The pyrethroids metabolite 3-phenoxybenzoic acid induces dopaminergic degeneration.

Exposure to pyrethroids, a significant class of the most widely used agricultural chemicals, has been associated with an increased risk of Parkinson's disease (PD). However, although many different pyrethroids induce roughly the same symptoms of Parkinsonism, the underlying mechanisms remain unknown. To find the shared key features among these mechanisms, we focused on 3-phenoxybenzoic acid (3-PBA), a common and prominent metabolite of most pyrethroids produced via hydrolysis by CEs in mammals. To determine the contribution of 3-PBA to the initiation and progression of PD, we performed in vivo and in vitro experiments, respectively, and found that 3-PBA not only accumulates in murine brain tissues over time but also further induces PD-like pathologies (increased α-syn and phospho-S129, decreased TH) to the same or even greater extent than the precursor pyrethroid. A before-after study of PET-DAT in the same mice revealed that low concentrations of 3-PBA (0.5 mg/kg) could paradoxically cause DAT to increase (22.46% higher than pre-drug test). The intervention of DAT inhibitors and activators respectively alleviated and enhanced the dopaminergic toxicity of 3-PBA, indicating that 3-PBA interacts with DAT. In particular, low concentrations of 3-PBA increase the DAT, which in turn induces 3-PBA to enter the dopaminergic neurons to exert toxic effects. Finally, we described a mechanism underlying this potential role of 3-PBA in the pathological aggregation of α-syn. Specifically, 3-PBA was found to dysregulate C/EBP ß levels and further anomalously activate AEP in vivo and in vitro, accompanied by increased accumulation of pathologically cleaved α-syn (N103 fragments) and accelerated α-syn aggregation. All these results suggest that 3-PBA exposure could mimic the pathological and pathogenetic features of PD, showing that this metabolite is a key pathogenic compound in pyrethroid-related pathological effects and a possible dopamine neurotoxin. Additionally, our findings provide a crucial reference for the primary prevention of PD.

Toxicity of benzyl benzoate as a food additive and pharmaceutical agent.

Benzyl benzoate (BB), one of the benzyl derivates, is a component of brown aromatic resin in cinnamon oil and cough syrups and it is widely used in various fields in the perfume, pharmaceutical, and food industries. It is absorbed and hydrolyzed to benzoic acid and benzyl alcohol. Two different doses of BB (25 mg kg-1 body weight and 100 mg kg-1 body weight) were orally administered to 5-week old male rats for 90 days. Histopathological, morphological, hematological, and biochemical assays were performed in toxicological evaluations. Initial/final body weights, relative organ weights, and food and water consumptions of rats did not change significantly. There were statistically significant differences in terms of monocyte, neutrophil, lymphocyte %, and serum AST levels in control and BB treatment groups. Several histopathological findings were observed in liver, kidney, thymus, prostate, and epididymis tissues of the rats in the treatment groups. Immunohistochemical examinations were also performed in the tissues for fibronectin (FN), type IV collagen, transforming growth factor ß (TGF-ß), matrix metalloproteinase-2 (MMP-2), and tissue inhibitor of metalloproteinase-2 (TIMP-2). Alterations in immunolocalization of these markers were observed between the control and the treatment groups. No changes were detected in the sperm count, daily sperm production, and sperm morphology.

A biologically based model to quantitatively assess the role of the nuclear receptors liver X (LXR), and pregnane X (PXR) on chemically induced hepatic steatosis.

Hepatic steatosis is characterized by the intracellular increase of free fatty acids (FFAs) in the form of triglycerides in hepatocytes. This hepatic adverse outcome can be caused by many factors, including exposure to drugs or environmental toxicants. Mechanistically, accumulation of lipids in the liver can take place via several mechanisms such as de novo synthesis and/or uptake of FFAs from serum via high fat content diets. De novo synthesis of FFAs within the liver is mediated by the liver X receptor (LXR), and their uptake into the liver is mediated through the pregnane X receptor (PXR). We investigated the impact of chemical exposure on FFAs hepatic content via activation of LXR and PXR by integrating chemical-specific physiologically based pharmacokinetic (PBPK) models with a quantitative toxicology systems (QTS) model of hepatic lipid homeostasis. Three known agonists of LXR and/or PXR were modeled: T0901317 (antagonist for both receptors), GW3965 (LXR only), and Rifampicin (PXR only). Model predictions showed that T0901317 caused the most FFAs build-up in the liver, followed by Rifampicin and then GW3965. These modeling results highlight the importance of PXR activation for serum FFAs uptake into the liver while suggesting that increased hepatic FAAs de novo synthesis alone may not be enough to cause appreciable accumulation of lipids in the liver under normal environmental exposure levels. Moreover, the overall PBPK-hepatic lipids quantitative model can be used to screen chemicals for their potential to cause in vivo hepatic lipid content buildup in view of their in vitro potential to activate the nuclear receptors and their exposure levels.

