Toxicology Mechanisms and Methods最新文献

Objectives: This study, aimed to determine and compare DNA damage in e-cigarette and HTP (IQOS) users by assessing DNA-adducts, which are biomarkers of various DNA alkylation and oxidation.

Methods: For the evaluation of DNA alkylation, N³-Ethyladenine (N³-EtA) and N³-Methyladenine (N³-MeA) adducts were used. DNA oxidation was assessed using, 8-hydroxy-2'-deoxyguanosine(8-OHdG). The urinary cotinine, N³-MeA, N³-EtA, and 8-OHdG concentrations of the cigarette smokers (n:39), e-cigarette users (n:28), IQOS users (n:20), passive smokers (n:32), and nonsmokers(n:41) who lived Ankara, Turkiye were determined using, liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Results: In light of the detected 8-OHdG levels, e-cigarette (3.19 ng/g creatinine) and IQOS (4.38 ng/g creatinine) users had higher oxidative DNA damage than healthy nonsmokers (2.51 ng/g creatinine). Alkylated DNA-adducts were identified in the urine of e-cigarette (N³-MeA: 3.92 ng/g creatinine; N³-EtA: 0.23 ng/g creatinine) and IQOS (N³-MeA: 7.54 ng/g creatinine; N³-EtA: 0.29 ng/g creatinine) users. In the generation of N³-MeA adducts, a significant difference was found between IQOS users and e-cigarette users (p < 0.05). Also, DNA alkylation in flavored e-cigarette users (N³-MeA: 4.51 ng/g creatinine; N³-EtA: 0.27 ng/g creatinine) was higher than in non-flavored e-cigarette users (N³-MeA: 2.27 ng/g creatinine; N³-EtA: 0.06 ng/g creatinine). The highest cotinine levels were found in cigarette smokers (16.1316 ng/g creatinine). No significant difference was found when e-cigarette (1163.02 ng/g creatinine) and IQOS smokers were compared (1088.3 ng/g creatinine).

Conclusion: People who use e-cigarettes and IQOS may be at higher risk of genotoxicity than those who do not use and are not exposed to any tobacco products. Furthermore, the usage of flavoring additives in e-cigarettes contributed to additional genotoxic damage risks.

Benzene exposure is known to cause serious damage to the human hematopoietic system. However, recent studies have found that chronic benzene exposure may also cause neurological damage, but there were few studies in this issue. The aim of this study was to investigate the mechanism of damage to the central nervous system (CNS) by chronic benzene exposure with a multi-omics analysis. We established a chronic benzene exposure model in C57BL/6J mice by gavage of benzene-corn oil suspension, identified the differentially expressed proteins (DEPs) and differentially expressed genes (DEGs) in mice brain using 4D Label-free proteomic and RNA-seq transcriptomic. We observed that the benzene exposure mice had a significant loss of body weight, reduction in complete blood counts, abnormally high MRI signals in brain white matter, as well as extensive brain edema and neural demyelination. 162 DEPs were identified by the proteome, including 98 up-regulated and 64 down-regulated proteins. KEGG pathway analysis of DEPs showed that they were mainly involved in the neuro-related signaling pathways such as metabolic pathways, pathways of neurodegeneration, chemical carcinogenesis, Alzheimer disease, and autophagy. EPHX1, GSTM1, and LIMK1 were identified as important candidate DEGs/DEPs by integrated proteomic and transcriptomic analyses. We further performed multiple validation of the above DEGs/DEPs using fluorescence quantitative PCR (qPCR), parallel reaction monitoring (PRM), immunohistochemistry, and immunoblotting to confirm the reliability of the multi-omics study. The functions of these DEGs/DEPs were further explored and analyzed, providing a theoretical basis for the mechanism of nerve damage caused by benzene exposure.

The SH-SY5Y human neuroblastoma cell line is a standard in vitro experimental model of neuronal-like cells used in neuroscience and toxicological research. These cells can be differentiated into mature neurons, most commonly using retinoic acid (RA). Despite differences in characteristics, both undifferentiated and differentiated SH-SY5Y cells are used in research. However, due to uncertainties regarding the expression of specific markers of neural function in each culture, there is no definite conclusion on which culture is better suited for (neuro)toxicological and/or neuroscience investigations. To address this dilemma, we investigated the basal expression/activity of the key elements of acetylcholine, dopamine, serotonin, and GABA neurotransmitter pathways, along with the elements involved in exocytosis of neurotransmitters, and neuron electrophysiological activity in undifferentiated and in RA-differentiated SH-SY5Y cells using a six-day differentiation protocol. Our findings revealed that both SH-SY5Y cell types are functionally active. While undifferentiated SH-SY5Y cells exhibited greater multipotency in the expression of tested markers, most of those markers expressed in both cell types showed higher expression levels in RA-differentiated SH-SY5Y cells. Our results suggest that the six-day differentiation protocol with RA induces maturation, but not differentiation of the cells into specific neuron phenotype. The greater multipotency of undifferentiated cells in neural markers expression, together with their higher sensitivity to xenobiotic exposure and more simple cultivation protocols, make them a better candidate for high throughput toxicological screenings. Differentiated neurons are better suited for neuroscience researches that require higher expression of more specific neural markers and the specific types of neural cells.

