Limits of the regulatory evaluation of resorcinol as a thyroid disruptor: When limited experimental data challenge established effects in humans.

Severe hypothyroidism has been reported in humans during resorcinol therapeutic use. However, available data highlight differences in the severity of resorcinol-induced thyroid effects between humans and rodents, leading to a debate on the relevance of human data for its classification as a thyroid disruptor. The aim of this review is to illustrate some of the limitations of the evaluation framework for thyroid disrupters using resorcinol as a case study of a chemical with clear thyroid-disrupting properties in humans that could not have been identified solely from regulatory studies on animals. The reliability of human data has been called into question due to the specific exposure patterns in humans and the paucity of robust toxicokinetic data. In humans, therapeutic use of resorcinol induces severe hypothyroidism, but in rodents, thyroid disruption is limited to decreased thyroxine concentrations and histological changes in the thyroid. The adverse effects of thyroid disruption, such as impaired neurodevelopment, have not been sufficiently investigated, and experimental neurobehavioral data for resorcinol remain scarce and inconclusive. Although regulatory toxicological evaluations have not included in-depth investigations of thyroid regulation and related adverse effects, they have been used to challenge the relevance of human data. Resorcinol is an emblematic example of how the framework for regulatory evaluations of thyroid disruptors relies almost exclusively on animal studies which may not be suitable for assessing thyroid disruption. This review highlights the need to revise regulatory guidelines and to adopt strategies based on up-to-date, scientifically sound approaches to identify thyroid disruptors. The limits of the current regulatory framework for identifying thyroid disruptors can lead to opposing positions between regulatory bodies. The French Agency for Food, Environmental and Occupational Health & Safety (ANSES)'proposal to identify resorcinol as a "substance of very high concern" due to its ED properties has not been adopted by the European instances.

Study on the differences in sludge toxicity and microbial community structure caused by catechol, resorcinol and hydroquinone with metagenomic analysis.

The aerobic biodegradation rate, organic toxicity and microbial community structure of activated sludge acclimated by catechol, resorcinol and hydroquinone were investigated, to study the relationship between microbial structure and sludge organic toxicity caused by phenolic compounds. At the stable operation stage, the degradation rates of the dihydroxy benzenes in a single sequencing batch reactor (SBR) cycle were followed the order: resorcinol (89.71%) > hydroquinone (85.64%) > catechol (59.62%). Sludge toxicity bioassay indicated that the toxicity of sludge was catechol (45.63%) > hydroquinone (40.28%) > resorcinol (38.15%). The accumulation of secondary metabolites such as 5-10 kDa tryptophan and tyrosine protein substances caused the differential sludge toxicity. Microbial metagenomic analysis showed that the toxicity of sludge was significantly related to the microbial community structure. Thauera, Azoarcus, Pseudomonas and other Proteobacteria formed in the sludge during acclimation. Catechol group had the least dominant bacteria and loop ring opening enzyme genes (catA, dmpB, dxnF, hapD) numbers. Therefore, the degradation of catechol was the most difficult than resorcinol and hydroquinone, resulting the highest sludge toxicity.

AUY922 induces retinal toxicity through attenuating TRPM1.

