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Differences in endocrine and reproductive responses to substance exposure across generations: highlighting the importance of complementary findings 各代人的内分泌和生殖系统对药物接触反应的差异:强调补充性研究结果的重要性。
IF 4.8 2区 医学 Q1 TOXICOLOGY Pub Date : 2024-07-18 DOI: 10.1007/s00204-024-03813-3
Ingo Bichlmaier

This article analyzes the results from 112 Extended One-Generation Reproductive Toxicity studies. The objective was to determine if test animals show consistent endocrine and reproductive effects within the same and across different generations and life stages. The analysis, grounded in a comprehensive Binary Matrix, included 530 observed effects and 193 unique, statistically significant associations. Associations’ strength was quantified using Jaccard (J) coefficients to measure effect co-occurrence in the same study. Associated effects co-occur infrequently across the whole dataset (median J = 0.231). However, specific patterns emerged: associations of same effects across generations exhibited a higher strength (median J = 0.400) compared to associations of different effects (median J = 0.222). Notably, associations with effects observed in both the parental animals of the adult first filial generation (P1) and developing second filial generations (dF2) demonstrated J coefficients (with medians ranging from 0.300 to 0.430) that were approximately twofold higher than those of other associations. Consistently, equivalent life stage associations across generations revealed statistically significant higher association strengths for the P1 and dF2 generations (medians of 0.375 and 0.333, respectively) compared to other generations (medians of 0.200 and 0.174), possibly due to longer exposure duration and altered cross-talk between pregnant P1 dam and its conceptus. Overall, it is concluded that co-occurrence of associated effects in the same study is rather infrequent and that associations with effects in P1 and dF2 are stronger than all other associations. In general, the findings underscore the importance of independently analyzing each effect per generation due to the generally low co-occurrence rates of associated effects, challenging traditional expectations of generational continuity in toxic effects.

本文分析了 112 项单代生殖毒性扩展研究的结果。目的是确定试验动物在同一代和不同代以及不同生命阶段是否表现出一致的内分泌和生殖效应。该分析以全面的二进制矩阵为基础,包括 530 种观察到的效应和 193 种独特的、具有统计学意义的关联。相关性的强度采用 Jaccard (J) 系数进行量化,以衡量同一研究中效应的共现情况。在整个数据集中,相关效应共现的频率很低(中位数 J = 0.231)。不过,也出现了一些特定的模式:与不同效应的关联(中位数 J = 0.222)相比,跨代相同效应的关联表现出更高的强度(中位数 J = 0.400)。值得注意的是,在成年第一代孝子(P1)和发育中的第二代孝子(dF2)的亲代动物中观察到的效应关联的 J 系数(中位数在 0.300 到 0.430 之间)比其他关联高出约两倍。与其他世代(中位数分别为 0.200 和 0.174)相比,P1 和 dF2 世代(中位数分别为 0.375 和 0.333)的等效生命阶段关联在统计学上显示出更高的关联强度。总之,在同一项研究中,相关效应同时出现的情况并不多见,而且与 P1 和 dF2 的效应相关性强于所有其他相关性。总体而言,由于相关效应的共同出现率普遍较低,这些发现强调了对每一代的每种效应进行独立分析的重要性,这对传统的毒性效应世代连续性预期提出了挑战。
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引用次数: 0
Identification of reversible OATP1B1 and time-dependent CYP3A4 inhibition as the major risk factors for drug-induced cholestasis (DIC) 确定可逆性 OATP1B1 和时间依赖性 CYP3A4 抑制是药物性胆汁淤积症 (DIC) 的主要风险因素。
IF 4.8 2区 医学 Q1 TOXICOLOGY Pub Date : 2024-07-18 DOI: 10.1007/s00204-024-03794-3
Vlasia Kastrinou-Lampou, Raquel Rodríguez-Pérez, Birk Poller, Felix Huth, Zsuzsanna Gáborik, Beáta Mártonné-Tóth, Csilla Temesszentandrási-Ambrus, Heiko S. Schadt, Gerd A. Kullak-Ublick, Michael Arand, Gian Camenisch

