Ammonia-induced excess ROS causes impairment and apoptosis in porcine IPEC-J2 intestinal epithelial cells.

Ammonia is one of the most important toxic metabolites in the intestine of animals. It can cause intestinal damage and associated intestinal diseases through different endogenous or exogenous stimuli. However, the definition of harmful ammonia concentration and the molecular mechanism of ammonia - induced intestinal epithelial injury remain unclear. In this study, we found that the viability of porcine IPEC-J2 intestinal epithelial cells significantly decreased with the increase of NH4Cl dose (20-80 mM). Ammonia (40 mM NH4Cl) increased the expression level of ammonia transporter RHCG and disrupted the intestinal barrier function of IPEC-J2 cells by reducing the expression levels of the tight junction molecules ZO-1 and Claudin-1. Ammonia caused elevated levels of ROS and apoptosis in IPEC-J2 cells. This was manifested by decreased activity of antioxidant enzymes SOD and GPx, decreased mitochondrial membrane potential, and increased cytoplasmic Ca2+ concentration. In addition, the expression levels of apoptosis-related molecules Caspase-9, Caspase-3, Fas, Caspase-8, p53 and Bax were increased, the expression level of anti-apoptotic molecule Bcl-2 was decreased. Moreover, the antioxidant NAC (N-acetyl-L-cysteamine) effectively alleviated ammonia-induced cytotoxicity, reduced ROS level, Ca2+ concentration, and the apoptosis of IPEC-J2 cells. The results suggest that ammonia-induced excess ROS triggered apoptosis through mitochondrial pathway, death receptor pathway and DNA damage. This study can provide reference and theoretical basis for the definition of harmful ammonia concentration in pig intestine and the effect and mechanism of ammonia on pig intestinal health.

Assessment of long-term risks of secondary cancer in paediatric patients with brain tumours after boron neutron capture therapy.

This study firstly explored the risks of secondary cancer in healthy organs of Chinese paediatric patients with brain tumours after boron neutron capture therapy (BNCT). Three neutron beam irradiation geometries (i.e. right lateral, top to bottom, posterior to anterior) were adopted in treating patients with brain tumours under the clinical environment of BNCT. The concerned organs in this study were those with high cancer morbidity in China (e.g. lung, liver and stomach). The equivalent doses for these organs were calculated using Monte Carlo and anthropomorphic paediatric phantoms with Chinese physiological features. The risk of secondary cancer, characterised by the lifetime attributable risk (LAR) factor given in the BEIR VII report, was compared among the three irradiation geometries. The results showed that the LAR was lower with the PA irradiation geometry than with the two other irradiation geometries when the 2 cm diameter tumour was at a depth of 6 cm on the right side of the brain. Under the PA irradiation geometry, the LAR in the organs increased with increasing tumour volume and depth because of the long irradiation time. As the patients aged from 10-15 years old, the LAR decreased, which was related to the increased patient height and shortened life expectancy. Female patients had a relatively higher risk of secondary cancer than male patients in this study, which could be due to the thinner body thickness and the weaker protective effect on the internal organs of the female patients. In conclusion, the risks of secondary cancer in organs were related to irradiation geometries, gender, and age, indicating that the risk of secondary cancer is a personalised parameter that needs to be evaluated before administering BNCT, especially in patients with large or deep tumours.

Glabrol impurity exacerbates glabridin toxicity in zebrafish embryos by increasing myofibril disorganization.

ETHNOPHARMACOLOGICAL RELEVANCE: Glabridin, extracted from Glycyrrhiza glabra L., is widely used for the treatment of hyperpigmentation because of its anti-inflammatory and antioxidant activities and its ability to inhibit melanin synthesis. This led to the strict regulation of its quality and safety. However, traditional quality control methods used for plant extracts cannot reflect the product quality owing to multiple unknown impurities, which necessitates the further analysis of impurities. AIM OF THE STUDY: The study identified the toxic impurities of glabridin and their toxicological mechanism. MATERIALS AND METHODS: In total, 10 glabridin samples from different sources were quantified using high-performance liquid chromatography. Sample toxicities were evaluated using zebrafish and cell models. To identify impurities, samples with different toxicity were analyzed by ultra-high-performance liquid chromatography coupled with quadrupole-Orbitrap mass spectrometry. The toxicity of related impurities was verified in the zebrafish model. Phalloidin stain was used to evaluate subtle changes in myofibril alignment. RESULTS: Although glabridin content in the samples was similar, there were significant differences in toxicity. The results were verified using four different mammalian cell lines. Higher contents of glabrone and glabrol were identified in the sample with the highest toxicity. In the zebrafish model, the addition of glabrol reduced the LC50 of glabridin to 9.224, 6.229, and 5.370 µM at 48, 72, and 96 h post-fertilization, respectively, whereas glabrone did not have any toxic effect. Phalloidin staining indicated that a glabrol impurity exacerbates the myotoxicity of glabridin in zebrafish embryos. CONCLUSION: Glabrol, but not glabrone, was identified as a key impurity that increased glabridin toxicity. This finding indicates that controlling glabrol content is necessary during glabridin product production.