In vitro and in silico approach to study the hormonal activities of the alternative plasticizer tri-(2-ethylhexyl) trimellitate TEHTM and its metabolites.

Tri-(2-ethylhexyl) trimellitate (TEHTM) is a plasticizer for polyvinyl chloride (PVC) material used in medical devices. It is an alternative to di-(2-ethylhexyl) phthalate (DEHP), a well-known reprotoxic and endocrine disruptor. As plasticizers are known to easily migrate when in contact with fatty biological fluids, patient exposure to TEHTM is highly probable. However, there is currently no data on the potential endocrine-disrupting effects of its human metabolites. To evaluate the effects of TEHTM metabolites on endocrine activity, they were first synthesized and their effects on estrogen, androgen and thyroid receptors, as well as steroid synthesis, were investigated by combining in vitro and in silico approaches. Among the primary metabolites, only 4-MEHTM (4-mono-(2-ethylhexyl) trimellitate) showed agonist activities on ERs and TRs, while three diesters were TR antagonists at non-cytotoxic concentrations. These results were completed by docking experiments which specified the ER and TR isoforms involved. A mixture of 2/1-MEHTM significantly increased the estradiol level and reduced the testosterone level in H295R cell culture supernatants. The oxidized secondary metabolites of TEHTM had no effect on ER, AR, TR receptors or on steroid hormone synthesis. Among the fourteen metabolites, these data showed that two of them (4-MEHTM and 2/1-MEHTM) induced effect on hormonal activities in vitro. However, by comparing the concentrations of the primary metabolites found in human urine with the active concentrations determined in bioassays, it can be suggested that the metabolites will not be active with regard to estrogen, androgen, thyroid receptors and steroidogenesis-mediated effects.

Eltrombopag second-line therapy in adult patients with primary immune thrombocytopenia in an attempt to achieve sustained remission off-treatment: results of a phase II, multicentre, prospective study.

Up to 30% immune thrombocytopenia (ITP) patients achieve a sustained remission off-treatment (SROT) after discontinuation of thrombopoietin receptor agonists (TPO-RAs). Factors predictive of response are lacking. Patients aged ≥18 years with newly diagnosed or persistent ITP were treated with eltrombopag for 24 weeks. Primary end-point was SROT: the proportion of responders that were able to taper and discontinue eltrombopag maintaining the response during a period of observation (PO) of six months. Secondary end-points included the association between some immunological parameters (TPO serum levels, cytokines and lymphocyte subsets) and response. Fifty-one patients were evaluable. Primary end-point was achieved in 13/51 (25%) treated patients and 13/34 (38%) patients who started the tapering. Baseline TPO levels were not associated with response at week 24 nor with SROT. Higher baseline levels of IL-10, IL-4, TNF-α and osteopontin were negative factors predictive of response (P = 0·001, 0·008, 0·02 and 0·03 respectively). This study confirms that SROT is feasible for a proportion of ITP patients treated with eltrombopag. Some biological parameters were predictive of response.

An integrated study of metabolomics and transcriptomics to reveal the anti-primary dysmenorrhea mechanism of Akebiae Fructus.

