X‐ray Structures and Feasibility Assessment of CLK2 Inhibitors for Phelan–McDermid Syndrome

CLK2 inhibition has been proposed as a potential mechanism to improve autism and neuronal functions in Phelan–McDermid syndrome (PMDS). Herein, the discovery of a very potent indazole CLK inhibitor series and the CLK2 X‐ray structure of the most potent analogue are reported. This new indazole series was identified through a biochemical CLK2 Caliper assay screen with 30k compounds selected by an in silico approach. Novel high‐resolution X‐ray structures of all CLKs, including the first CLK4 X‐ray structure, bound to known CLK2 inhibitor tool compounds (e.g., TG003, CX‐4945), are also shown and yield insight into inhibitor selectivity in the CLK family. The efficacy of the new CLK2 inhibitors from the indazole series was demonstrated in the mouse brain slice assay, and potential safety concerns were investigated. Genotoxicity findings in the human lymphocyte micronucleus test (MNT) assay are shown by using two structurally different CLK inhibitors to reveal a major concern for pan‐CLK inhibition in PMDS.     

Mechanisms of Oxidative Processes in Meat and Toxicity Induced by Postprandial Degradation Products: A Review

Antioxidant system loss after slaughtering, reactive species production, cell disruption, contact with oxygen and light, heme and nonheme iron, and irradiation starts up mainly by 2 related oxidative processes: lipid peroxidation and protein oxidation. Products generated in these processes are responsible for meat quality loss, and some of them are suspected to be toxic to humans. This review article is focused on reactive species implicated in oxidative processes in meat, on lipid peroxidation mechanisms, heme protein, and nonheme protein oxidation, and on some toxic oxidation and digestion products. Nonenzymatic fatty acid peroxidation is exemplified by an arachidonic acyl group, and the initiation of chain reaction can be described by 3 pathways: singlet oxygen, hydroxyl radical from the Fenton reaction, and perferrylmyoglobin. Enzymatic oxidation of fatty acids is exemplified using linoleic acid, and the main characteristics of lipoxygenase are also presented. Heme protein oxidation is described in an interrelation with lipid peroxidation and the significance for food quality is shown. For protein oxidation, 3 different mechanism types are described: oxidation of amino acid residues, oxidation of protein backbone, and reactions of proteins with carbonyl compounds from lipid peroxidation. The effects of oxidative damage on protein properties and bioavailability are also shown. At the end of each oxidative process, the postprandial toxicity induced by oxidation products and the dietary degradation products are presented. Also discussed are reports by some researchers who suggest that dietary lipid and protein oxidation products and heme iron from red meat are in part cytotoxic and/or genotoxic.     

Comparison of Cytotoxic,Genotoxic, and DNA-Protective Effects of Skyrin on Cancerous vs. Non-Cancerous Human Cells

Secondary metabolites as a potential source of anticancer therapeutics have been the subject of many studies. Since hypericin, a metabolite isolated from Hypericum perforatum L., shows several biomedical properties applicable in oncology, the aim of our study was to investigate its potential precursor skyrin in terms of genotoxic and DNA-protective effects. These skyrin effects were analyzed by cell-free methods, and cytotoxicity was estimated by an MTT assay and by a trypan blue exclusion test, while the genotoxic/antigenotoxic potential was examined by comet assay using non-cancerous human lymphocytes and the HepG2 cancer cell line. Skyrin did not show DNA-damaging effects but rather exhibited DNA-protectivity using a DNA-topology assay. However, we observed only weak antioxidant and chelating skyrin properties in other cell-free methods. Regarding the cytotoxic activity of skyrin, HepG2 cells were more prone to skyrin-induced death in comparison to human lymphocytes. Skyrin in non-cytotoxic concentrations did not exhibit elevated genotoxicity in both cell types. On the other hand, skyrin displayed moderate DNA-protective effects that were more noticeable in the case of non-cancerous human lymphocytes. The potential genotoxic effects of skyrin were not observed, and its DNA-protective capacity was more prominent in non-cancerous cells. Therefore, skyrin might be a promising agent used in anticancer therapy.     

Preliminary evaluation of the genotoxic potential of a hydrophilic polymer with three preservation systems

This study compared the genotoxic potential of a polymeric associative thickener used in topically applied emulsions preserved with three different preservative systems. The method used for the assessment of genotoxicity is the in vitro micronucleus test [Organization for Economic Cooperation and Development (OECD) guideline number 487]. When changing an additive such as a preservation system in a raw material, it is crucial to re-evaluate its toxicity potential because this change may significantly alter its properties. This study shows that at the levels tested neither of the systems evaluated demonstrated any cytotoxic or genotoxic effects. Skin exposure must take into consideration factors such as duration, skin condition and metabolism, but most importantly concentration. Although preservatives can be toxic at high concentrations, they are usually safe at the concentrations used in cosmetic raw materials and formulations. If used to preserve raw materials, they undergo further dilution when added to the formulation.     