Hepatic bile acid regulation is a multifaceted process modulated by several hepatic transporters and enzymes. Drug-induced cholestasis (DIC), a main type of drug-induced liver injury (DILI), denotes any drug-mediated condition in which hepatic bile flow is impaired. Our ability in translating preclinical toxicological findings to human DIC risk is currently very limited, mainly due to important interspecies differences. Accordingly, the anticipation of clinical DIC with available in vitro or in silico models is also challenging, due to the complexity of the bile acid homeostasis. Herein, we assessed the in vitro inhibition potential of 47 marketed drugs with various degrees of reported DILI severity towards all metabolic and transport mechanisms currently known to be involved in the hepatic regulation of bile acids. The reported DILI concern and/or cholestatic annotation correlated with the number of investigated processes being inhibited. Furthermore, we employed univariate and multivariate statistical methods to determine the important processes for DILI discrimination. We identified time-dependent inhibition (TDI) of cytochrome P450 (CYP) 3A4 and reversible inhibition of the organic anion transporting polypeptide (OATP) 1B1 as the major risk factors for DIC among the tested mechanisms related to bile acid transport and metabolism. These results were consistent across multiple statistical methods and DILI classification systems applied in our dataset. We anticipate that our assessment of the two most important processes in the development of cholestasis will enable a risk assessment for DIC to be efficiently integrated into the preclinical development process.

肝脏胆汁酸调节是一个由多种肝脏转运体和酶调节的多方面过程。药物性胆汁淤积症(DIC)是药物性肝损伤(DILI)的一种主要类型,是指任何药物介导的肝脏胆汁流动受损的情况。目前,我们将临床前毒理学研究结果转化为人类 DIC 风险的能力非常有限,这主要是由于物种间存在重大差异。因此,由于胆汁酸平衡的复杂性,利用现有的体外或硅学模型预测临床 DIC 也具有挑战性。在此,我们评估了 47 种已上市药物的体外抑制潜力,这些药物的 DILI 严重程度各不相同,它们对目前已知参与肝脏胆汁酸调节的所有代谢和转运机制都有抑制作用。报告的 DILI 关注度和/或胆汁淤积注释与被抑制的研究过程数量相关。此外,我们还采用了单变量和多变量统计方法来确定鉴别 DILI 的重要过程。我们发现细胞色素 P450 (CYP) 3A4 的时间依赖性抑制 (TDI) 和有机阴离子转运多肽 (OATP) 1B1 的可逆性抑制是胆汁酸转运和代谢相关机制中 DIC 的主要风险因素。这些结果在我们数据集中应用的多种统计方法和 DILI 分类系统中都是一致的。我们预计,我们对胆汁淤积症发生过程中两个最重要环节的评估将使 DIC 风险评估有效地融入临床前开发过程。
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引用次数: 0
The relative nature of the standards for proof of safety: a review of FDA’s safety standards for various consumer products 安全证明标准的相对性:食品和药物管理局对各种消费品安全标准的审查。
IF 4.8 2区 医学 Q1 TOXICOLOGY Pub Date : 2024-07-17 DOI: 10.1007/s00204-024-03816-0
George A. Burdock, Erik Hedrick

Are all food ingredients, dietary supplement ingredients and even foods, required to meet the same safety standards? Are they all equally safe? If so, then why do the various categories have different expressions describing their safety, such as “reasonable certainty of no harm” for food ingredients and “reasonable expectation of no harm” for dietary supplement ingredients? The basis for these different expressions is that they are not standards of safety, but standards of proof of safety. Just as in criminal vs. civil courts, the threshold for proving guilt or fault is different, so too are there differences between various categories of consumer products regulated by the US Food and Drug Administration. This manuscript describes the threshold requirements for each standard, as well as to the identity of the decision makers on what is safe, their credentials as decision makers and the databases mandated for their use.

