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3-Monochloropropane-1,2-diol (3-MCPD), 2-monochloropropane-1,2-diol (2-MCPD) and 2,3-epoxy-1-propanol (glycidol), in their free form or esterified to fatty acids, are food contaminants formed during the refinement of oils and fats. We conducted a survey to quantify the levels of these compounds in 130 food items, in order to assess the exposure to them in food and the consequent health risk for consumers. Food samples, including infant formula, were analysed by gas-chromatography mass spectrometry with the indirect method, and we used the latest open access food consumption database for the Italian population for a probabilistic assessment of exposure. We adopted an in silico approach to fill the gap for the toxicity of 2-MCPD. The occurrence values for the three contaminants in food were in most cases lower than or comparable to those reported in previous surveys. Exposure assessment for the most exposed individuals (95 th percentiles of consumers only) of different age groups, gave values below the tolerable daily intake recommended by the European Food Safety Authority for 3-MCPD and below the simulated or predicted toxicity thresholds for 2-MCPD, indicating a negligible risk due to dietary exposure to these contaminants. For glycidol, however, estimated exposure indicated a non-negligible increase in cancer risk, and a margin of exposure <25,000 for younger population groups, indicating a potential health concern., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

The urinary mercapturic acids N-acetyl-S-(2-carbamoylethyl)-L-cysteine (AAMA) and N-acetyl-S-(2-carbamoyl-2-hydroxyethyl)-L-cysteine (GAMA) are short-term biomarkers of exposure from acrylamide and its metabolite glycidamide, respectively. The medium-term exposure to acrylamide and glycidamide is monitored by the adducts N-(2-carbamoylethyl)-Val (AA-Val) and N-(2-carbamoyl-2-hydroxyethyl)-Val (GA-Val) in hemoglobin (Hb), respectively. Three questions were addressed by application of these biomarkers in two diet studies including 36 omnivores, 36 vegans and 16 strict raw food eaters (abstaining from any warmed or heated food for at least four months): first, what is the internal acrylamide exposure following a vegan or a raw food diet in comparison to that in omnivores? Second, did the exposure change between 2017 and 2021? And third, what is the stability over time of AAMA/GAMA excretion compared to that of AA-Val/GA-Val levels in Hb between both time points? Median urinary AAMA excretion per day in non-smoking omnivores, vegans and raw food eaters were 62.4, 85.4 and 15.4 µg/day, respectively; the corresponding median AA-Val levels were 27.7, 39.7 and 13.3 pmol/g Hb, respectively. Median levels in strict raw food eaters were about 25% (AAMA excretion) and 48% (AA-Val) of those in omnivores. In comparison to 2017, AAMA and GAMA excretion levels were hardly altered in 2021, however, levels of AA-Val and GA-Val in 2021 slightly increased. There was a weak correlation between AAMA excretion levels determined four years apart (r S  = 0.30), and a moderate correlation between levels of AA-Val (r S  = 0.55) in this timeframe. Our data in strict raw food eaters confirm a significant endogenous formation to acrylamide in a size range, which is-based on the levels of AA-Val-distinctly higher than reported previously based on levels of urinary AAMA excretion. The relatively lower AAMA excretion in raw food eaters likely represents a lower extent of glutathione conjugation due to missing hepatic first-pass metabolism in case of endogenous formation of acrylamide, which leads to a higher systemic exposure., (© 2024. The Author(s).)

