Genetic toxicity assessment using liver cell models: past,present, and future

ABSTRACT

Genotoxic compounds may be detoxified to non-genotoxic metabolites while many pro-carcinogens require metabolic activation to exert their genotoxicity in vivo. Standard genotoxicity assays were developed and utilized for risk assessment for over 40 years. Most of these assays are conducted in metabolically incompetent rodent or human cell lines. Deficient in normal metabolism and relying on exogenous metabolic activation systems, the current in vitro genotoxicity assays often have yielded high false positive rates, which trigger unnecessary and costly in vivo studies. Metabolically active cells such as hepatocytes have been recognized as a promising cell model in predicting genotoxicity of carcinogens in vivo. In recent years, significant advances in tissue culture and biological technologies provided new opportunities for using hepatocytes in genetic toxicology. This review encompasses published studies (both in vitro and in vivo) using hepatocytes for genotoxicity assessment. Findings from both standard and newly developed genotoxicity assays are summarized. Various liver cell models used for genotoxicity assessment are described, including the potential application of advanced liver cell models such as 3D spheroids, organoids, and engineered hepatocytes. An integrated strategy, that includes the use of human-based cells with enhanced biological relevance and throughput, and applying the quantitative analysis of data, may provide an approach for future genotoxicity risk assessment.     

Inactivated influenza vaccine formulated with single-stranded RNA-based adjuvant confers mucosal immunity and cross-protection against influenza virus infection

Influenza vaccination is considered the most valuable means to prevent and control seasonal influenza infections, which causes various clinical symptoms, ranging from mild cough and fever to even death. Among various influenza vaccine types, the inactivated subunit type is known to provide improved safety with reduced reactogenicity. However, there are some drawbacks associated with inactivated subunit type vaccines, with the main ones being its low immunogenicity and the induction of Th2-biased immune responses. In this study, we investigated the role of a single-stranded RNA (ssRNA) derived from the intergenic region in the internal ribosome entry site of the Cricket paralysis virus as an adjuvant rather than the universal vaccine for a seasonal inactivated subunit influenza vaccine. The ssRNA adjuvant stimulated not only well-balanced cellular (indicated by IgG2a, IFN-γ, IL-2, and TNF-α) and humoral (indicated by IgG1 and IL-4) immune responses but also a mucosal immune response (indicated by IgA), a key protector against respiratory virus infections. It also increases the HI titer, the surrogate marker of influenza vaccine efficacy. Furthermore, ssRNA adjuvant confers cross-protective immune responses against heterologous influenza virus infection while promoting enhanced viral clearance. Moreover, ssRNA adjuvant increases the number of memory CD4⁺ and CD8⁺ T cells, which can be expected to induce long-term immune responses. Therefore, this ssRNA-adjuvanted seasonal inactivated subunit influenza vaccine might be the best influenza vaccine generating robust humoral and cellular immune responses and conferring cross-protective and long-term immunity.     

广州地区无偿献血人群人类疱疹病毒8型血清流行病学调查

目的：了解广州地区无偿献血者HHV-8的感染情况，为制定预防策略提供依据。方法：采用ELISA法检测3135名无偿献血者血浆HHV-8IgG抗体。结果：3135名献血者中6名血浆标本被检出HHV-8IgG抗体阳性，均为汉族、男性献血员，总阳性率为0．19％。不同年龄和性别组HHV-8感染率差异无统计学意义。结论：广州地区无偿献血人群HHV-8的感染率较低。     

二乙酰二脱水卫矛醇对小鼠白血病L1210细胞增殖的影响

目的　研究二乙酰二脱水卫矛醇 (1 ,2 :5 ,6 dianhydro 3 ,4 diacetylgalactitol,DADAG)的抗脑白血病作用及机制。方法　用小鼠脑内移植瘤模型、MTT法、DNA掺入法、流式细胞仪和Westernblot法 ,观察DADAG对小鼠脑内移植瘤和体外白血病L1 2 1 0 细胞的作用 ,并探讨作用机制。结果　DADAG对DBA/ 2小鼠脑内移植白血病L1 2 1 0 有明显的抑制作用 ;对体外白血病L1 2 1 0 细胞同样有很强的抗增殖作用 ,其IC50 值为 2 4 6mg·L- 1 。DADAG不可逆地抑制L1 2 1 0 细胞内DNA的生物合成。DADAG 2 4mg·L- 1 处理L1 2 1 0 细胞 6h后 ,细胞发生G2 /M周期阻滞 ,2 4h后达最高峰。细胞周期素B1 蛋白水平在DADAG处理 2 4h后开始下降 ,而磷酸化的细胞周期依赖性激酶CDK1在DADAG处理 6h后开始上调 ,并呈时间依赖性。结论　DADAG的抗脑白血病作用与其抑制白血病细胞的增殖密切相关     

Oxidative metabolism of lung macrophages exposed to sodium arsenite

Arsenic pollution has become increasingly severe. It occurs as the result of geological processes and different human activities. Arsenic toxicity at the respiratory level occurs mainly by inhalation of products of coal combustion. The aim of this study was to evaluate sodium arsenite (As³⁺) toxicity in murine alveolar macrophages (AMs) in vitro and its association with the alterations in cell metabolism.

No changes in viability, apoptosis or cell area were detected in AMs treated with As³⁺ concentrations up to 2 μM for 24–96 h. A marked decrease in these end-points was observed for As³⁺ concentrations ranging from 2.5 μM to 10 μM.