是否所有食品成分、膳食补充剂成分甚至食品都必须符合相同的安全标准?它们是否都同样安全?如果是,那么为什么不同的类别有不同的描述其安全性的表述,如食品成分的 "合理确定无害 "和膳食补充剂成分的 "合理预期无害"?这些不同表述的依据是,它们不是安全标准,而是证明安全的标准。正如在刑事法庭和民事法庭中,证明有罪或过错的阈值不同一样,美国食品药品管理局监管的各类消费品之间也存在差异。本手稿介绍了每种标准的阈值要求,以及决定什么是安全的决策者的身份、他们作为决策者的资质和规定使用的数据库。
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引用次数: 0
Integrated systematic functional screen and fine-mapping decipher the role and genetic regulation of RPS19 in colorectal cancer development 整合系统功能筛选和精细图谱,破解 RPS19 在结直肠癌发展中的作用和遗传调控。
IF 4.8 2区 医学 Q1 TOXICOLOGY Pub Date : 2024-07-16 DOI: 10.1007/s00204-024-03822-2
Can Chen, Wenzhuo Wang, Caibo Ning, Zequn Lu, Ming Zhang, Ying Zhu, Jianbo Tian, Haijie Li, Yue Ge, Beifang Yang, Xiaoping Miao

Despite genome-wide association studies (GWAS) have identified more than 200 risk loci associated with colorectal cancer (CRC), the causal genes or risk variants within these loci and their biological functions remain not fully revealed. Recently, the genomic locus 19q13.2, with the lead SNP rs1800469 was identified as a crucial CRC risk locus in Asian populations. However, the functional mechanism of this region has not been fully elucidated. Here we employed an RNA interfering-based on-chip approach to screen for the genes essential for cell proliferation in the CRC risk locus 19q13.2. Notably, we found that RPS19 exhibited the most significant effect among the identified genes and acted as a critical oncogene facilitating CRC cell proliferation. Subsequently, combining integrative fine-mapping analysis and a large-scale population study consisting of 6027 cases and 6099 controls, we prioritized rs1025497 as a potential causal candidate for CRC risk, demonstrating that rs1025497[A] allele significantly reduced the risk of CRC (OR 0.70, 95% confidence interval = 0.56–0.83, P = 1.12 × 10–6), which was further validated in UK Biobank cohort comprising 5,313 cases and 21,252 controls. Mechanistically, we experimentally elucidated that variant rs1025497 might acted as an allele-specific silencer, inhibiting the expression level of oncogene RPS19 mediated by the transcription suppressive factor HBP1. Taken together, our sturdy unveils the significant role of RPS19 during CRC pathogenesis and delineates its distal regulatory mechanism mediated by rs1025497, advancing our understanding of the etiology of CRC and provided new insights into the personalized medicine of human cancer.

尽管全基因组关联研究(GWAS)发现了 200 多个与结直肠癌(CRC)相关的风险位点,但这些位点中的致病基因或风险变异及其生物学功能仍未完全揭示。最近,以 SNP rs1800469 为首的基因组位点 19q13.2 被确定为亚洲人群中一个关键的 CRC 风险位点。然而,该区域的功能机制尚未完全阐明。在这里,我们采用了基于 RNA 干扰的芯片方法来筛选 CRC 风险位点 19q13.2 中对细胞增殖至关重要的基因。值得注意的是,我们发现 RPS19 在已发现的基因中具有最显著的影响,是促进 CRC 细胞增殖的关键癌基因。随后,结合综合精细图谱分析和由 6027 例病例和 6099 例对照组成的大规模人群研究,我们优先选择 rs1025497 作为 CRC 风险的潜在因果候选基因,结果表明 rs1025497[A] 等位基因可显著降低 CRC 风险(OR 0.70,95% 置信区间 = 0.56-0.83,P = 1.12 × 10-6),这一结果在由 5313 例病例和 21252 例对照组成的英国生物库队列中得到了进一步验证。从机理上讲,我们通过实验阐明了变异体rs1025497可能作为等位基因特异性沉默因子,抑制由转录抑制因子HBP1介导的癌基因RPS19的表达水平。综上所述,我们的研究揭示了 RPS19 在 CRC 发病过程中的重要作用,并阐明了 rs1025497 介导的远端调控机制,从而推进了我们对 CRC 病因学的理解,并为人类癌症的个体化医疗提供了新的见解。
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引用次数: 0
Sirtuin 3-activated superoxide dismutase 2 mediates fluoride-induced osteoblastic differentiation in vitro and in vivo by down-regulating reactive oxygen species Sirtuin 3激活的超氧化物歧化酶2通过下调活性氧介导氟化物诱导的体外和体内成骨细胞分化。
IF 4.8 2区 医学 Q1 TOXICOLOGY Pub Date : 2024-07-16 DOI: 10.1007/s00204-024-03819-x
Liu Yang, Qiao Li, Sa Wang, Yi Ji, Xinbo Ma, Ming Qin, Yanhui Gao, Yanmei Yang