ETHNOPHARMACOLOGICAL RELEVANCE: Akebiae Fructus, a Tujia minority folk medicine and a well-known traditional Chinese medicine for soothing the liver, regulating Qi, promoting blood circulation and relieving pain, is widely used in the treatment of primary dysmenorrhea. However, little is known about its underlying mechanism. AIM OF THE STUDY: To explore the effect of Akebiae Fructus on primary dysmenorrhea model induced by estradiol benzoate and oxytocin, and to provide better understanding of the mechanism of Akebiae Fructus for primary dysmenorrhea treatment. MATERIALS AND METHODS: The primary dysmenorrhea mouse model was used in this study. Except for the control group and the normal administration group, the mice of other groups were subcutaneously injected with estradiol benzoate (10 mg/kg/d) for 10 consecutive days. From the 5th day of the ten-day model period, the positive control groups were given 0.075 g/kg ibuprofen and 7.5 g/kg Leonurus granule, the drug groups were given 0.2 g/kg, 0.4 g/kg, 0.8 g/kg Akebiae Fructus extract, the normal administration group was given 0.8 g/kg Akebiae Fructus extract, and the same volume saline was given in the control group. On the tenth day, oxytocin (10 U/kg) was peritoneally injected after estradiol benzoate injected 1 h. After the oxytocin injection, writhing behavior was observed for 30 min. Then the uterine tissue was collected to measure the level of PGF2α and PGE2, and for histological analysis and transcriptomics analysis. Meanwhile, plasma and urine samples were collected for metabolomic analysis. RESULTS: Akebiae Fructus inhibited the writhing, decreased the PGF2α level and ameliorated the morphological changes. 32 potential metabolic biomarkers in plasma and 17 in urine were found for primary dysmenorrhea, and after Akebiae Fructus treatment, 25 metabolites in plasma and 14 in urine were restored. These altered metabolites were mainly involved in lipid, amino acid and organic acid metabolism. For the transcriptomic study, a total of 2244 differentially expressed genes (1346 up-regulated and 898 down-regulated) were obtained between the control and model group, and 148 differentially expressed genes (DEGs) were found related with Akebiae Fructus treatment of primary dysmenorrhea. Correlation analysis was carried out based on the transcriptomic and metabolomic data. 5 differentially expressed genes (Plpp3, Sgpp2, Arg1, Adcy8, Ak5) were found related with the enrichment metabolic pathways. The mechanism by which Akebiae Fructus ameliorates primary dysmenorrhea may account for the regulation of the gene expression to control the key enzymes in the sphingolipid metabolism, arginine and proline metabolism, glycerophospholipid metabolism and purine metabolism, inhibiting the abnormal secretion of PGF2α, alleviating the uterine contraction and reducing inflammation and pain. CONCLUSIONS: Akebiae Fructus could effectively alleviate the symptoms of primary dysmenorrhea, regulate metabolic disorders, and control the related gene expression in primary dysmenorrhea. The study may provide clues for further study of Akebiae Fructus treatment on primary dysmenorrhea.

Evaluation of potential miticide toxicity to Varroa destructor and honey bees, Apis mellifera, under laboratory conditions.

The honey bee, Apis mellifera L., is the world's most important managed pollinator of agricultural crops, however, Varroa mite, Varroa destructor Anderson and Trueman, infestation has threatened honey bee survivorship. Low efficacy and development of Varroa mite resistance to currently used Varroacides has increased the demand for innovative, effective treatment tool options that exhibit high efficacy, while minimizing adverse effects on honey bee fitness. In this investigation, the toxicity of 16 active ingredients and 9 formulated products of registered miticides for use on crops from 12 chemical families were evaluated in comparison to amitraz on Varroa mites and honey bees using contact surface and topical exposures. It was found that fenpyroximate (93% mortality), spirotetramat (84% mortality) and spirodiclofen (70% mortality) had greater toxicity to Varroa mites, but high dose rates caused high bee mortality (> 60%). With this in mind, further research is needed to investigate other options to minimize the adverse effect of these compounds on bees. The results also found high toxicity of fenazaquin and etoxazole against Varroa mites causing 92% and 69% mortality, respectively; and were found to be safe on honey bees. Collectively, it is recommended that fenazaquin and etoxazole are candidates for a potential Varroacide and recommended for further testing against Varroa mites at the colony level.

Benzoyl-CoA conjugate accumulation as an initiating event for male reprotoxic effects in the rat? Structure-activity analysis, species specificity, and in vivo relevance.