Triptolide (TP) is a major bioactive compound derived from Tripterygium wilfordii Hook. F. (TwHF) known for its medicinal properties, but it also exhibits potential toxic effects. It has been demonstrated to induce severe male reproductive toxicity, yet the precise mechanism behind this remains unclear, which limits its broad clinical application. This study aimed to investigate the mechanisms underlying testicular damage and spermatogenesis dysfunction induced by TP in mice, using both mouse models and the spermatocyte-derived cell line GC-2spd. In the present study, it was found that TP displayed significant testicular microstructure damaged and spermatogenesis defects including lower concentration and abnormal morphology by promoting ROS formation, MDA production and restraining GSH level, glutathione peroxidase 4 (GPX4) expression in vivo. Furthermore, Ferrostatin-1 (FER-1), a ferroptosis inhibitor, was found to significantly reduce the accumulation of lipid peroxidation, alleviate testicular microstructural damage, and enhance spermatogenic function in mice. Besides, notably decreased cell viability, collapsed mitochondrial membrane potential, and elevated DNA damage were observed in vitro. The above-mentioned phenomenon could be reversed by pre-treatment of FER-1, indicating that ferroptosis participated in the TP-mediated spermatogenesis dysfunction. Mechanistically, TP could enhance GPX4 ubiquitin degradation via triggering K63-linked polyubiquitination of GPX4, thereby stimulating ferroptosis in spermatocytes. Functionally, GPX4 deletion intensified ferroptosis and exacerbated DNA damage in GC-2 cells, while GPX4 overexpression mitigated ferroptosis induced by TP. Overall, these findings for the first time indicated a vital role of ferroptosis in TP induced-testicular injury and spermatogenic dysfunction through promoting GPX4 K63-linked polyubiquitination, which hopefully offers a potential therapeutic avenue for TP-related male reproductive damage. In addition, this study also provides a theoretical foundation for the improved clinical application of TP or TwHF in the future., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Triptolide (TP) is a major active and toxic composition of the Chinese medicine Tripterygium wilfordii Hook. F. (TWHF), exhibiting various therapeutic bioactivities. Among the toxic effects, the hepatotoxicity of TP deserves serious attention. Previously, our research group proposed a new view of TP-related hepatotoxicity: hepatic hypersensitivity under lipopolysaccharide (LPS) stimulation. However, the mechanism of TP/LPS-induced hepatic hypersensitivity remains unclear. In this study, we investigated the mechanism underlying TP/LPS-induced hypersensitivity from the perspective of the inhibition of proteasome activity, activated endoplasmic reticulum stress (ERS)-related apoptosis, and the accumulation of reactive oxygen species (ROS). Our results showed that N-acetylcysteine (NAC), a common ROS inhibitor, decreased the expression of cleaved caspase-3 and cleaved PARP, which are associated with FLIP enhancement. Moreover, 4-phenylbutyric acid (4-PBA), an ERS inhibitor, was able to alleviate TP/LPS-induced hepatotoxicity by reducing ERS-related apoptosis protein expression (GRP78, p-eIF2α/eIF2α, ATF4, CHOP, cleaved caspase-3 and cleaved PARP) and ROS levels, with ATF4 being an indispensable mediator. In addition, the proteasome activity inhibitor MG-132 further aggravated ERS-related apoptosis, which indicated that the inhibition of proteasome activity also plays an important role in TP/LPS-related liver injuries. In summary, we propose that TP/LPS may upregulate the activation of ERS-associated apoptosis by inhibiting proteasome activity and enhancing ROS production through ATF4., (© 2024. The Author(s).)

The aim of the study was to assess non-occupational and occupational exposure to bisphenol compounds in Finland. The participants were 151 non-occupationally exposed volunteers and 15 potentially exposed employees of a sewage-pipe relining company and a floor-coating company. The following chemicals were measured in the urine samples: bisphenol A (BPA), bisphenol F (BPF), bisphenol A diglycidyl ether (BADGE), bisphenol F diglycidyl ether (BFDGE), and the metabolites of the latter two [bisphenol A (2,3-dihydroxypropyl) glycidyl ether (BADGE·H 2 O), bisphenol A bis(2,3-dihydroxypropyl) ether (BADGE·2 H 2 O), bisphenol A (3-chloro-2-hydroxypropyl) (2,3-dihydroxypropyl) ether (BADGE·HCl·H 2 O), bisphenol A (3-chloro-2-hydroxypropyl) glycidyl ether (BADGE·HCl), and bisphenol A bis(3-chloro-2-hydroxypropyl) ether (BADGE·2HCl) and bisphenol F bis(2,3-dihydroxypropyl) ether (BFDGE·2 H 2 O), and bisphenol F bis(3-chloro-2-hydroxypropyl) ether (BFDGE·2HCl)]. BADGE and BFDGE were also measured in breathing zone air samples and hand-wipe samples of the sewage-pipe relining and floor-coating workers. Non-occupational exposure to BPA has decreased in Finland. The BPF level of the non-occupationally exposed was higher than the respective levels reported in the recent literature. BPA and BPF concentrations in the workers' urine samples were in the same range as those in the corresponding concentrations of the non-occupationally exposed population. Higher concentrations of BADGE and BFDGE metabolites were found in some of the workers' urine samples. Elevated urine concentrations were also