Skeletal fluorosis is a chronic metabolic bone disease caused by long-term excessive fluoride intake. Abnormal differentiation of osteoblasts plays an important role in disease progression. Research on the mechanism of fluoride-mediated bone differentiation is necessary for the prevention and treatment of skeletal fluorosis. In the present study, a rat model of fluorosis was established by exposing it to drinking water containing 50 mg/L F. We found that fluoride promoted Runt-related transcription factor 2 (RUNX2) as well as superoxide dismutase 2 (SOD2) and sirtuin 3 (SIRT3) expression in osteoblasts of rat bone tissue. In vitro, we also found that 4 mg/L sodium fluoride promoted osteogenesis-related indicators as well as SOD2 and SIRT3 expression in MG-63 and Saos-2 cells. In addition, we unexpectedly discovered that fluoride suppressed the levels of reactive oxygen species (ROS) and mitochondrial reactive oxygen species (mtROS) in osteoblasts. When SOD2 or SIRT3 was inhibited in MG-63 cells, fluoride-decreased ROS and mtROS were alleviated, which in turn inhibited fluoride-promoted osteogenic differentiation. In conclusion, our results suggest that SIRT3/SOD2 mediates fluoride-promoted osteoblastic differentiation by down-regulating reactive oxygen species.

氟骨症是一种慢性代谢性骨病,由长期过量摄入氟引起。成骨细胞的异常分化在疾病进展中起着重要作用。研究氟介导骨分化的机制对于预防和治疗骨骼氟中毒非常必要。本研究通过将大鼠暴露于含 50 mg/L 氟的饮用水中,建立了氟中毒大鼠模型。我们发现氟促进了大鼠骨组织成骨细胞中 Runt 相关转录因子 2(RUNX2)以及超氧化物歧化酶 2(SOD2)和 sirtuin 3(SIRT3)的表达。在体外,我们还发现 4 毫克/升的氟化钠能促进成骨相关指标以及 SOD2 和 SIRT3 在 MG-63 和 Saos-2 细胞中的表达。此外,我们还意外地发现氟抑制了成骨细胞中活性氧(ROS)和线粒体活性氧(mtROS)的水平。当抑制 MG-63 细胞中的 SOD2 或 SIRT3 时,氟减少的 ROS 和 mtROS 得到缓解,这反过来又抑制了氟促进的成骨分化。总之,我们的研究结果表明,SIRT3/SOD2 通过下调活性氧介导氟化物促进的成骨细胞分化。
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引用次数: 0
A new 3D model of L929 fibroblasts microtissues uncovers the effects of Bothrops erythromelas venom and its antivenom 一种新的 L929 成纤维细胞微组织三维模型揭示了红腹锦鸡毒液及其抗毒血清的作用。
IF 4.8 2区 医学 Q1 TOXICOLOGY Pub Date : 2024-07-15 DOI: 10.1007/s00204-024-03824-0
F. R. S. Andrade, E. L. da Silva, A. D. Marinho, A. C. X. Oliveira, D. Sánchez-Porras, F. Bermejo-Casares, R. C. Montenegro, V. Carriel, H. S. A. Monteiro, R. J. B. Jorge