BACKGROUND: Ocular adverse events are common dose-limiting toxicities in cancer patients treated with HSP90 inhibitors, such as AUY922; however, the pathology and molecular mechanisms that mediate AUY922-induced retinal toxicity remain undescribed. METHODS: The impact of AUY922 on mouse retinas and cell lines was comprehensively investigated using isobaric tags for relative and absolute quantitation (iTRAQ)­based proteomic profiling and pathway enrichment analysis, immunohistochemistry and immunofluorescence staining, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, MTT assay, colony formation assay, and western blot analysis. The effect of AUY922 on the Transient Receptor Potential cation channel subfamily M member 1 (TRPM1)-HSP90 chaperone complex was characterized by coimmunoprecipitation. TRPM1-regulated gene expression was analyzed by RNAseq analysis and gene set enrichment analysis (GSEA). The role of TRPM1 was assessed using both loss-of-function and gain-of-function approaches. RESULTS: Here, we show that the treatment with AUY922 induced retinal damage and cell apoptosis, dysregulated the photoreceptor and retinal pigment epithelium (RPE) layers, and reduced TRPM1 expression. Proteomic profiling and functional annotation of differentially expressed proteins reveals that those related to stress responses, protein folding processes, regulation of apoptosis, cell cycle and growth, reactive oxygen species (ROS) response, cell junction assembly and adhesion regulation, and proton transmembrane transport were significantly enriched in AUY922-treated cells. We found that AUY922 triggered caspase-3-dependent cell apoptosis, increased ROS production and inhibited cell growth. We determined that TRPM1 is a bona fide HSP90 client and characterized that AUY922 may reduce TRPM1 expression by disrupting the CDC37-HSP90 chaperone complex. Additionally, GSEA revealed that TRPM1-regulated genes were associated with retinal morphogenesis in camera-type eyes and the JAK-STAT cascade. Finally, gain-of-function and loss-of-function analyses validated the finding that TRPM1 mediated the cell apoptosis, ROS production and growth inhibition induced by AUY922. CONCLUSIONS: Our study demonstrates the pathology of AUY922-induced retinal toxicity in vivo. TRPM1 is an HSP90 client, regulates photoreceptor morphology and function, and mediates AUY922-induced cytotoxicity.

Comparison of the metabolism of 10 cosmetics-relevant chemicals in EpiSkin™ S9 subcellular fractions and in vitro human skin explants.

An understanding of the bioavailability of topically applied cosmetics ingredients is key to predicting their local skin and systemic toxicity and making a safety assessment. We investigated whether short-term incubations with S9 from the reconstructed epidermal skin model, EpiSkin™, would give an indication of the rate of chemical metabolism and produce similar metabolites to those formed in incubations with human skin explants. Both have advantages: EpiSkin™ S9 is a higher-throughput assay, while the human skin explant model represents a longer incubation duration (24 hours) model integrating cutaneous distribution with metabolite formation. Here, we compared the metabolism of 10 chemicals (caffeine, vanillin, cinnamyl alcohol, propylparaben, 4-amino-3-nitrophenol, resorcinol, 4-chloroaniline, 2-amino-3-methyl-3H-imidazo[4,5-F]quinoline and 2-acetyl aminofluorene) in both models. Both models were shown to have functional Phase 1 and 2 enzymes, including cytochrome P450 activities. There was a good concordance between the models with respect to the level of metabolism (stable vs. slowly vs. extensively metabolized chemicals) and major early metabolites produced for eight chemicals. Discordant results for two chemicals were attributed to a lack of the appropriate cofactor (NADP+ ) in S9 incubations (cinnamyl alcohol) and protein binding influencing chemical uptake in skin explants (4-chloroaniline). These data support the use of EpiSkin™ S9 as a screening assay to provide an initial indication of the metabolic stability of a chemical applied topically. If required, chemicals that are not metabolized by EpiSkin™ S9 can be tested in longer-term incubations with in vitro human explant skin to determine whether it is slowly metabolized or not metabolized at all.

Bioactive cytosporone derivatives isolated from the mangrove-derived fungus Dothiorella sp. ML002.

Three new cytosporone derivatives dothiorelones K-M (1, 2 and 7), together with six known ones (3-6, 8 and 9) were isolated from the mangrove-derived fungus Dothiorella sp. ML002. Their structures were determined by comprehensive 1D, 2D NMR spectroscopic and HR-ESI-MS spectroscopic data. Compounds 1, 2 and 5 displayed inhibitory activities against α-glucosidase with the IC50 values of 22.0, 77.9 and 5.4 µg/mL, respectively. Additionally, compounds 1, 2, and 5 also exhibited antibacterial activities against Staphylococcus aureus (ATCC 6538) with the same MIC values of 50 µg/mL, respectively. The results indicated that cytosporone derivatives will be useful to as diabetes control agents.

Simultaneous removal of dihydroxybenzenes and toxicity reduction by Penicillium chrysogenum var. halophenolicum under saline conditions.