Per- and polyfluoroalkyl substances (PFASs), one of the persistent organic pollutants, have immunosuppressive effects. The evaluation of this effect has been the focus of regulatory toxicology. In this investigation, 146 PFASs (immunosuppressive or nonimmunosuppressive) and corresponding concentration gradients were collected from literature, and their structures were characterized by using Dragon descriptors. Feature importance analysis and stepwise feature elimination are used for feature selection. Three machine learning (ML) methods, namely Random Forest (RF), Extreme Gradient Boosting Machine (XGB), and Categorical Boosting Machine (CB), were utilized for model development. The model interpretability was explored by feature importance analysis and correlation analysis. The findings indicated that the three models developed have exhibited excellent performance. Among them, the best-performing RF model has an average AUC score of 0.9720 for the testing set. The results of the feature importance analysis demonstrated that concentration, SpPosA_X, IVDE, R2s, and SIC2 were the crucial molecular features. Applicability domain analysis was also performed to determine reliable prediction boundaries for the model. In conclusion, this study is the first application of ML models to investigate the immunosuppressive activity of PFASs. The variables used in the models can help understand the mechanism of the immunosuppressive activity of PFASs, allow researchers to more effectively assess the immunosuppressive potential of a large number of PFASs, and thus better guide environmental and health risk assessment efforts.

Sanguinarine (SAN) is an alkaloid with multiple biological activities, mainly extracted from Sanguinaria canadensis or Macleaya cordata. The low bioavailability of SAN limits its utilization. At present, the nature and mechanism of SAN intestinal absorption are still unclear. The pharmacokinetics, single-pass intestinal perfusion test (SPIP), and equilibrium solubility test of SAN in rats were studied. The absorption of SAN at 20, 40, and 80 mg/L in different intestinal segments was investigated, and verapamil hydrochloride (P-gp inhibitor), celecoxib (MPR2 inhibitor), and ko143 (BCRP inhibitor) were further used to determine the effect of efflux transporter proteins on SAN absorption. The equilibrium solubility of SAN in three buffer solutions (pH 1.2, 4.5 and 6.8) was investigated. The oral pharmacokinetic results in rats showed that SAN was rapidly absorbed (T_max=0.5 h), widely distributed (Vz/F = 134 L/kg), rapidly metabolized (CL = 30 L/h/kg), and had bimodal phenomena. SPIP experiments showed that P-gp protein could significantly affect the effective permeability coefficient (P_eff) and apparent absorption rate constant (Ka) of SAN. Equilibrium solubility test results show that SAN has the best solubility at pH 4.5. In conclusion, SAN is a substrate of P-gp, and its transport modes include efflux protein transport, passive transport and active transport.

Precision-cut kidney slices (PCKS) provide a powerful model to close the gap between in vivo and in vitro research. Publications by various authors favor different incubation conditions, media, and antibiotics, that have not yet been compared in a standardized manner. After preparation, rat-PCKS were incubated in a total of nine combinations of incubation media and antibiotics for four days. We found that a combination of DMEM/F-12 and gentamicin showed the highest levels of viability. Utilizing both qualitative and quantitative methods, we observed stable levels of cellular viability for 10 days when incubated in the most suitable medium combination of DMEM and gentamicin. Additionally, a calcein acetoxymethyl/ethidium homodimer-1 based live/dead staining, analysis of total protein content and lactate dehydrogenase (LDH) were explored to assess both short- and long-term tissue viability. PCKS showed a significant decrease in total protein content, leveling off at around 60% over the duration of 10 days. To be able to evaluate viability irrespective of decreases in total protein detected, we chose to utilize the alamarBlue Cell Viability Assay. Quantifying both intra- and extracellular activity of LDH, while using different concentrations of ethanol as a positive control, we explored enzyme content as a parameter for cell membrane damage and cytotoxicity in PCKS. Overall, we showed that PCKS are suitable for both short- and long-term observation by optimizing incubation parameters, with numerous possibilities for other assays and methods in future studies.

In medical practice, iodine contrast media are necessary for diagnostic techniques. However, it comes with a potential risk to the patient in the form of allergic reactions. The aim of this research is to study the effects of iodine contrast media on endothelial cells in an in vitro system at various concentrations, specifically investigating their impact on cell viability, cell cycle, and apoptosis in the treated cells within the field of diagnostic radiology. Our results showed that in iodine contrast media, when the concentration was within the range of 2.5-50 mgI/ml, cell viability decreased by 50%. Conversely, exposure to ioversol at concentrations between 12.5 and 50.0 mgI/ml resulted in a notable increase in the percentage of total apoptotic cells, including both early and late apoptosis. In conclusion, our in vitro investigation sheds light on the effect of iodinated contrast media on endothelial cell viability, cell cycle progression, and apoptosis. These findings contribute valuable insights to ensure the safety of their use, aligning with guidelines in radiological procedures. Further research and adherence to established guidelines are crucial for refining our understanding and promoting the safe application of iodinated contrast media in the field of radiology.

