Unveiling complex responses at the molecular level: Transcriptional alterations by mixtures of bisphenol A, octocrylene, and 2'-ethylhexyl 4- (dimethylamino)benzoate on Chironomus riparius.

Living organisms are exposed to mixtures of pollutants in the wild. Inland aquatic ecosystems contain many compounds from different sources that pollute the water column and the sediment. However, majority of toxicological research is focused on the effects of single exposures to toxicants. Furthermore, studies have been principally oriented toward ecologically relevant effects of intoxication, and lack an analysis of the cellular and molecular mechanisms involved in the response to toxicants. Effects of single, binary, and ternary mixtures of three compounds, bisphenol A, octocrylene, and 2'-ethylhexyl 4- (dimethylamino)benzoate, were assessed using a Real-Time PCR array. Forty genes, and additional six reference genes, were included in the array. The genes were selected based on their association with hormone responses, detoxification mechanisms, the stress response, DNA repair, and the immune system. The study was performed on Chironomus riparius, a benthic dipteran with an essential role in the food web. Transcriptional responses were assessed both 24 and 96 h post-exposure, to determinate short- and medium-term cellular responses. Individual fourth instar larvae were exposed to 0.1 and 1 mg/L of each of the toxic compounds and compound mixtures. A weak response was detected at 24 h, which was stronger in larvae exposed to mixtures than to individual toxicants. The response at 96 h was complex and principally involved genes related to the endocrine system, detoxification mechanisms, and the stress response. Furthermore, exposure to mixtures of compounds altered the expression patterns of an increased number of genes than did individual compound exposures, which suggested complex interactions between compounds affected the regulation of transcriptional activity. The results obtained highlight the importance of analyzing the mechanisms involved in the response to mixtures of compounds over extended periods and offer new insights into the basis of the physiological responses to pollution.

The PDE4 inhibitor CHF6001 affects keratinocyte proliferation via cellular redox pathways.

Psoriasis is a skin disease characterized by abnormal keratinocyte proliferation and inflammation. Currently, there are no cures for this disease, so the goal of treatment is to decrease inflammation and slow down the associated rapid cell growth and shedding. Recent advances have led to the usage of phosphodiesterase 4 (PDE4) inhibitors for treatment of this condition. For example, apremilast is an oral, selective PDE4 inhibitor that is able to reduce skin inflammation and is Food and Drug Administration (FDA)-approved to treat adults with moderate to severe psoriasis and/or psoriatic arthritis. However, common target-related adverse events, including diarrhea, nausea, headache, and insomnia limit the usage of this drug. To circumvent these effects, the usage of PDE4 inhibitors specifically designed for topical treatment, such as CHF6001, may combine local anti-inflammatory activity with limited systemic exposure, improving tolerability. In this study, we showed that CHF6001, currently undergoing clinical development for COPD, suppresses human keratinocyte proliferation as assessed via BrdU incorporation. We also observed decreased re-epithelialization in a scratch-wound model after CHF6001 treatment. At the molecular level, CHF6001 inhibited translocation of phosphorylated NF-κB subunit p65, promoting loss of nuclear cyclin D1 accumulation and an increase of cell cycle inhibitor p21. Furthermore, CHF6001 decreased oxidative stress, measured by assessing lipid peroxidation (4-HNE adduct formation), through the inactivation of the NADPH oxidase. These results suggest that CHF6001 has the potential to treat skin disorders associated with hyperproliferative keratinocytes, such as psoriasis by targeting oxidative stress, abnormal re-epithelization, and inflammation.

Design and development of novel p-aminobenzoic acid derivatives as potential cholinesterase inhibitors for the treatment of Alzheimer's disease.

Based on the quantitative structure-activity relationship (QSAR), some novel p-aminobenzoic acid derivatives as promising cholinesterase enzyme inhibitors were designed, synthesized, characterized and evaluated to enhance learning and memory. The in vitro enzyme kinetic study of the synthesized compounds revealed the type of inhibition on the respective acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes. The in vivo studies of the synthesized compounds exhibited significant reversal of cognitive deficits in the animal models of amnesia as compared to standard drug donepezil. Further, the ex vivo studies in the specific brain regions like the hippocampus, hypothalamus, and prefrontal cortex regions also exhibited AChE inhibition comparable to standard donepezil. The in silico molecular docking and dynamics simulations studies of the most potent compound 22 revealed the consensual interactions at the active site pocket of the AChE.

Effects of single exposure and binary mixtures of ultraviolet filters octocrylene and 2-ethylhexyl 4-(dimethylamino) benzoate on gene expression in the freshwater insect Chironomus riparius.