The dihydroxybenzenes are widely found in wastewater and usually more than one of these aromatic compounds co-exist as pollutants of water resources. The current study investigated and compared the removal efficiency of hydroquinone, catechol and resorcinol in binary substrate systems under saline conditions by Penicillium chrysogenum var. halophenolicum, to clarify the potential of this fungal strain to degrade these aromatic compounds. Since P. chrysogenum is a known penicillin producer, biosynthetic penicillin genes were examined and antibiotic was quantified in mono and binary dihydroxybenzene systems to elucidate the carbon flux of dihydroxybenzenes metabolism in the P. chrysogenum var. halophenolicum to the secondary metabolism. In binary substrate systems, the three assayed dihydroxybenzene compounds were found to be co-metabolized by fungal strain. The fungal strain preferentially degraded hydroquinone and catechol. Resorcinol was degraded slower and supports higher antibiotic titers than either catechol or hydroquinone. Dihydroxybenzenes were faster removed in mixtures compared to mono substrate systems, except for the case of hydroquinone. In this context, the expression of penicillin biosynthetic gene cluster was not related to the removal of dihydroxybenzenes. Penicillin production was triggered simultaneously or after dihydroxybenzene degradation, but penicillin yields, under these conditions, did not compromise dihydroxybenzene biological treatment. To investigate the decrease in dihydroxybenzenes toxicity due to the fungal activity, viability tests with human colon cancer cells (HCT116) and DNA damage by alkaline comet assays were performed. For all the conditions assays, a decrease in saline medium toxicity was observed, indicating its potential as detoxification agent.

A novel method to generate monocyte-derived dendritic cells during coculture with HaCaT facilitates detection of weak contact allergens in cosmetics.

The in vitro sensitization assay LCSA (Loose-fit Coculture-based Sensitization Assay) has proved reliable for the detection of contact sensitizers in the past. However, the coculture of human monocyte-derived dendritic cells (DCs) with primary human keratinocytes (KCs) in serum-free medium is relatively complex compared to other sensitization assays which use continuous cell lines. To facilitate high-throughput screening of chemicals, we replaced KCs with the HaCaT cell line under various culture conditions. Coculture of HaCaT with peripheral blood mononuclear cells in serum-supplemented medium leads to generation of CD1a+/CD1c+ DCs after addition of GM-CSF, IL-4, and TGF-ß1 (as opposed to CD1a-/CD1c- DCs which arise in the "classic" LCSA coculture). These cells resemble monocyte-derived DCs generated in monoculture, but, unlike those, they show a marked upregulation CD86 after treatment with contact allergens. All of the nine sensitizers in this study were correctly identified by CD1a+/CD1c+ DCs in coculture with HaCaT. Among the substances were weak contact allergens such as propylparaben (which is false negative in the local lymph node assay in mice) and resorcinol (which was not detected by CD1a-/CD1c- DCs in the "classic" LCSA). The level of CD86 upregulation on CD1a+/CD1c+ DCs was higher for most allergens compared to CD1a-/CD1c- DCs, thus improving the assay's discriminatory power. Three out of four non-sensitizers were also correctly assessed by the coculture assay. A false-positive reaction to caprylic (octanoic) acid confirms earlier results that some fatty acids are able to induce CD86 on DC in vitro. In conclusion, change of the LCSA protocol led to reduction of time and cost while even increasing the assay's sensitivity and discriminatory power.

Ocular toxicity of AUY922 in pigmented and albino rats.

AUY922, a heat shock protein 90 inhibitor is associated with ocular adverse events (AEs). To provide a better understanding of ocular AEs in patients, 4 investigative studies were performed in a step-wise approach to assess retinal structure and function in pigmented (Brown Norway) and albino (Wistar) rats. In rats administered 30mg/kg of AUY922, the AUC0-24h and Cmax are comparable to that in patients at 70mg/m(2). AUY922 at ≥30mg/kg was poorly tolerated by rats with morbidity or mortality generally after the third weekly treatment. Electroretinography (ERG) changes were observed at doses ≥30mg/kg. The ERG changes were dose dependent, consistent with an effect on the photoreceptors, and fully reversible. The ERG effects could not be minimized by decreasing the Cmax while maintaining AUC. Histopathological changes were seen mainly when rats were administered AUY922 at 100mg/kg. The 2-hour infusion of AUY922 at 100mg/kg caused disorganization of the outer segment photoreceptor morphology in male Brown Norway rats; the severity of the disorganization increased with the number of administrations, but was reversible during a 4-week posttreatment period. There was no major difference in ocular response between Brown Norway and Wistar rats. No changes in serum iron levels, and no changes in rhodopsin, PDE6α, ß-transducin concentrations, or retinal pigment epithelium-specific protein RPE65 expression were observed after single and multiple infusions of AUY922 at 100mg/kg compared to vehicle-treated controls. AUY922 retinal toxicity in rats recapitulates and further characterizes that reported in patients and is shown to be reversible, while a precise molecular mechanism for the effect was not determined.