observed in the samples collected the next morning. Some of the urinary BADGE and BFDGE metabolite results correlated with the hand-wipe results. The results show that occupational exposure to BADGE and BFDGE may occur in sewage-pipe relining and floor-coating work. They also indicate that dermal contamination plays a role in total exposure. Although the measured urinary levels indicate that the absorption of these bisphenol compounds are unlikely to pose a systemic health risk, the risk of dermal sensitization remains., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Despite the known reproductive toxicity induced by triptolide (TP) exposure, the regulatory mechanism underlying testicular vacuolization injury caused by TP remains largely obscure., Methods: Male mice were subjected to TP at doses of 15, 30, and 60 μg/kg for 35 consecutive days. Primary Sertoli cells were isolated from 20-day-old rat testes and exposed to TP at concentrations of 0, 40, 80, 160, 320, and 640 nM. A Biotin tracer assay was conducted to assess the integrity of the blood-testis barrier (BTB). Transepithelial electrical resistance (TER) assays were employed to investigate BTB function in primary Sertoli cells. Histological structures of the testes and epididymides were stained with hematoxylin and eosin (H&E). The expression and localization of relevant proteins or pathways were assessed through Western blotting or immunofluorescence staining., Results: TP exposure led to dose-dependent testicular injuries, characterized by a decreased organ coefficient, reduced sperm concentration, and the formation of vacuolization damage. Furthermore, TP exposure disrupted BTB integrity by reducing the expression levels of tight junction (TJ) proteins in the testes without affecting basal ectoplasmic specialization (basal ES) proteins. Through the TER assay, we identified that a TP concentration of 160 nM was optimal for elucidating BTB function in primary Sertoli cells, correlating with reductions in TJ protein expression. Moreover, TP exposure induced changes in the distribution of the BTB and cytoskeleton-associated proteins in primary Sertoli cells. By activating the AKT/mTOR signaling pathway, TP exposure disturbed the balance between mTORC1 and mTORC2, ultimately compromising BTB integrity in Sertoli cells., Conclusion: This investigation sheds light on the impacts of TP exposure on testes, elucidating the mechanism by which TP exposure leads to testicular vacuolization injury and offering valuable insights into comprehending the toxic effects of TP exposure on testes., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

For endocrine disrupting chemicals (EDC) the existence of "safe exposure levels", that is exposure levels that do not present an appreciable risk to human health is most controversially discussed, as is the existence of health-based reference values. Concerns have been especially raised that EDCs might not possess a threshold level such that no exposure level to EDCs can be considered safe. To explore whether or not threshold levels can be identified, we performed a screening exercise on 14 pesticidal and biocidal active substances previously identified as EDCs in the European Union. The respective substances are ideal subjects for case studies to review for endocrine activity and disruptive potential following well-defined regulatory assessment based on solid data to effectually establish adversity as consequence of endocrine disruption. Dimethomorph, metiram and propiconazole for which the weight of evidence demonstrating endocrine disruption was the strongest were used as subjects for further study. Epoxiconazole was additionally selected as its effects on the endocrine system are extensive. For all four substances, analysis of the toxicological data clearly indicated thresholds of adversity below which no adverse effects mediated through an endocrine mechanism were observed. Particular emphasis was placed on mechanistic considerations including homeostasis and the concept of adversity. As a proof of concept this study provides evidence that like other substances of toxicological concern EDCs have threshold levels for adversity. While for some EDCs the respective thresholds might indeed be very low this shows that, data allowing, for other EDCs sufficiently protective reference values can be derived., (© 2024. The Author(s).)

Amitriptyline (ATL), a tricyclic antidepressant, has been reported to cause various adverse effects, particularly hepatotoxicity. The mechanisms of ATL-induced hepatotoxicity remain unknown. The study was performed to identify the olefin epoxidation metabolite of ATL and determine the possible toxicity mechanism. Two glutathione (GSH) conjugates (M1 and M2) and two N- acetylcysteine (NAC) conjugates (M3 and M4) were detected in rat liver microsomal incubations supplemented with GSH and NAC, respectively. Moreover, M1/M2 and M3/M4 were respectively found in ATL-treated rat primary hepatocytes and in bile and urine of rats given ATL. Recombinant P450 enzyme incubations demonstrated that CYP3A4 was the primary enzyme involved in the olefin epoxidation of ATL. Treatment of hepatocytes with ATL resulted in significant cell death. Inhibition of CYP3A attenuated the susceptibility to the observed cytotoxicity of ATL. The metabolic activation of ATL most likely participates in the cytotoxicity of ATL.