In Brazil, around 80% of snakebites are caused by snakes of the genus Bothrops. A three-dimensional culture model was standardized and used to perform treatments with Bothrops erythromelas venom (BeV) and its antivenom (AV). The MRC-5 and L929 cell lines were cultured at increasing cell densities. Morphometric parameters were evaluated through images obtained from an inverted microscope: solidity, circularity, and Feret diameter. L929 microtissues (MT) showed better morphometric data, and thus they were used for further analysis. MT viability was assessed using the acridine orange and ethidium bromide staining method, which showed viable cells in the MT on days 5, 7, and 10 of cultivation. Histochemical and histological analyses were performed, including hematoxylin/eosin staining, which showed a good structure of the spheroids. Alcian blue staining revealed the presence of acid proteoglycans. Immunohistochemical analysis with ki-67 showed different patterns of cell proliferation. The MT were also subjected to pharmacological tests using the BeV, in the presence or absence of its AV. The results showed that the venom was not cytotoxic, but it caused morphological changes. The MT showed cell detachment, losing their structure. The antivenom was able to partially prevent the venom activities.

在巴西,大约 80% 的蛇咬伤是由 Bothrops 属蛇引起的。我们对一种三维培养模型进行了标准化,并利用该模型对两栖类红腹锦蛇毒液(BeV)及其抗蛇毒血清(AV)进行治疗。MRC-5 和 L929 细胞系的培养细胞密度不断增加。通过倒置显微镜获得的图像对形态计量参数进行了评估:坚实度、圆度和 Feret 直径。L929 显微组织(MT)显示出更好的形态计量数据,因此被用于进一步分析。采用吖啶橙和溴化乙锭染色法评估 MT 的存活率,结果显示 MT 在培养第 5、7 和 10 天有存活细胞。进行了组织化学和组织学分析,包括苏木精/伊红染色,结果显示球体结构良好。阿尔新蓝染色显示存在酸性蛋白聚糖。用 ki-67 进行的免疫组化分析显示了不同的细胞增殖模式。还利用 BeV 对 MT 进行了药理测试,无论是否存在其 AV。结果表明,毒液没有细胞毒性,但会引起形态变化。MT 出现细胞脱落,失去结构。抗蛇毒血清能够部分阻止蛇毒的活动。
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引用次数: 0
Comparison of DNA damage in fresh and frozen blood samples: implications for the comet assay in human biomonitoring studies 新鲜和冷冻血液样本中 DNA 损伤的比较:彗星试验在人类生物监测研究中的意义。
IF 4.8 2区 医学 Q1 TOXICOLOGY Pub Date : 2024-07-14 DOI: 10.1007/s00204-024-03823-1
Katarina Matković, Marko Gerić, Luka Kazensky, Mirta Milić, Vilena Kašuba, Ante Cvitković, Mandica Sanković, Antun Šumanovac, Peter Møller, Goran Gajski

The use of the comet assay in large biomonitoring studies may present logistical and technical challenges because of the processing of numerous samples. Proper sample preservation becomes imperative to prevent spurious DNA breakage. Previous research has shown the feasibility of conducting the comet assay on frozen blood samples, highlighting the potential of freezing at – 80 °C in preserving DNA integrity. Nonetheless, this approach presents challenges, including potential DNA damage during freezing and thawing, variability in processing, and the need for standardized protocols. Our objective was to evaluate whether there are comparable results in DNA migration assessed by the comet assay between fresh and frozen blood samples on a larger scale (N = 373). In our findings, elevated DNA migration was evident in frozen samples relative to fresh ones. Additionally, smoking, alcohol consumption, and season were linked to increased DNA damage levels in whole blood cells. Based on our results and available literature, conducting the comet assay on frozen blood samples emerges as a practical and efficient approach for biomonitoring and epidemiological research. This method enables the assessment of DNA damage in large populations over time, with samples, if properly cryopreserved, that may be used for years, possibly even decades. These observations hold significant implications for large-scale human biomonitoring and long-term epidemiological studies, particularly when samples are collected during fieldwork or obtained from biobanks. Continued method optimization and validation efforts are essential to enhance the utility of this approach in environmental and occupational health studies, emphasizing caution when comparing data obtained between fresh and frozen blood samples.