A comparison of potency differences among thyroid peroxidase (TPO) inhibitors to induce developmental toxicity and other thyroid gland-linked toxicities in humans and rats.

The potencies of resorcinol, 6-propylthiouracil (PTU) and methimazole (MMI) for inducing developmental toxicity and neurotoxicity were compared in pregnant rats, regarded as valid model for human thyroid toxicity. Profound differences on maternal thyroid hormone levels (THs), maternal toxicity as well as developmental and neurotoxicity sequelae occurred. Resorcinol affected none of those end points. PTU and MMI caused significant effects. Therapy with either PTU or MMI during the first trimester of human pregnancy can cause reductions of maternal THs, accompanied by disruptions of prenatal development. Clinical MMI studies show sporadic evidence of teratogenic effects, with equivocal relation to thyroid peroxidase (TPO) inhibition. In recent decades no MMI associated prenatal toxicity has been reported, an outcome possibly related to carefully managed therapy. Orally administered resorcinol was rapidly absorbed, metabolized and excreted and was undetectable in the thyroid. In contrast, PTU or MMI accumulated. Resorcinol's potency to inhibit TPO was profoundly lower than that of PTU or MMI. Quantum chemical calculations may explain low resorcinol reactivity with TPO. Thus, distinctions in the target organ and the TPO inhibitory potency between these chemicals are likely contributing to different reductions of maternal THs levels and affecting the potency to cause developmental toxicity and neurotoxicity.

Synthesis and biological evaluation of 3,5-disubstituted-4-alkynylisoxozales as a novel class of HSP90 inhibitors.

A series of 3,5-disubstitute-4-alkynylisoxazole derivatives were designed and synthesized through palladium(II)-copper(I) catalyzed Sonogashira cross-coupling reaction of an alkynyl moiety and an isoxazole scaffold as novel HSP90 inhibitors. The resultant compounds displayed moderate to potent binding affinity to HSP90 proteins, and also demonstrated good cell growth inhibitory activity against various cancer cell lines (A549, K562, MCF-7, DU145 and Hela). Some compounds (18d, 18e, 19a, 19d, 20c and 20q) show similar or better binding affinity towards HSP90α and HSP90ß comparing to NVP-AUY922. In addition, compounds 18e, 19a and 20q exhibited potent inhibitory activity against various human cancer cell lines.

In vitro human metabolism of the flame retardant resorcinol bis(diphenylphosphate) (RDP).

Resorcinol bis(diphenylphosphate) (RDP) is widely used as a flame retardant in electrical/electronic products and constitutes a suitable alternative to decabrominated diphenyl ether. Due to its toxicity and its recently reported ubiquity in electronics and house dust, there are increasing concerns about human exposure to this emerging contaminant. With the aim of identifying human-specific biomarkers, the in vitro metabolism of RDP and its oligomers was investigated using human liver microsomes and human liver cytosol. Mono- and dihydroxy-metabolites, together with glucuronidated and sulfated metabolites, were detected. Regarding RDP oligomers, only a hydroxy-metabolite of the dimer could be detected. RDP and its oligomers were also readily hydrolyzed, giving rise to a variety of compounds, such as diphenyl phosphate, para-hydroxy-triphenyl phosphate, and para-hydroxy RDP, which were further metabolized. These degradation products or impurities are possibly of environmental importance in future studies.

Hair dyes resorcinol and lawsone reduce production of melanin in melanoma cells by tyrosinase activity inhibition and decreasing tyrosinase and microphthalmia-associated transcription factor (MITF) expression.

