were investigated after exposing 28 days. The obtained consequences indicated that 0.01 mg kg−1·BW was the maximal no-effective dose to intestinal flora. This study provided a clear safe threshold for investigating the potential adverse effects of low-dose NPs exposures on human health.

Mitochondrial dysfunction and oxidative stress contribute to cross-generational toxicity of benzo(a)pyrene in Danio rerio

The potential for polycyclic aromatic hydrocarbons (PAHs) to have adverse effects that persist across generations is an emerging concern for human and wildlife health. This study evaluated the role of mitochondria, which are maternally inherited, in the cross-generational toxicity of benzo(a)pyrene (BaP), a model PAH and known mitochondrial toxicant. Mature female zebrafish (F0) were fed diets containing 0, 12.5, 125, or 1250 μg BaP/g at a feed rate of 1% body weight twice/day for 21 days. These females were bred with unexposed males, and the embryos (F1) were collected for subsequent analyses. Maternally-exposed embryos exhibited altered mitochondrial function and metabolic partitioning (i.e. the portion of respiration attributable to different cellular processes), as evidenced by in vivo oxygen consumption rates (OCRs). F1 embryos had lower basal and mitochondrial respiration and ATP turnover-mediated OCR, and increased proton leak and reserve capacity. Reductions in mitochondrial DNA (mtDNA) copy number, increases in mtDNA damage, and alterations in biomarkers of oxidative stress were also found in maternally-exposed embryos. Notably, the mitochondrial effects in offspring occurred largely in the absence of effects in maternal ovaries, suggesting that PAH-induced mitochondrial dysfunction may manifest in subsequent generations. Maternally-exposed larvae also displayed swimming hypoactivity. The lowest observed effect level (LOEL) for maternal BaP exposure causing mitochondrial effects in offspring was 12.5 µg BaP/g diet (nominally equivalent to 250 ng BaP/g fish). It was concluded that maternal BaP exposure can cause significant mitochondrial impairments in offspring.

Multi-endpoint analysis of cadmium chloride-induced genotoxicity shows role for reactive oxygen species and p53 activation in DNA damage induction, cell cycle irregularities, and cell size aberrations.

Cadmium chloride (CdCl2) is a known genotoxic carcinogen, with a mechanism of action thought to partly involve the generation of reactive oxygen species (ROS). We applied here a multi-endpoint approach in vitro to explore the impact of CdCl2 on both the genome and on wider cell biology pathways relevant to cancer. Multi-endpoint approaches are believed to offer greater promise in terms of understanding the holistic effects of carcinogens in vitro. This richer understanding may help better classification of carcinogens as well as allowing detailed mechanisms of action to be identified. We found that CdCl2 caused DNA damage [micronuclei (MN)] in both TK6 and NH32 cells in a dose-dependent manner after 4 h exposure (plus 23 h recovery), with lowest observable effect levels (LOELs) for MN induction of 1 μM (TK6) and 1.6 μM (NH32). This DNA damage induction in TK6 cells was ROS dependent as pretreatment with the antioxidant N-Acetyl Cysteine (1 mM), abrogated this effect. However, 2',7'-dichlorofluorescin diacetate was not capable of detecting the ROS induced by CdCl2. The use of NH32 cells allowed an investigation of the role of p53 as they are a p53 null cell line derived from TK6. NH32 showed a 10-fold increase in MN in untreated cells and a similar dose-dependent effect after CdCl2 treatment. In TK6 cells, CdCl2 also caused activation of p53 (accumulation of total and phosphorylated p53), imposition of cell cycle checkpoints (G2/M) and intriguingly the production of smaller and more eccentric (elongated) cells. Overall, this multi-endpoint study suggests a carcinogenic mechanism of CdCl2 involving ROS generation, oxidative DNA damage and p53 activation, leading to cell cycle abnormalities and impacts of cell size and shape. This study shows how the integration of multiple cell biology endpoints studied in parallel in vitro can help mechanistic understanding of how carcinogens disrupt normal cell biology.

Diclofenac prodrugs nanoparticles: An alternative and efficient treatment for rheumatoid arthritis?

We have synthesized new lipidic prodrugs of diclofenac by grafting aliphatic chains (C10, C12, C16 and C18) to diclofenac through an ester bond. Their molecular formulas were confirmed through HR-MS and the formation of ester bond by FTIR and NMR spectroscopy. Nanoparticles of the different prodrugs were successfully formulated using emulsion evaporation method and DSPE-PEG2000 as the only excipient. All nanoparticles were spherical and had a size between 110 and 150 nm, PdI ≤ 0.2 and negative Zeta potential values from −30 to −50 mV. In addition, they were stable upon storage at 4 °C up to 30–35 days. The encapsulation efficiency of the prodrug was above 90 % independently of the aliphatic chain length grafted. Nanoparticles did not induce any toxicity on LPS-activated THP-1 cells up to a concentration of 100 μg/mL (equivalent diclofenac) whereas diclofenac sodium salt IC50 was around 20 μg/mL. Following incubation of nanoparticles with LPS-activated THP-1 cells, a dose dependent inhibition of TNF-α was observed comparable to standard diclofenac sodium. Based on in vitro studies representative nanoparticles, Prodrug 3 NPs (C16 aliphatic chain) were selected for further in vitro and in vivo studies. Upon incubation in murine plasma, Prodrug 3 NPs underwent an enzymatic cleavage and almost 70 % of diclofenac was released from nanoparticles in 8 h. In vivo studies on a collagen induced arthritis murine model showed contrasted results: on one hand Prodrug 3 NPs led to a significant decrease of arthritis score and of paw volume compared to PBS after the second injection, on the other hand the third injection induced an important hepatic toxicity with the death of half of the mice from the NP group. To promote the reduction of inflammation while avoiding hepatic toxicity using NPs would require to precisely study the No Observable Adverse Effect Level and the schedule of administration in the future.

The effect of long-term exposure to nonylphenol at environmentally relevant levels on mouse liver and adipose tissue

Exposure to the xenoestrogen nonylphenol (NP) during critical windows of development leads to metabolic abnormalities in adult life. However, less is known about NP exposure outside the developmental period on metabolic outcomes. We investigated the effect of prolonged exposure to NP after sexual maturity and at environmentally relevant concentrations below the ‘no observable adverse effects level’ (0.5 and 2.5 mg/kg/d). Male Swiss mice fed a normal-fat diet exposed to 2.5 mg/kg/d NP showed reduced weight gain and hepatic fat content. In male and female C57BL/6 mice fed a high-fat diet, NP exposure modified the mRNA levels of estrogen receptor α (Esr1) and adipose lineage markers in a sexually dimorphic and adipose depot-dependent pattern. Moreover, in primary female but not male stromal vascular cells from C57BL/6 mouse inguinal WAT induced to differentiate into adipocytes, NP upregulated Fabp4 expression. Low-level exposure to NP outside critical developmental windows may affect the metabolic phenotype distinctly. Data availability statementAll data not included in the manuscript, such as raw results, are available upon request and should be addressed to AAA.