在大型生物监测研究中使用彗星试验可能会面临后勤和技术方面的挑战,因为需要处理大量样本。为了防止假性 DNA 断裂,必须妥善保存样本。先前的研究表明,在冷冻血液样本上进行彗星测定是可行的,这突出表明了在零下 80 °C 的温度下冷冻样本在保持 DNA 完整性方面的潜力。然而,这种方法也面临着挑战,包括冷冻和解冻过程中 DNA 的潜在损伤、处理过程中的可变性以及对标准化方案的需求。我们的目的是在更大范围内(N = 373)评估通过彗星试验评估的新鲜和冷冻血液样本的 DNA 迁移结果是否具有可比性。根据我们的研究结果,与新鲜样本相比,冷冻样本的 DNA 迁移率明显升高。此外,吸烟、饮酒和季节也与全血细胞中 DNA 损伤水平的增加有关。根据我们的研究结果和现有文献,对冷冻血液样本进行彗星测定是一种实用、高效的生物监测和流行病学研究方法。这种方法可以评估大量人群在一段时间内的 DNA 损伤情况,如果样本冷冻保存得当,可以使用数年甚至数十年。这些观察结果对大规模人类生物监测和长期流行病学研究具有重要意义,尤其是在实地工作中收集样本或从生物库中获取样本时。要提高这种方法在环境和职业健康研究中的实用性,必须继续进行方法优化和验证工作,同时强调在比较新鲜和冷冻血液样本之间获得的数据时要小心谨慎。
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引用次数: 0
Synthesis and in vitro assessment of the reactivation profile of clinically available oximes on the acetylcholinesterase model inhibited by A-230 nerve agent surrogate 合成和体外评估临床可用的肟类化合物对受 A-230 神经毒剂替代物抑制的乙酰胆碱酯酶模型的再激活情况。
IF 4.8 2区 医学 Q1 TOXICOLOGY Pub Date : 2024-07-14 DOI: 10.1007/s00204-024-03821-3
Leandro B. Bernardo, Caio V. N. Borges, Pedro A. G. Buitrago, Kamil Kuča, Samir F. A. Cavalcante, Roberto B. Sousa, Antônio L. S. Lima, Daniel A. S. Kitagawa

The risk of the use of toxic chemicals for unlawful acts has been a matter of concern for different governments and multilateral agencies. The Organisation for the Prohibition of Chemical Weapons (OPCW), which oversees the implementation of the Chemical Weapons Convention (CWC), considering recent events employing chemical warfare agents as means of assassination, has recently included in the CWC “Annex on Chemicals” some organophosphorus compounds that are regarded as acting in a similar fashion to the classical G- and V-series of nerve agents, inhibiting the pivotal enzyme acetylcholinesterase. Therefore, knowledge of the activity of the pyridinium oximes, the sole class of clinically available acetylcholinesterase reactivators to date, is plainly justified. In this paper, continuing our research efforts in medicinal chemistry on this class of toxic chemicals, we synthesized an A-230 nerve agent surrogate and applied a modified Ellman’s assay to evaluate its ability to inhibit our enzymatic model, acetylcholinesterase from Electrophorus eel, and if the clinically available antidotes are able to rescue the enzyme activity for the purpose of relating the findings to the previously disclosed in silico data for the authentic nerve agent and other studies with similar A-series surrogates. Our experimental data indicates that pralidoxime is the most efficient compound for reactivating acetylcholinesterase inhibited by A-230 surrogate, which is the opposite of the in silico data previously disclosed.