Magnesium oxide nanoparticles (MgO NPs) have gained significant importance in biomedicine and variety of nanotechnology-based materials used in the agriculture and biomedical industries. However, the release of different nanowastes in the water ecosystem becomes a serious concern. Therefore, this study was executed to evaluate the toxic impacts of MgO NPs on grass carp. A total of 60 grass carp were randomly divided in three groups (G0, G1, and G2). Fish reared in group G0 were kept as control while fish of groups G1 and G2 were exposed to 0.5 mg/L and 0.7 mg/L MgO NPs, respectively, mixed in water for 21 days. The 96h median lethal concentration (LC₅₀) of MgO NPs was found to be 4.5 mg/L. Evaluation of oxidative stress biomarkers, antioxidant enzymes, DNA damage in different visceral organs and the presence of micronuclei in erythrocytes were determined on days 7, 14, and 21 of the trial. Results revealed dose- and time-dependent significantly increased values of reactive oxygen species, lipid peroxidation product, DNA damage in multiple visceral organs and formation of micronuclei in the erythrocytes of treated fish (0.7 mg/L). The results on antioxidant profile exhibited significantly lower amounts of total proteins, catalase, superoxide dismutase, and peroxidase in visceral organs of the fish exposed to MgO NPs (0.5 and 0.7 mg/L) at day 21 of trial compared to control group. In conclusion, it has been recorded that MgO NPs severely influence the normal physiological functions of the grass carp even at low doses.

New Approach Methodologies (NAMs) are being widely used to reduce, refine, and replace, animal use in studying toxicology. For respiratory toxicology, this includes in silico and in vitro alternatives using air:liquid interface (ALI) exposures to replace traditional in vivo inhalation studies. In previous studies using 1,3-dichloropropene (1,3-DCP), a 5-day 4 h repeat exposures of MucilAir^™ nasal cell culture models caused, dose-dependent cytotoxicity, depletion of GSH, changes in differential gene expression and histopathological transitions in cellular morphology from pseudostratified columnar epithelium to squamous epithelium. In this report we attempted to extend these studies using 15-day 1,3-DCP 4 h exposures to using MucilAir^™ nasal cultures as outlined by an US EPA recent task order (US EPA 2023). For the 15-day repeat exposure, there were severe histopathologic changes in the MucilAir^™ nasal mock-treatment (air-only) VITROCELL^® chamber controls compared to incubator controls preventing any further analysis. The histopathological transitions in cellular morphology from pseudostratified columnar epithelium to squamous epithelium observed in the air only control in this study and previously with 1,3-DCP in MucilAir^™ nasal cultures is also a hallmark of chemically induced cytotoxic responses in vivo in the respiratory tract. Histopathology assessments of 3D respiratory tract models used in ALI exposures can provide the linkage between in vitro to in vivo outcomes as part of the validation efforts of ALI use in regulatory toxicology. This report indicates that importance of histopathological assessments of incubator and mock-treatment (air-only) controls from each ALI exposure experiment along with exposed cell based model.

From the past to the present, many chemicals have been used for the purpose of flame retardant. Due to PBDEs' (Polybrominated diphenyl ether) lipophilic and accumulative properties, some of them are banned from the market. As an alternative to these chemicals, OPFRs (organophosphorus flame retardants) have started to be used as flame retardants. In this article, acute toxicity profiles, mutagenicity, carcinogenicity, blood-brain barrier permeability, ecotoxicity and nutritional toxicity as also AHR, ER affinity and MMP, aromatase affinity, CYP2C9, CYP3A4 interaction of the of 16 different compounds of the OPFRs were investigated using a computational toxicology method; ProTox- 3.0. According to our results, eight compounds were found to be active in terms of carcinogenic effect, whereas two compounds were found to be active for mutagenicity. On the other hand, all compounds were found to be active in terms of blood-barrier permeability. Fourteen compounds and four compounds are found to have ecotoxic and nutritional toxic potency, respectively. Eight compounds were determined as active to AhR, and four chemicals were found to be active in Estrogen Receptor alpha. Eight chemicals were found to be active in terms of mitochondrial membrane potency. Lastly, three chemicals were found to be active in aromatase enzymes. In terms of CYP interaction potencies, eight compounds were found to be active in both CYP2C9 and CYP3A4. This research provided novel insights into the potential toxic effects of OPFRs. However, further studies are needed to evaluate their toxicity. Moreover, these findings lay the groundwork for in vitro and in vivo toxicity research.