Hair coloring products are one of the most important cosmetics for modern people; there are three major types of hair dyes, including the temporary, semi-permanent and permanent hair dyes. The selected hair dyes (such as ammonium persulfate, sodium persulfate, resorcinol and lawsone) are the important components for hair coloring products. Therefore, we analyzed the effects of these compounds on melanogenesis in B16-F10 melanoma cells. The results proved that hair dyes resorcinol and lawsone can reduce the production of melanin. The results also confirmed that resorcinol and lawsone inhibit mushroom and cellular tyrosinase activities in vitro. Resorcinol and lawsone can also downregulate the protein levels of tyrosinase and microphthalmia-associated transcription factor (MITF) in B16-F10 cells. Thus, we suggest that frequent use of hair dyes may have the risk of reducing natural melanin production in hair follicles. Moreover, resorcinol and lawsone may also be used as hypopigmenting agents to food, agricultural and cosmetic industry in the future.

Exposure to resorcinol bis (diphenyl phosphate) induces colonization of alien microorganisms with potential impacts on the gut microbiota and metabolic disruption in male zebrafish.

Organophosphate esters (OPEs) have been demonstrated to induce various forms of toxicity in aquatic organisms. However, a scarcity of evidence impedes the conclusive determination of whether OPEs manifest sex-dependent toxic effects. Here, we investigated the effects of tris (1-chloro-2-propyl) phosphate (TCPP) and resorcinol bis (diphenyl phosphate) (RDP) on the intestines of both female and male zebrafish. The results indicated that, in comparison to TCPP, RDP induced more pronounced intestinal microstructural damage and oxidative stress, particularly in male zebrafish. 16S rRNA sequencing and metabolomics revealed significant alterations in the species richness and oxidative stress-related metabolites in the intestinal microbiota of zebrafish under exposure to both TCPP and RDP, manifesting gender-specific effects. Based on differential species analysis, we defined invasive species and applied invasion theory to analyze the reasons for changes in the male fish intestinal community. Correlation analysis demonstrated that alien species may have potential effects on metabolism. Overall, this study reveals a pronounced gender-dependent impact on both the intestinal microbiota and metabolic disruptions of zebrafish due to OPEs exposure and offers a novel perspective on the influence of pollutants on intestinal microbial communities and metabolism.

De novo tyrosinase inhibitor: 4-(6,7-dihydro-5H-indeno[5,6-d]thiazol-2-yl)benzene-1,3-diol (MHY1556).

In this study, we have synthesized and studied de novo tyrosinase inhibitor, MHY1556, which showed significantly better efficacy than other pre-existing tyrosinase inhibitors in vitro experiments. The IC50 value of MHY1556 was 0.50µM which was significantly lower than that of kojic acid (IC50=53.95µM), which is a well-known tyrosinase inhibitor and was used as a positive control in this study. We predicted the tertiary structure of tyrosinase, simulated the docking with compound MHY1556 and confirmed that the compound strongly interacts with mushroom tyrosinase residues. Substitutions with a hydroxy group at both R1 and R3 of the phenyl ring indicated that these groups play a major role in the high binding affinity to tyrosinase, especially through the hydrogen bonding interaction of the hydroxyl group at R1 with GLY281. In addition, MHY1556 showed concentration-dependent inhibitory effects in melanin content assay where B16F10 melanoma cells were treated with α-melanocyte stimulating hormone (α-MSH), and also there is no significant cytotoxicity of this compound in cell viability assay conducted in B16F10 melanoma cells. The tyrosinase activity assay results with MHY1556 also support its potent inhibitory effects. Therefore, our data strongly suggest MHY1556 suppresses the melanogenesis via a tyrosinase inhibitory effect.

The defensive function of trichocysts in Paramecium tetraurelia against metazoan predators compared with the chemical defense of two species of toxin-containing ciliates.

The time-honored assumption about the defensive function of trichocysts in Paramecium against predators was recently verified experimentally against different species of unicellular predators. In the present study, we examined the defensive function of trichocysts against three metazoan predators, Cephalodella sp. (Rotifera), Eucypris sp. (Arthropoda), and Stenostomum sphagnetorum (Platyhelminthes). The results confirmed the defensive function of trichocysts against two of these metazoan predators (Cephalodella sp. and Eucypris sp.), while they seem ineffective against S. sphagnetorum. We also compared the defensive efficiency of the trichocysts of P. tetraurelia with that of toxin-containing extrusomes of two ciliates.