In order to study the toxic effect and mechanism of triptolide(TP) on the reproductive system of female rats with Ⅱ type collagen induced arthritis(CIA), 50 SD rats were randomly divided into normal control group, CIA model group, and three groups receiving TP tablets at clinically equivalent doses of 0. 5, 1, and 2 times, respectively(with TP dosages of 3. 75, 7. 5, and 15 μg·kg~(-1)·d~(-1)), each comprising 10 rats. Intragastric administration was started on the day after the first immunization, once a day, for 42 days.The results were taken on the 21st and 42nd days to calculate the uterine and ovarian organ indexes; pathological and morphological changes in uterus and ovaries were observed under a light microscope; and the levels of estradiol(E&#95;2) and cytochrome P450A1(aromatase,CYP19A1) in ovarian homogenate were detected by ELISA. Furthermore, immunohistochemistry was employed to detect the expression levels of transforming growth factor β3( TGFβ3) pathway-related proteins, mothers against decapentaplegic homolog 3(Smad3) and steroidogenic factor-1(SF-1) in ovarian tissues. In vitro, the mouse Chinese hamster ovary(CHO) cell line was established, and after 24 hours of TP administration(30, 60, 120 nmol·L~(-1)), cell proliferation was detected by the thiazolyl blue tetrazolium bromide(MTT) method, apoptosis by the flow cytometry, and TGFβ3, Smad3 and SF-1 protein expression in cells by the Western blot method, and the nuclear entry of SF-1 was detected by immunofluorescence. The results showed that compared with the CIA model group, all TP administration groups showed decreased number of uterine glands, total follicles, mature follicles, and corpus luteum on days 21 and 42 of administration, but there was no statistical difference, and only the administration of 2 times the clinically equivalent dose of TP could significantly increase the number of atretic follicles at 42 days of administration. TP at 3. 75 μg·kg-1·d-1significantly reduced the level of E&#95;2 at 21 days of administration and the expression of TGFβ3 and Smad3 factors in ovarian tissues,but had no significant effect on the rate-limiting enzyme in estrogen synthesis CYP19A1. TP at 7. 5 and 15 μg·kg~(-1)·d~(-1) significantly reduced the expression of SF-1 regardless of administration for 21 days or 42 days. TP can significantly promote ovarian cell apoptosis in vitro, with apoptosis mainly concentrated in the late stage of apoptosis after 24 hours of administration. In addition, 60 nmol·L~(-1) TP significantly reduced the protein expression of TGFβ3, Smad3 and SF-1 in a dose-dependent manner. In summary, intragastric administration of TP at less than 2 times the clinically equivalent dose for 21 days and 42 days did not cause obvious reproductive damage to the uterus and ovarian tissues of CIA rats, and the number of atretic follicles changed significantly only when the 2 times the clinically equivalent dose was administered for 42 days. TP exerted reproductive toxicity in vivo on reproductive target organs and in vitro on ovarian cells by inhibiting the expression of TGFβ3/Smad3/SF-1 pathway.

