Application of the adverse outcome pathway framework to genotoxic modes of action

In May 2017, the Health and Environmental Sciences Institute's Genetic Toxicology Technical Committee hosted a workshop to discuss whether mode of action (MOA) investigation is enhanced through the application of the adverse outcome pathway (AOP) framework. As AOPs are a relatively new approach in genetic toxicology, this report describes how AOPs could be harnessed to advance MOA analysis of genotoxicity pathways using five example case studies. Each of these genetic toxicology AOPs proposed for further development includes the relevant molecular initiating events, key events, and adverse outcomes (AOs), identification and/or further development of the appropriate assays to link an agent to these events, and discussion regarding the biological plausibility of the proposed AOP. A key difference between these proposed genetic toxicology AOPs versus traditional AOPs is that the AO is a genetic toxicology endpoint of potential significance in risk characterization, in contrast to an adverse state of an organism or a population. The first two detailed case studies describe provisional AOPs for aurora kinase inhibition and tubulin binding, leading to the common AO of aneuploidy. The remaining three case studies highlight provisional AOPs that lead to chromosome breakage or mutation via indirect DNA interaction (inhibition of topoisomerase II, production of cellular reactive oxygen species, and inhibition of DNA synthesis). These case studies serve as starting points for genotoxicity AOPs that could ultimately be published and utilized by the broader toxicology community and illustrate the practical considerations and evidence required to formalize such AOPs so that they may be applied to genetic toxicity evaluation schemes. Environ. Mol. Mutagen. 61:114–134, 2020. © 2019 Wiley Periodicals, Inc.     

The synergistic antitumor activity of 3-(2-nitrophenyl) propionic acid-paclitaxel nanoparticles (NPPA-PTX NPs) and anti-PD-L1 antibody inducing immunogenic cell death

Cancer immunotherapy is a strategy that is moving to the frontier of cancer treatment in the current decade. In this study, we show evidence that 3-(2-nitrophenyl) propionic acid-paclitaxel nanoparticles (NPPA-PTX NPs), act as immunogenic cell death (ICD) inducers, stimulating an antitumor response which results in synergistic antitumor activity by combining anti-PD-L1 antibody (aPD-L1) in vivo. To investigate the antitumor immunity induced by NPPA-PTX NPs, the expression of both ICD marker calreticulin (CRT) and high mobility group box 1 (HMGB1) were analyzed. In addition, the antitumor activity of NPPA-PTX NPs combined with aPD-L1 in vivo was also investigated. The immune response was also measured through quantitation of the infiltration of T cells and the secretion of pro-inflammatory cytokines. The results demonstrate that NPPA-PTX NPs induce ICD of MDA-MB-231 and 4T1 cells through upregulation of CRT and HMGB1, reactivating the antitumor immunity via recruitment of infiltrating CD3+, CD4+, CD8+ T cells, secreting IFN-γ, TNF-α, and the enhanced antitumor activity by combining with aPD-L1. These data suggest that the combined therapy has a synergistic antitumor activity and has the potential to be developed into a novel therapeutic regimen for cancer patients.     

Combined effects of irritants and allergens. Synergistic effects of nickel and sodium lauryl sulfate in nickel- sensitized individuals

Knowledge of the combined effects of irritants and allergens is of interest with respect to accurate risk assessment. The threshold for elicitation of allergic contact dermatitis in previously sensitized individuals may theoretically be markedly influenced by the simultaneous presence of irritants and allergens. Combined exposures have, however, only been studied infrequently. In the present study, the combined effect of an irritant and an allergen was evaluated in a dose-response designed experimental study. 20 nickel-sensitized subjects were exposed to patch testing with varying concentrations of NiCl2 (nickel chloride) and sodium lauryl sulfate (SLS) alone and in combination. Evaluation of skin reactions was performed by colorimetry, measurement of transepidermal water loss and clinical evaluation, and the data were analyzed by logistic dose-response models. A synergistic effect was found of combined exposure to NiCl2 and SLS, as compared to each of the substances applied separately, as evaluated by colorimetry and clinical scoring. This means that the effect produced by the combined exposure was substantially greater than the effect produced by either of the substances alone. A synergistic effect of combined exposure on skin barrier impairment was not found, since the barrier function is significantly influenced by SLS-exposure only and not by NiCl2. Concentration limits are used by industry and government agencies to protect consumers. The present results clearly illustrate that elicitation thresholds and concentration limits may be influenced considerably by combined exposure to allergens and irritants.     