体外微核高内涵筛选法在食品毒理学遗传毒性评价中应用研究

目的探讨基于高内涵筛选(high-content screening, HCS)技术的体外微核(in vitro micronucleus, IVMN)检测方法应用于食品毒理学遗传毒性评价的可行性。方法采用IVMN和HCS法,分别对10种化合物进行遗传毒性评价,其中5种已知遗传毒性化合物(染色体断裂剂和非整倍体诱发剂)、1种已知非遗传毒性化合物以及4种食品原料,每个受试物至少设置3个剂量组,每个剂量组设2个复孔,同时设置阴性对照组(无血清最小必需培养基)和阳性对照组(+S9为环磷酰胺20μg/ml、-S9为丝裂霉素C 1.0μg/ml)。以中国仓鼠肺细胞为细胞模型,在有和/或无代谢活化系统条件下,依次对上述受试物采用短时处理(4 h)后进行微核检测,并分析微核细胞率。结果经IVMN和HCS法得到的IVMN试验结果显示:2.5～10μg/ml苯并芘[B(a)P]、5～20μg/ml甲磺酸甲酯(MMS)、0.01～0.04μg/ml 4-硝基喹啉-N-氧化物(4NQO)、0.25～1.0μg/ml秋水仙碱(COL)和0.5～2.0μg/ml硫酸长春碱(VB)在各自浓度范围内,在有或无代谢活化系统的条件下,诱导产生的微核细胞率随着受试物浓度的升高而增加,呈明显剂量-反应关系,且微核细胞率与阴性对照组比较,差异均有统计学意义(P0.05),试验结果为阳性;1 250～5 000μg/ml氯化钠(NaCl)、1 250～5 000μg/ml食品原料A、1 250～5 000μg/ml食品原料B、312.5～1 250μg/ml食品原料C和156.25～625μg/ml食品原料D在各自浓度范围内,在有和无代谢活化系统的条件下,微核细胞率虽呈现一定的剂量依赖性增加趋势,但均维持在较低的水平,且与阴性对照组比较,差异均无统计学意义(P0.05),试验结果为阴性。结论本试验条件下,两种方法对10种物质的检测结果均一致,提示将IVMN HCS法应用于食品毒理学遗传毒性评价具有一定的可行性。     

魔芋葡甘低聚糖毒理学及肠道益生性评价

目的：研究以半干法酶解制备的魔芋葡甘低聚糖（konjac oligosaccharides,KOS）的毒理学特性和肠道益生性。方法：以小鼠急性毒性实验,小鼠骨髓细胞微核实验和小鼠精子畸形实验对KOS急性毒性和遗传毒性进行研究、评价;通过小鼠盲肠内容物体外厌氧发酵评价KOS的肠道益生性。结果：KOS对雌、雄小鼠急性经口半致死量（LD50）均大于21 500 mg/kg,属无毒级;小鼠骨髓细胞微核实验及小鼠精子畸形实验均呈阴性;体外厌氧发酵实验表明KOS可被肠道菌群有效利用,明显增加肠道益生菌（双歧杆菌、乳酸菌）和短链脂肪酸的数量,而对大肠杆菌的增殖效果不明显。结论：KOS的安全性高,是一种功能显著的肠道益生元。     

Nonclinical Safety Assessment of Morus alba L. Fruits: Study of 90‐D Toxicity in Sprague Dawley Rats and Genotoxicity in Salmonella

Morus alba L. is a traditional herb with a long history of consumption, both as an edible fruit and as medicine. However, its safety evaluation has not yet been established. The objective of this study was to evaluate subchronic oral toxicity and genotoxicity of M. alba L. fruits (MFE). The subchronic toxicity after daily oral administration of MFE at 0, 40, 200, and 1000 mg/kg for 90 d was examined in Sprague Dawley (SD) rats. MFE administration did not lead to death, adverse effects, change in food and water consumption, and body weight gain. Significant toxic effects were not found within the parameters of organ weight, biochemical values, and hematological and urine analysis between the control and the MFE group. The genotoxicity of MFE was assayed by Ames test in Salmonella typhimurium strains TA98, TA102, and TA1535. No genotoxicity was found in all the tested strains. Thus in this study, a no‐observed‐adverse‐effect level for MFE in 90 d repeated oral toxicity study in rats was determined to be greater than 1000 mg/kg regardless of gender. The results also suggested that MFE does not have a genotoxicity potential.     