Tripterygium wilfordii (TW) preparations have been utilized in China for treating rheumatoid arthritis and autoimmune diseases. However, their clinical use is limited due to reproductive toxicity, notably premature ovarian failure (POF). Our study aimed to investigate the effect and mechanism of bergenin in attenuating POF induced by triptolide in mice. POF was induced in female ICR mice via oral triptolide administration (50 μg/kg) for 60 days. Mice received bergenin (25, 50, 100 mg/kg, i.g.) or estradiol valerate (EV) (0.1 mg/kg, i.g.) daily, 1 h before triptolide treatment. In vitro, ovarian granulosa cells (OGCs) were exposed to triptolide (100 nM) and bergenin (1, 3, 10 μM). Antioxidant enzyme activity, protein expression, apoptosis rate, and reactive oxygen species (ROS) levels were assessed. The results showed that triptolide-treated mice exhibited evident atrophy, along with an increase in atretic follicles. Bergenin (50, 100 mg/kg) and EV (0.1 mg/kg), orally administered, exerted significant anti-POF effect. Bergenin and EV also decreased apoptosis in mouse ovaries. In vitro, bergenin (1, 3, 10 μM) attenuated triptolide-induced OGCs apoptosis by reducing levels of apoptosis-related proteins. Additionally, bergenin reduced oxidative stress through downregulation of antioxidant enzymes activity and overall ROS levels. Moreover, the combined use with Sh-Nrf2 resulted in a reduced protection of bergenin against triptolide-induced apoptosis of OGCs. Together, bergenin counteracts triptolide-caused POF in mice by inhibiting Nrf2-mediated oxidative stress and preventing OGC apoptosis. Combining bergenin with TW preparations may effectively reduce the risk of POF., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Triptolide (TP) is the major bioactive component of traditional Chinese medicine Tripterygium wilfordii Hook. F., a traditional Chinese medicinal plant categorized within the Tripterygium genus of the Celastraceae family. It is recognized for its therapeutic potential in addressing a multitude of diseases. Nonetheless, TP is known to exhibit multi-organ toxicity, notably hepatotoxicity, which poses a significant concern for the well-being of patients undergoing treatment. The precise mechanisms responsible for TP-induced hepatotoxicity remain unresolved. In our previous investigation, it was determined that TP induces heightened hepatic responsiveness to exogenous lipopolysaccharide (LPS). Additionally, natural killer (NK) cells were identified as a crucial effector responsible for mediating hepatocellular damage in this context. However, associated activating receptors and the underlying mechanisms governing NK cell represented innate lymphoid cell (ILC) activation remained subjects of inquiry and were not yet investigated. Herein, activating receptor Killer cell lectin like receptor K1 (NKG2D) of group 1 ILCs was specifically upregulated in TP- and LPS-induced acute liver failure (ALF), and in vivo blockade of NKG2D significantly reduced group 1 ILC mediated cytotoxicity and mitigated TP- and LPS-induced ALF. NKG2D ligand UL16-binding protein-like transcript 1 (MULT-1) was found upregulated in liver resident macrophages (LRMs) after TP administration, and LRMs did exhibit NK cell activating effect. Furthermore, M1 polarization of LRMs cells was observed, along with an elevation in intracellular tumor necrosis factor (TNF)-α levels. In vivo neutralization of TNF-α significantly alleviated TP- and LPS-induced ALF. In conclusion, the collaborative role of group 1 ILCs and LRMs in mediating hepatotoxicity was confirmed in TP- and LPS-induced ALF. TP-induced MULT-1 expression in LRMs was the crucial mechanism in the activation of group 1 ILCs via MULT-1-NKG2D signal upon LPS stimulation, emphasizing the importance of infection control after TP administration., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Triptolide (TP) is a remarkable anti-inflammatory and immunosuppressive component separated from Tripterygium wilfordii Hook. F. However, its hepatotoxicity limits its application in the clinical. Our group has proposed a new perspective on TP-induced hepatotoxicity, in which TP enhances liver hypersensitivity upon lipopolysaccharide (LPS) stimulation. Because the cause of the disease is unknown, there is currently no uniform treatment available. In this study, we attempted to determine whether the GSK-3β-JNK pathway affects liver damage and its regulatory mechanism in response to TP/LPS costimulation. In addition, we investigated the effect of CsA or the GSK 3β inhibitor CHIR-98014 on TP/LPS-induced hepatotoxicity. The results showed that the TP/LPS cotreatment mice exhibited obvious hepatotoxicity, as indicated by a remarkable increase in the serum ALT and AST levels, glycogen depletion, GSK 3β-JNK upregulation, and increased apoptosis. Instead of the specific knockdown of JNK1, the specific knockdown of JNK2 had a protective effect. Additionally, 40 mg/kg of CsA and 30 mg/kg of CHIR-98014 might provide protection. In summary, CHIR-98014 could protect against TP/LPS- or TP/TNF-α-induced activation of the GSK 3β-JNK pathway and mitochondria-dependent apoptosis, improving the indirect hepatotoxicity induced by TP.