将有毒化学品用于非法行为的风险一直是各国政府和多边机构关注的问题。负责监督《化学武器公约》(CWC)执行情况的禁止化学武器组织(OPCW)考虑到最近发生的使用化学战剂作为暗杀手段的事件,最近在《化学武器公约》的 "化学品附件 "中列入了一些有机磷化合物,这些化合物被认为具有与传统的 G 和 V 系列神经毒剂类似的作用,可抑制关键酶乙酰胆碱酯酶。因此,我们有必要了解吡啶肟类化合物的活性,这是迄今为止唯一一类可用于临床的乙酰胆碱酯酶再活化剂。在本文中,我们继续对这一类有毒化学品进行药物化学研究,合成了一种 A-230 神经毒剂替代物,并采用改良的埃尔曼试验来评估其对我们的酶模型--来自电鳗鲡的乙酰胆碱酯酶--的抑制能力,以及临床上可用的解毒剂是否能够挽救酶的活性,以便将研究结果与之前披露的真实神经毒剂的硅学数据及其他类似 A 系列替代物的研究结果联系起来。我们的实验数据表明,普拉唑肟是重新激活被 A-230 代用品抑制的乙酰胆碱酯酶最有效的化合物,这与之前披露的硅学数据相反。
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引用次数: 0
A multi-biomarker micronucleus assay using imaging flow cytometry 利用成像流式细胞仪进行多生物标记微核检测。
IF 4.8 2区 医学 Q1 TOXICOLOGY Pub Date : 2024-07-12 DOI: 10.1007/s00204-024-03801-7
Danielle S. G. Harte, Anthony M. Lynch, Jatin Verma, Paul Rees, Andrew Filby, John W. Wills, George E. Johnson

Genetic toxicity testing assesses the potential of compounds to cause DNA damage. There are many genetic toxicology screening assays designed to assess the DNA damaging potential of chemicals in early drug development aiding the identification of promising drugs that have low-risk potential for causing genetic damage contributing to cancer risk in humans. Despite this, in vitro tests generate a high number of misleading positives, the consequences of which can lead to unnecessary animal testing and/or the abandonment of promising drug candidates. Understanding chemical Mode of Action (MoA) is vital to identifying the true genotoxic potential of substances and, therefore, the risk translation into the clinic. Here we demonstrate a simple, robust protocol for staining fixed, human-lymphoblast p53 proficient TK6 cells with antibodies against ɣH2AX, p53 and pH3S28 along with DRAQ5™ DNA staining that enables analysis of un-lysed cells via microscopy approaches such as imaging flow cytometry. Here, we used the Cytek® Amnis® ImageStream®X Mk II which provides a high-throughput acquisition platform with the sensitivity of flow cytometry and spatial morphological information associated with microscopy. Using the ImageStream manufacturer’s software (IDEAS® 6.2), a masking strategy was developed to automatically detect and quantify micronucleus events (MN) and characterise biomarker populations. The gating strategy developed enables the generation of a template capable of automatically batch processing data files quantifying cell-cycle, MN, ɣH2AX, p53 and pH3 populations simultaneously. In this way, we demonstrate how a multiplex system enables DNA damage assessment alongside MN identification using un-lysed cells on the imaging flow cytometry platform. As a proof-of-concept, we use the tool chemicals carbendazim and methyl methanesulphonate (MMS) to demonstrate the assay’s ability to correctly identify clastogenic or aneugenic MoAs using the biomarker profiles established.

遗传毒性测试评估化合物造成 DNA 损伤的可能性。有许多遗传毒理学筛选试验旨在评估早期药物开发过程中化学品的 DNA 损伤潜力,以帮助识别有潜力的药物,这些药物在导致人类癌症风险的遗传损伤方面风险较低。尽管如此,体外试验仍会产生大量误导性阳性结果,其后果可能导致不必要的动物试验和/或放弃有前途的候选药物。了解化学作用模式(MoA)对于确定物质的真正遗传毒性潜力,从而将风险转化到临床中至关重要。在这里,我们展示了一种简单、稳健的方案,即用针对ɣH2AX、p53 和 pH3S28 的抗体对固定的、具有 p53 能力的人类淋巴细胞 TK6 细胞进行染色,同时进行 DRAQ5™ DNA 染色,从而通过成像流式细胞仪等显微镜方法对未裂解的细胞进行分析。在这里,我们使用了 Cytek® Amnis® ImageStream®X Mk II,它提供了一个高通量采集平台,具有流式细胞仪的灵敏度和显微镜的空间形态信息。利用 ImageStream 制造商的软件(IDEAS® 6.2),开发了一种屏蔽策略,用于自动检测和量化微核事件(MN),并描述生物标记群的特征。所开发的屏蔽策略能够生成一个模板,自动批量处理数据文件,同时量化细胞周期、MN、ɣH2AX、p53 和 pH3 群体。通过这种方式,我们展示了多重系统如何在成像流式细胞仪平台上使用未裂解细胞进行 DNA 损伤评估和 MN 鉴定。作为概念验证,我们使用工具化学品多菌灵和甲基甲磺酸甲酯(MMS)来证明该检测方法有能力利用所建立的生物标记图谱正确识别致克隆性或非遗传性MoAs。
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引用次数: 0
A genetic variant in the TAPBP gene enhances cervical cancer susceptibility by increasing m6A modification TAPBP 基因中的一个遗传变异会通过增加 m6A 修饰来提高宫颈癌的易感性。
IF 4.8 2区 医学 Q1 TOXICOLOGY Pub Date : 2024-07-12 DOI: 10.1007/s00204-024-03820-4
Jing Hu, Shizhi Wang, Xing Zhang, Wenjing Yan, Haohan Liu, Xue Chen, Yamei Nie, Fengying Liu, Yun Zheng, Yiran Lu, Hua Jin