Constructing of Bacillus subtilis-Based Lux-Biosensors with the Use of Stress-Inducible Promoters

Here, we present a new lux-biosensor based on Bacillus subtilis for detecting of DNA-tropic and oxidative stress-causing agents. Hybrid plasmids pNK-DinC, pNK-AlkA, and pNK-MrgA have been constructed, in which the Photorhabdus luminescens reporter genes luxABCDE are transcribed from the stress-inducible promoters of B. subtilis: the SOS promoter PdinC, the methylation-specific response promoter PalkA, and the oxidative stress promoter PmrgA. The luminescence of B. subtilis-based biosensors specifically increases in response to the appearance in the environment of such common toxicants as mitomycin C, methyl methanesulfonate, and H₂O₂. Comparison with Escherichia coli-based lux-biosensors, where the promoters PdinI, PalkA, and Pdps were used, showed generally similar characteristics. However, for B. subtilis PdinC, a higher response amplitude was observed, and for B. subtilis PalkA, on the contrary, both the amplitude and the range of detectable toxicant concentrations were decreased. B. subtilis PdinC and B. subtilis PmrgA showed increased sensitivity to the genotoxic effects of the 2,2′-bis(bicyclo [2.2.1] heptane) compound, which is a promising propellant, compared to E. coli-based lux-biosensors. The obtained biosensors are applicable for detection of toxicants introduced into soil. Such bacillary biosensors can be used to study the differences in the mechanisms of toxicity against Gram-positive and Gram-negative bacteria.     

Effectiveness of steamed and cooked broccoli to attenuate bone marrow injury and suppressed haemopoiesis in male rats exposed to petrol vapours

The protective potential of broccoli against petrol-induced haematological disorders was evaluated in adult male rats. Animals were exposed to petrol vapours 6 h/6 days/week, for 12 weeks, while broccoli (steamed and cooked) was given orally (1.5 g/kg b.wt) for the same period. Petrol exposure significantly reduced red blood cell (RBC) indices, total and differential white blood cells (WBCs) and serum immunoglobulins: IgA; IgG, with elevated neutrophils%; and IgM level, indicating haematotoxicity. Meanwhile, significant elevation in levels of malondialdehyde and hydrogen peroxide, decreased antioxidants (superoxide dismutase, catalase and reduced glutathione) and total antioxidant capacity were observed in lysed RBCs and bone marrow of exposed rats. The levels of pro-inflammatory cytokines, interleukin (IL)-2, IL-6 and tumour necrosis factor-alpha (TNF-α), were significantly elevated with consequent bone marrow DNA damage detected via comet assay. Histologically, bone marrow revealed structural abnormalities of increased adipocytes and abnormal megakaryocytes with separated or hypo-lobulated nuclei. Consumption of broccoli during petrol exposure attenuated oxidative stress, inflammation and haematotoxicity, through preserving bone marrow integrity.     

Antioxidant,Cytotoxic, Genotoxic,and DNA-Protective Potential of 2,3-Substituted Quinazolinones: Structure—Activity Relationship Study

The evaluation of antioxidant compounds that counteract the mutagenic effects caused by the direct action of reactive oxygen species on DNA molecule is of considerable interest. Therefore, a series of 2,3-substituted quinazolinone derivatives (Q1–Q8) were investigated by different assays, and the relationship between their biological properties and chemical structure was examined. Genotoxicity and the potential DNA-protective effects of Q1–Q8 were evaluated by comet assay and DNA topology assay. Antioxidant activity was examined by DPPH-radical-scavenging, reducing-power, and total antioxidant status (TAS) assays. The cytotoxic effect of compounds was assessed in human renal epithelial cells (TH-1) and renal carcinoma cells (Caki-1) by MTT assay. Analysis of the structure–activity relationship disclosed significant differences in the activity depending on the substitution pattern. Derivatives Q5–Q8, bearing electron-donating moieties, were the most potent members of this series. Compounds were not genotoxic and considerably decreased the levels of DNA lesions induced by oxidants (H₂O₂, Fe²⁺ ions). Furthermore, compounds exhibited higher cytotoxicity in Caki-1 compared to that in TH-1 cells. Substantial antioxidant effect and DNA-protectivity along with the absence of genotoxicity suggested that the studied quinazolinones might represent potential model structures for the development of pharmacologically active agents.