A number of para-substituted benzoic acids (p-BA) and chemicals metabolized to p-BA have been found to confer adverse effects in male rats on sperm viability, motility, and morphology. These effects are putatively associated with the metabolism of p-BA to toxic intermediates. We had shown that p-BA lead to accumulation of high levels of p-alkyl-benzoyl-CoA conjugates in plated primary rat hepatocytes. Here we further investigated the relevance of this metabolic pathway for the reprotoxic effects in rats and rabbits. We extended the structure-activity relationship to a set of 19 chemicals (nine reprotoxic and ten non-reprotoxic) and confirmed a very strong correlation between p-alkyl-benzoyl-CoA accumulation in rat hepatocytes and the toxic outcome. Species specificity was probed by comparing rat, rabbit and human hepatocytes, and p-benzoyl-CoA accumulation was found to be specific to the rat hepatocytes, not occurring in human hepatocytes. There was also very limited accumulation in hepatocytes from rabbits that are a non-responder species in in vivo studies. Tissues of rats treated with 3-(4-isopropylphenyl)-2-methylpropanal were analysed and p-isopropyl-benzoyl-CoA conjugates were detected in the liver and in the testes in animals at toxic doses indicating that the metabolism observed in vitro is relevant to the in vivo situation and the critical metabolite does also occur in the reproductive tissue. These multiple lines of evidence further support benzoyl-CoA accumulation as a key initiating event for a specific group of male reproductive toxicants, and indicate a species-specific effect in the rat.

Genotoxic Potential Assessment of the Herbicide Bispyribac-Sodium in a Fresh Water Fish Clarias batrachus (Linn.).

Genotoxic potential of herbicide bispyribac-sodium was evaluated in fish Clarias batrachus using micronucleus (MN) test and comet assay. Fish were exposed to three environmentally relevant test concentrations of the herbicide for 20, 25 and 30 days. Significant effects (p < 0.05) for both concentration and duration of exposure were observed in herbicide exposed fish. Similar trend of DNA damage was observed through MN test and comet assay. Maximum DNA damage was observed in fish exposed to highest concentration of herbicide at all duration. Maximum damage was observed on day 25 at all concentrations followed by a decline. This study established C. batrachus as an ecotoxicological model for bispyribac-sodium induced genotoxicity testing. It further confirmed that both MN test and comet assay are useful tool for assessment of genotoxicity induced by water pollutants.

An Evaluation of the Human Relevance of the Liver Tumors Observed in Female Mice Treated With Permethrin Based on Mode of Action.

In 2-year studies, the nongenotoxic pyrethroid insecticide permethrin produced hepatocellular tumors in CD-1 mice but not in Wistar rats. Recently, we demonstrated that the mode of action (MOA) for mouse liver tumor formation by permethrin involves activation of the peroxisome proliferator-activated receptor alpha (PPARα), resulting in a mitogenic effect. In the present study, the effects of permethrin and 2 major permethrin metabolites, namely 3-phenoxybenzoic acid and trans-dichlorochrysanthemic acid, on cytochrome P450 mRNA levels and cell proliferation (determined as replicative DNA synthesis) were evaluated in cultured CD-1 mouse, Wistar rat, and human hepatocytes. Permethrin and 3-phenoxybenzoic acid induced CYP4A mRNA levels in both mouse and human hepatocytes, with trans-dichlorochrysanthemic acid also increasing CYP4A mRNA levels in mouse hepatocytes. 3-Phenoxybenzoic acid induced CYP4A mRNA levels in rat hepatocytes, with trans-dichlorochrysanthemic acid increasing both CYP4A mRNA levels and replicative DNA synthesis. Permethrin, 3-phenoxybenzoic acid, and trans-dichlorochrysanthemic acid stimulated replicative DNA synthesis in mouse hepatocytes but not in human hepatocytes, demonstrating that human hepatocytes are refractory to the mitogenic effects of permethrin and these 2 metabolites. Thus, although some of the key (eg, PPARα activation) and associative (eg, CYP4A induction) events in the established MOA for permethrin-induced mouse liver tumor formation could occur in human hepatocytes at high doses of permethrin, 3-phenoxybenzoic acid, and/or trans-dichlorochrysanthemic acid, increased cell proliferation (an essential step in carcinogenesis by nongenotoxic PPARα activators) was not observed. These results provide additional evidence that the established MOA for permethrin-induced mouse liver tumor formation is not plausible for humans.