Ultraviolet filters are used extensively in the production of many personal care and industrial products. These products can inadvertently pollute the environment through recreational activities. They have been associated with endocrine disruption in vertebrates but their effects in invertebrates are poorly understood. Chironomus riparius is a species of the dipteran order, with aquatic larvae that are frequently used in toxicity tests. Previously, we showed that octocrylene (OC) and 2-ethylhexyl 4-(dimethylamino) benzoate (OD-PABA) differentially affected the mRNA levels of the ecdysone receptor and Hsp70 genes. For a better understanding of their mode of action, transcriptional activity by real-time PCR was analyzed in fourth instar larvae exposed to OC, OD-PABA, or a binary mixture of both. We studied 16 genes related to the endocrine system, stress, the immune system, and biotransformation mechanisms to elucidate the putative interactions between these compounds. No response was observed for the genes involved in biotransformation, suggesting that enzymes other than cytochromes P450 and glutathione-S-transferases (GSTs) could get involved in transformation of these compounds. Similarly, no response was observed for endocrine-related genes while the stress gene HYOU1 was inhibited by OD-PABA, suggesting an effect in response to hypoxia. In addition, no significant interactions were observed following exposure to a binary mixture of these compounds. Overall, the results suggest a weak, acute response in different metabolic pathways and a lack of interaction between the compounds. Finally, new genes are identified in this organism, opening the possibility to analyze new cellular pathways as targets of toxicants.

Synthesis, Structural Characterization, and Antiangiogenic Activity of Polyfluorinated Benzamides.

The introduction of fluorine into bioactive molecules is a matter of importance in medicinal chemistry. In this study, representatives of various chemical entities of fluoroaromatic compounds were synthesized. Depending on the reaction conditions, either tetrafluorophthalimides or ammonium tetrafluorophthalamates are accessible from tetrafluorophthalic anhydride and primary amines. Tetrafluorophthalamic acids undergo thermal decarboxylation to yield tetrafluorobenzamides. These could be successfully converted upon treatment with primary amines, in the course of an aromatic nucleophilic substitution, to 2,3,5-trifluorobenzamides with respective amino substituents at the 4-position. The five structure types were characterized by means of spectroscopic and crystallographic methods. The synthesized compounds were evaluated as inhibitors of angiogenesis by measuring microvessel outgrowth in a rat aortic ring assay. The biological activity was maintained throughout these different polyfluorinated chemotypes.

Evaluation of the endocrine-disrupting effects of homosalate (HMS) and 2-ethylhexyl 4-dimethylaminobenzoate (OD-PABA) in rat pups during the prenatal, lactation, and early postnatal periods.

Homosalate (HMS) and 2-ethylhexyl 4-dimethylaminobenzoate (OD-PABA) are ultraviolet filters. We aimed to investigate the effects of dermal exposure to HMS and OD-PABA during the prenatal, lactation, and early infancy periods on pubertal development and thyroid function in male and female rats. The thyroid glands, uteri, testes, prostate glands, and seminal vesicles were excised and weighed, the reproductive organs were analyzed histologically, and the serum hormone levels were measured. In the prenatal period, the thyroxine (T4) levels increased in the female rats in the exposed groups ( p < 0.05); the thyroid weights, reproductive organ weights, and gonadal hormone levels were not altered. In males, the testosterone levels decreased ( p < 0.05), but the thyroid weights, T4 levels, prostate, and testis weights were not changed. In the lactation period, the weights of the thyroid glands increased in the exposed female groups ( p < 0.05), but the T4, gonadal hormone levels, and reproductive organ weights were not changed. In the males, the thyroid gland weights, T4 levels, reproductive organ weights, and gonadal hormone levels were not changed. During infancy, the thyroid gland weights increased in the female rats in the exposed groups ( p < 0.05), but the T4 levels, gonadal hormone levels, and reproductive organ weights were not affected. In the male rats in the exposed groups, the T4 levels were increased ( p < 0.05), but the thyroid and reproductive organ weights, gonadal hormone levels were not affected. Organ histopathology was not affected in all groups. HMS and OD-PABA do not have endocrine disruptor effects on thyroid function and the pubertal development of female and male rats.

Ecotoxicological Evaluation of the UV Filters Ethylhexyl Dimethyl p-Aminobenzoic Acid and Octocrylene Using Marine Organisms Isochrysis galbana, Mytilus galloprovincialis and Paracentrotus lividus.

The growing concern regarding the negative effects of solar radiation on the skin has led to a drastic increase in the use of sunscreens containing in its composition up to 10% of aromatic chemicals, such as ethylhexyl dimethyl p-aminobenzoic acid (OD-PABA) and octocrylene (OC). The objective of this study was to evaluate the toxicity and to assess the environmental risk posed by these two ultraviolet filters, widely used in cosmetics and as plastic additives, in the marine environment. Several ecotoxicological bioassays were performed with three model organisms belonging to different trophic levels: the microalgae Isochrysis galbana, the mussel Mytilus galloprovincialis, and the sea urchin Paracentrotus lividus. The results show remarkable toxicity to marine species for both OD-PABA (EC10 values range 26,5-127 µg L-1) and OC (EC10 range 103-511 µg L-1). The cell division in the microalgae I. galbana was the most sensitive endpoint tested. To determine the environmental risk of these substances, the risk coefficient (RQ) was calculated. Due to the higher concentrations reported, OC showed remarkable risk (RQ = 0.27), whereas for OD-PABA the risk was low (RQ = 0.007).