Regarding the dynamics of the endo-exocytic process triggered by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell incorporation, no variations were detected for As³⁺ concentrations lower than 2 μM while higher concentrations markedly modified this response.

MTT specific activity, as a measure of cell metabolic activity, was not modified irrespective of the As³⁺ concentration assayed. However, nitroblue tetrazolium (NBT) specific activity, as a measure of superoxide anion generation, is responsive but only to low As³⁺ doses.

Although this study focuses on lung macrophages, the effects of As³⁺ described herein may also apply to the response of macrophages residing in other organs.

Arsenite modifies the metabolic and the oxidative status of AMs in vitro. When macrophages are in an As³⁺ rich medium, they exhibit a reduction in respiratory burst levels and lose their intrinsic capacity to respond to toxicants.     

Presence of beta human papillomaviruses in nonmelanoma skin cancer from organ transplant recipients and immunocompetent patients in the West of Scotland

Background  Nonmelanoma skin cancer (NMSC) has been linked to cutaneous human papillomaviruses of the genus beta (betaPV).
Objectives  We sought to assess the presence of betaPV in NMSC biopsies from a group of Scottish skin cancer patients, both immunocompetent (IC) patients and immunosuppressed (IS) organ transplant recipients.
Methods  One hundred and twenty-one paraffin-embedded skin tumours (27 actinic keratosis, 41 intraepidermal carcinoma, 53 squamous cell carcinoma) and 11 normal skin samples were analysed for the presence of betaPV by a polymerase chain reaction–reverse hybridization assay designed to detect the presence of the 25 known betaPV genotypes.
Results  In IC patients, betaPV was detected in 30 of 59 (51%) tumours and two of 11 (18%) normal skin samples ( P  =   0·046). In IS patients, betaPV was found in 27 of 62 (44%) tumours; no normal skin samples were available for comparison. The most frequently found genotypes were HPV-24, HPV-15 and HPV-38. Of those tumours infected with betaPV, 28 of 57 (49%) were infected with more than one genotype (range 2–8). Tumours from IS patients were from a younger age group (mean age 57·4 years) than IC patients (mean age 73·8 years). Multiple infections were more common in tumours from IC patients (21 of 30; 70%) compared with those from IS patients (seven of 27; 26%) ( P  <   0·001). In the IC group, age did not appear to influence the distribution of single and multiple infections whereas in IS patients the proportion of multiple infections to single infections increased with age. There were no multiple infections in normal skin.
Conclusions  A wide spectrum of betaPV types was detected in our samples. Further characterization of betaPV in vivo is needed in order to determine the mechanisms by which the virus contributes to cutaneous carcinogenesis.     

新型纳米根管充填材料对成骨细胞生长的影响

通过体外培养的成骨细胞,采用二甲基噻唑二苯基四唑溴盐比色法和流式细胞术对新型纳米根管充填材料(nHA-PA66)作用下的成骨细胞生长情况的变化进行研究,评价其对成骨细胞生长的影响。以该材料的细胞培养基浸提液作用于实验组细胞,对照组采用培养基本身。实验组和对照组成骨细胞的生长情况和细胞周期无显著性差异,表明该新型纳米材料对成骨细胞的生长和细胞周期无不良影响。提示新型纳米根管充填材料的成骨细胞相容性较好,具有用作根充材料的基础。     

肝硬化患者凝血障碍与内毒素血症

本文通过多项止凝血实验指标和鲎试验对20例失代偿性肝硬化患者进行动态观察,发现血浆内毒素水平和ⅧR:Ag均增高.其他凝血因子和ATⅢ减低.纤维蛋白连接素减低.结果提示:(1)止凝血功能障碍与内毒素血症(ETM)并存,并与疾病的进展密切有关;(2)ETM的发生率和严重程度与肝硬化失代偿的程度有关;(3)ETM和止凝血功能异常的动态观察可作为判断预后的一项参考指标     

Sperm chromatin integrity in young men with no experiences of infertility and men from idiopathic infertility couples

Damage to the genetic component of spermatozoa seems to play the main role in a majority of cases where current approaches fail to reveal the specific cause of male infertility. In this study, we compared semen quality in men assigned to two defined groups: men from couples with unexplained infertility – idiopathic infertility (A) and young men with no experiences of infertility (B). All samples were examined by standard ejaculate analysis and sperm chromatin structure assay (SCSA). Sperm chromatin damage was significantly higher in men from group A than in those from group B. Similar results were obtained by comparison of men from group A (all men were normozoospermic) with normozoospermic men from group B. According to these results, we can suppose that chromatin disorders may be the causal factor of subfertility or infertility in some of these men. No evidence for a strong association between chromatin disorders and standard parameters of ejaculates was found. We failed to confirm a relationship between smoking and sperm quality in men from any of the investigated groups. SCSA is a method that facilitates the identification of infertile men who otherwise show normal semen variables.     

脂蛋白残粒RLP-c检测的临床意义

目的 探讨脂蛋白残粒RLP-c作为新的脂质指标的临床意义。方法 采用免疫分离法测定正常对照组(NC)、冠心病组(CHD)和2型糖尿病组(T2DM)的RLP-c水平,并比较RLP-c与其他脂质指标的相关性。结果 CHD组和T2DM组RLP-c水平明显高于NC组(P<0.01),其水平与甘油三脂(TG)和极低密度脂蛋白胆固醇(VLDL-c)高度相关(P<0.01)。结论 RLP-c可用于对动脉粥样硬化的危险性评估。