Ecotoxicity and screening level ecotoxicological risk assessment of five antimicrobial agents: triclosan, triclocarban, resorcinol, phenoxyethanol and p-thymol.

Acute and chronic (or sub-chronic) toxicity of five selected antimicrobial agents, including triclosan (TCS), triclocarban (TCC), resorcinol, phenoxyethanol and p-thymol, was investigated using the conventional three-aquatic-organism battery. These compounds are widely used in cosmetics and other personal care products and their ecological risk has recently become a significant concern. As results of toxicity tests, TCS was found to be most strongly toxic for green algae [e.g. 72 h no observed effect concentration (NOEC) of 0.50 µg l(-1) ] among the selected compounds, followed by TCC, while TCC was more toxic or similar to TCS for Daphnia and fish (e.g. Daphnia 8 day NOEC of 1.9 µg l(-1) ). Having compared the predicted no effect concentration (PNEC) determined from the toxicity data with measured environmental concentrations (MEC), the preliminary ecological risk assessment of these five antimicrobials was conducted. The MEC/PNEC ratios of TCS and TCC were over 1 for some monitoring data, especially in urban streams with watershed areas without sewage service coverage, and their potential risk for green algae and Daphnia might be at a level of concern, although the contribution of TCS/TCC on the total toxicity of the those sites needs to be further investigated. For the three other antimicrobials, the maximum MEC/PNEC ratio for resorcinol was 0.1-1, but those for phenoxyethanol and p-thymol were <0.1 and their risk to aquatic organisms is limited, although the additive effects with TCS, TCC and other antimicrobial agents, such as parabens, need to be further examined in future studies.

Emerging organophosphate ester resorcinol bis(diphenyl phosphate) exerts estrogenic effects via estrogen receptor pathways.

Environmental occurrence and human exposure of emerging organophosphate esters (eOPEs) have increased significantly in recent years. Resorcinol bis(diphenyl) phosphate (RDP) is one of the major eOPEs detected in indoor dust, but the knowledge on its toxicities and health risks is rather limited. In this study, we investigated the in vitro estrogenic effects and underlying mechanism of RDP in comparison with a legacy OPE triphenyl phosphate (TPHP). Our results showed that RDP promoted MCF-7 cell proliferation with the lowest effect concentration of 2.5 µM, and the maximum enhancement of 1.6 folds is greater than that of TPHP (1.3 folds). The effect was inhibited completely by an estrogen receptor (ER) antagonist, suggesting that ER activation was responsible for the enhancement. In luciferase reporter gene assays both RDP and TPHP activated ER transcriptional activity at 2.5 µM, but RDP activity was higher than TPHP. Competitive fluorescence binding assays showed that RDP bound to ER with an IC10 of 0.26 µM, which is 20 folds lower than TPHP (5.6 µM). Molecular docking simulation revealed that both RDP and TPHP interacted with ER at the binding pocket of estradiol, although the hydrogen bonds were different. Taken together, RDP exerted stronger estrogenic effects than TPHP through ER-mediated pathways and may pose more health risks.

Quantitative risk assessment for skin sensitisation: consideration of a simplified approach for hair dye ingredients.

With the availability of the local lymph node assay, and the ability to evaluate effectively the relative skin sensitizing potency of contact allergens, a model for quantitative-risk-assessment (QRA) has been developed. This QRA process comprises: (a) determination of a no-expected-sensitisation-induction-level (NESIL), (b) incorporation of sensitization-assessment-factors (SAFs) reflecting variations between subjects, product use patterns and matrices, and (c) estimation of consumer-exposure-level (CEL). Based on these elements an acceptable-exposure-level (AEL) can be calculated by dividing the NESIL of the product by individual SAFs. Finally, the AEL is compared with the CEL to judge about risks to human health. We propose a simplified approach to risk assessment of hair dye ingredients by making use of precise experimental product exposure data. This data set provides firmly established dose/unit area concentrations under relevant consumer use conditions referred to as the measured-exposure-level (MEL). For that reason a direct comparison is possible between the NESIL with the MEL as a proof-of-concept quantification of the risk of skin sensitization. This is illustrated here by reference to two specific hair dye ingredients p-phenylenediamine and resorcinol. Comparison of these robust and toxicologically relevant values is therefore considered an improvement versus a hazard-based classification of hair dye ingredients.