Genetic variants can affect gene expression by altering the level of N6-methyladenosine (m6A) modifications. A better understanding of the association of these genetic variants with susceptibility to cervical cancer (CC) can promote advances in disease screening and treatment. Genome-wide identification of m6A-associated functional SNPs for CC was performed using the TCGA and JENGER databases, incorporating the data from RNA-seq and MeRIP-seq. The screened risk-associated SNP rs1059288 (A>G), which is located in the 3′ UTR of TAPBP, was further validated in a case–control study involving 921 cases and 1077 controls. The results revealed a significant association between rs1059288 and the risk of CC (OR 1.48, 95% CI 1.13–1.92). Mechanistically, the presence of the risk G allele of rs1059288 was associated with increased m6A modification of TAPBP compared with the A allele. This modification was facilitated by the m6A methyltransferase METTL14 and the reading protein YTHDF2. Immunohistochemical staining of tissue microarrays containing 61 CC and 45 normal tissues showed an overexpression of TAPBP in CC. Furthermore, the upregulation of TAPBP promoted the growth and migration of CC cells as well as tumor-forming ability, inhibited apoptosis, and conferred increased resistance to commonly used chemotherapeutic drugs such as bleomycin, cisplatin, and doxorubicin. Knockdown of TAPBP inhibited the JAK/STAT/MICB signaling pathway in CC cells and upregulated certain immune genes including ISG15, IRF3, PTPN6, and HLA-A. These findings offer insights into the involvement of genetic variations in TAPBP in the development and progression of CC.

基因变异可通过改变 N6-甲基腺苷(m6A)修饰水平来影响基因表达。更好地了解这些基因变异与宫颈癌(CC)易感性的关系,可以促进疾病筛查和治疗的进步。我们利用 TCGA 和 JENGER 数据库,结合 RNA-seq 和 MeRIP-seq 数据,对与宫颈癌相关的 m6A 功能 SNPs 进行了全基因组鉴定。筛选出的风险相关 SNP rs1059288(A>G)位于 TAPBP 的 3' UTR,在一项涉及 921 例病例和 1077 例对照的病例对照研究中得到了进一步验证。结果显示,rs1059288 与 CC 风险之间存在明显关联(OR 1.48,95% CI 1.13-1.92)。从机理上讲,与 A 等位基因相比,rs1059288 的 G 等位基因与 TAPBP 的 m6A 修饰增加有关。这种修饰由 m6A 甲基转移酶 METTL14 和阅读蛋白 YTHDF2 促进。对含有61个CC和45个正常组织的组织芯片进行免疫组化染色显示,TAPBP在CC中过度表达。此外,TAPBP的上调促进了CC细胞的生长和迁移以及肿瘤形成能力,抑制了细胞凋亡,并增强了对博来霉素、顺铂和多柔比星等常用化疗药物的耐药性。敲除 TAPBP 可抑制 CC 细胞中的 JAK/STAT/MICB 信号通路,并上调某些免疫基因,包括 ISG15、IRF3、PTPN6 和 HLA-A。这些发现有助于深入了解TAPBP基因变异对CC发病和进展的影响。
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引用次数: 0
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Archives of Toxicology
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