艾叶油对小鼠的遗传毒理学研究

目的:探讨艾叶油对成年小鼠及胚胎鼠的遗传毒性。方法:采用一次性小鼠灌胃急性毒性试验、小鼠骨髓嗜多染红细胞微核试验、小鼠胚胎肝转移微核试验、小鼠精子畸形试验。结果:艾叶油经小鼠口灌胃给药LD50为4.11ml/kg;艾叶油灌胃剂量2ml/kg时,孕鼠和雄鼠诱发的胚胎肝微核率、骨髓微核率和精子畸形率均较对照组显著升高(P<0.05)。艾叶油灌胃剂量为1ml/kg时,诱发的胚胎肝微核率较对照组显著升高(P<0.05),骨髓微核率与精子畸形率与对照组相比无显著性差异。艾叶油灌胃剂量为0.5ml/kg时,诱发的胚胎肝微核率、骨髓微核率、精子畸形率与对照组相比均无显著性差异。结论:一定剂量的艾叶挥发油对小鼠具有潜在的遗传毒性,并呈剂量-反应关系。     

复方乌骨藤胶囊对60Coγ照射S180荷瘤小鼠的增效减毒作用

目的:观察复方乌骨藤胶囊对60Coγ射线照射S180荷瘤小鼠的增效减毒作用。方法:小鼠72只,随机分为6组:荷瘤对照组,荷瘤+60Coγ照射组,荷瘤+60Coγ照射+贞芪扶正颗粒组,荷瘤+60Coγ照射+复方乌骨藤胶囊大、中、小剂量组。建立S180荷瘤小鼠模型,除荷瘤对照组外,其余各组于造模后第6d进行60Coγ射线照射,照射后即开始给药,1次/d,连续给药9d,末次给药后次日,检测体重、瘤重、脾指数、脾集落形成单位(CFU-S)、血常规和骨髓有核细胞数。结果:荷瘤+60Coγ照射+复方乌骨藤胶囊2.25g/kg组使瘤重明显下降,骨髓有核细胞数、CFU-S、网织红细胞明显升高;荷瘤+60Coγ照射+复方乌骨藤胶囊1.125g/kg组使瘤重明显下降,网织红细胞、CFU-S明显升高;荷瘤+60Coγ照射+复方乌骨藤胶囊0.5625g/kg组使瘤重明显下降。结论:复方乌骨藤胶囊对60Coγ照射的荷瘤小鼠有增效、减毒作用。     

臌胀片在HBeAg阳性慢性乙型病毒性肝炎治疗中的减毒增效作用

目的观察臌胀片在乙肝 e 抗原(HBeAg)阳性慢性乙型病毒性肝炎治疗中的减毒增效作用。方法将80例HBeAg阳性慢性乙型肝炎患者随机分为治疗组(臌胀片联合替比夫定)和对照组(替比夫定),均治疗1年,观察治疗前后2组患者丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)、乙肝病毒-DNA(HBV-DNA)阴转率、HBeAg阴转率、HBeAg/HBeAb血清学转换率、血清肌酸激酶(CK)水平及安全性。结果治疗48周后,治疗组ALT、AST、HBV DNA阴转率、HBeAg阴转率及HBeAg/HBeAb血清学转换率均优于对照组(P＜0.05)。2组患者均有CK升高,其中治疗组13例(32.5%),对照组24例(60.0%),2组比较差异有统计学意义(P＜0.05)。结论臌胀片与替比夫定联合用药后,其减毒增效作用显著。     

扶正固本类中药应用于大肠癌的临床研究进展

大肠癌是消化道常见恶性肿瘤之一,在我国发病率和死亡率位于第四位,在大肠癌治疗中采用多学科综合治疗方法已得到普遍认可。近年来大量临床及实验研究均表明,在大肠癌多学科综合治疗中运用中医药辅助治疗疗效显著,其中,扶正固本类中药占很大一部分比例。在手术、放疗、化疗前后应用扶正固本类中药,可以减轻放化疗后毒副反应,促使受创伤机体快速恢复,增强机体对放化疗的耐受性,以利于放疗、化疗的连续性,还可以提高机体免疫力,延长生存期,改善生存质量。本文就近几年扶正固本类中药在大肠癌综合治疗中的临床应用进展进行综述。     