Bioconcentration and multi-biomarkers of organic UV filters (BM-DBM and OD-PABA) in crucian carp.

Organic UV filters (OUV-Fs) are increasingly used in sunscreens and personal care products. In the present work, the bioconcentration and multi-biomarker effects of butyl methoxydibenzoylmethane (BM-DBM) and ethylhexyl dimethyl p-aminobenzoate (OD-PABA) were investigated in crucian carp (Carassius auratus). The fish were exposed to various concentrations of BM-DBM (3.88, 35.61, 181.85 and 337.15µg/L), OD-PABA (4.66, 53.83, 264.22 and 459.32µg/L) and their mixture (2.31+2.79, 23.69+26.18, 97.37+134.81 and 193.93+246.08µg/L) for 28 days. The maximal concentrations of two OUV-Fs were detected in the fish liver, followed by the brain, kidney, gill and muscle in most cases. The maximal BCF values of OD-PABA calculated in various exposure concentrations were 0.37 - 101.21 in single exposure groups and 0.11 - 31.09 in mixed exposure groups. Acetylcholinesterase (AChE) activity was significantly inhibited by BM-DBM as well as the mixtures at all of the exposure concentrations and by OD-PABA at higher concentrations (≥264.22µg/L) during 28 days of exposure. The maximal inhibition rates of AChE activity reached 64.04% for BM-DBM, 41.05% for OD-PABA and 61.50% for the mixtures at the highest concentration, which indicated that these two OUV-Fs might damage the central nervous system. Concerning oxidative stress status, BM-DBM and the mixtures significantly increased superoxide dismutase (SOD) and glutathione reductase (GR) activities and inhibited catalase (CAT) activity, while OD-PABA caused a significant increase of GR and CAT activities. AChE and GR activities seemed to be more sensitive biomarkers for BM-DBM and OD-PABA.

Mutagenicity and DNA-damaging potential of clenbuterol and its metabolite 4-amino-3,5-dichlorobenzoic acid in vitro.

The aim of this study was to evaluate in vitro toxicity of clenbuterol and its metabolite 4-amino-3,5-dichlorobenzoic acid. Cytotoxicity and pro-oxidative effect of both compounds were studied on human colon adenocarcinoma cell line SW 480. No significant cytotoxic effect of either compound was observed. Results of an Ames test on Salmonella typhimurium did not indicate mutagenic activity of clenbuterol on TA 98 and TA 100 strains, regardless of metabolic activation. Potential mutagenic effects of the highest clenbuterol concentration (2500 ng/ml) were observed on the TA 1535 strain. The obtained results of alkaline comet assay on isolated human lymphocytes suggested that both compounds induced an increase of primary DNA damage in a concentration-dependent manner. 4-ADBA was a slightly more potent inducer of primary DNA damage as compared to clenbuterol. Chromosomal aberration analysis showed that clenbuterol caused a statistically significant increase in the total number of aberrant cells only at the highest concentration tested (3% vs. 0.7% in the negative control). The results of this study might represent a solid frame for designing and planning future studies with both compounds, which should further clarify their mechanisms of action and genotoxic/cytogenetic effects relevant for human risk assessment.

Transdentinal cytotoxicity of experimental adhesive systems of different hydrophilicity applied to ethanol-saturated dentin.

The aim of this study was to evaluate the transdentinal cytotoxicity of experimental adhesive systems (EASs) with different hydrophilicity and dentin saturation solutions on odontoblast-like cells. One hundred 0.4-mm-thick dentin discs were mounted in in vitro pulp chambers and assigned to 10 groups. MDPC-23 cells were seeded onto the pulpal side of the discs, incubated for 48h. The EASs with increasing hydrophilicity (R1, R2, R3 and R4) were applied to the occlusal side after etching and saturation of etched dentin with water or ethanol. R0 (no adhesive) served as controls. R1 is a non-solvated hydrophobic blend, R2 is similar to a simplified etch-and-rinse adhesive system and R3 and R4 are similar to self-etching adhesives. After 24h, cell metabolism was evaluated by MTT assay (n=8 discs) and cell morphology was examined by SEM (n=2 discs). Type of cell death was identified by flow cytometry and the degree of monomer conversion (%DC) was determined by infrared spectroscopy (FTIR) after 10s or 20s of photoactivation. Data were analyzed by the Kruskal-Wallis and Mann-Whitney tests (α=0.05). Dentin saturation with ethanol resulted in higher necrotic cell death ratios for R2, R3 and R4 compared with water saturation, although R2 and R3 induced higher SDH production. Photoactivation for 20s significantly improved the %DC of all EASs compared with 10s. A significant positive correlation was observed between the degree of hydrophilicity and %DC. In conclusion, except for R1, dentin saturation with ethanol increased the cytotoxicity of EASs, as expressed by the induction of necrotic cell death.