生物与非生物诱导子协同作用对丹参毛状根培养生产丹参酮的影响

目的:考察生物诱导子yeast extract和不同非生物诱导子(Ag⁺,Co²⁺,α-氨基异丁酸)协同作用对丹参毛状根培养生产丹参酮的影响。方法:在丹参毛状根培养基中添加不同诱导子及诱导子组合,利用高效液相色谱法检测3种主要丹参酮(隐丹参酮、丹参酮Ⅰ和丹参酮ⅡA)的含量。结果:yeast extract与各非生物诱导子在不同浓度下的组合对丹参酮Ⅰ和丹参酮ⅡA都发挥了较好的协同诱导作用,而对隐丹参酮的协同诱导效果不显著。其中,yeast extract和Ag+(300μmol.L^-1)协同作用获得了最高的丹参酮Ⅰ产量,是对照组含量的13.9倍,诱导协同作用系数为3.0。yeast extract和Co²⁺(100μmol.L^-1)协同作用获得了最高的丹参酮ⅡA产量,是对照组含量的14.5倍,协同作用系数为2.1。yeast extract和α-氨基异丁酸(200μmol.L^-1)的组合是唯一一个比单个诱导子更有效地提高了隐丹参酮产量的组合,隐丹参酮的产量达到1.28 mg.g^-1,是对照组含量的30.3倍,诱导协同作用系数为1.3。结论:利用生物诱导子yeast extract和不同的非生物诱导子进行协同作用,其诱导效果比单独使用某一种诱导子的诱导效果更好,这种相互作用可以更有效地促进次生代谢产物的合成。     

Custom-Made Zirconium Dioxide Implants for Craniofacial Bone Reconstruction

Reconstruction of the facial skeleton is challenging for surgeons because of difficulties in proper shape restoration and maintenance of the proper long-term effect. ZrO₂ implant application can be a solution with many advantages (e.g., osseointegration, stability, and radio-opaqueness) and lacks the disadvantages of other biomaterials (e.g., metalosis, radiotransparency, and no osseointegration) or autologous bone (e.g., morbidity, resorption, and low accuracy). We aimed to evaluate the possibility of using ZrO₂ implants as a new application of this material for craniofacial bone defect reconstruction. First, osteoblast (skeleton-related cell) cytotoxicity and genotoxicity were determined in vitro by comparing ZrO₂ implants and alumina particle air-abraded ZrO₂ implants to the following: 1. a titanium alloy (standard material); 2. ultrahigh-molecular-weight polyethylene (a modern material used in orbital surgery); 3. a negative control (minimally cytotoxic or genotoxic agent action); 4. a positive control (maximally cytotoxic or genotoxic agent action). Next, 14 custom in vivo clinical ZrO₂ implants were manufactured for post-traumatologic periorbital region reconstruction. The soft tissue position improvement in photogrammetry was recorded, and clinical follow-up was conducted at least 6 years postoperatively. All the investigated materials revealed no cytotoxicity. Alumina particle air-abraded ZrO2 implants showed genotoxicity compared to those without subjection to air abrasion ZrO₂, which were not genotoxic. The 6-month and 6- to 8-year clinical results were aesthetic and stable. Skeleton reconstructions using osseointegrated, radio-opaque, personalized implants comprising ZrO₂ material are the next option for craniofacial surgery.     

IL‐28B (IFN‐λ3) and IFN‐α synergistically inhibit HCV replication

Genetic variation in the IL‐28B (interleukin‐28B; interferon lambda 3) region has been associated with sustained virological response (SVR) rates in patients with chronic hepatitis C treated with peginterferon‐α and ribavirin. However, the mechanisms by which polymorphisms in the IL‐28B gene region affect host antiviral responses are not well understood. Using the HCV 1b and 2a replicon system, we compared the effects of IFN‐λs and IFN‐α on HCV RNA replication. The anti‐HCV effect of IFN‐λ3 and IFN‐α in combination was also assessed. Changes in gene expression induced by IFN‐λ3 and IFN‐α were compared using cDNA microarray analysis. IFN‐λs at concentrations of 1 ng/mL or more exhibited concentration‐ and time‐dependent HCV inhibition. In combination, IFN‐λ3 and IFN‐α had a synergistic anti‐HCV effect; however , no synergistic enhancement was observed for interferon‐stimulated response element (ISRE) activity or upregulation of interferon ‐ stimulated genes (ISGs). With respect to the time course of ISG upregulation, the peak of IFN‐λ3‐induced gene expression occurred later and lasted longer than that induced by IFN‐α. In addition, although the genes upregulated by IFN‐α and IFN‐λ3 were similar to microarray analysis, interferon‐stimulated gene expression appeared early and was prolonged by combined administration of these two IFNs. In conclusion, IFN‐α and IFN‐λ3 in combination showed synergistic anti‐HCV activity in vitro. Differences in time‐dependent upregulation of these genes might contribute to the synergistic antiviral activity.