Quinolone antibacterial-agent-induced cutaneous phototoxicity: ear swelling reactions in Balb/c mice

The phototoxic potentials of quinolone derivatives and the possibility of free radical contribution to their phototoxicity were investigated by measuring increments in ear thickness. Balb/c mice, fasted overnight, were orally administered ofloxacin (OFLX), lomefloxacin (LMFX), enoxacin (ENX), ciprofloxacin (CPFX) and DR-3355 (the s-isomer of OFLX), and immediately exposed to ultraviolet-A light (UVA: 320-400 nm) for 4 h (21.6 joules/cm2). Measurement of ear thickness was carried out 0, 24 and 48 h after the end of irradiation. The time-course profiles of ear thickness varied with both the doses and the quinolone used, but linear dose-response curves were obtained from the data 24 h after irradiation ended. The 50% ear thickness increment-inducing doses of LMFX, ENX, OFLX, CPFX and DR-3355 were calculated as 24.8, 81.9, 428.0, 457.9 and 526.6 mg/kg, respectively. The phototoxic potential of these quinolones coincided with the data obtained previously by measuring the incidence of erythema on the ears. Pretreatment with butylated hydroxtoluene, a free radical scavenger, almost completely prevented all swelling reactions induced by the quinolones. These results suggest that the degree of phototoxicity induced by the quinolones used could depend on the balance between the generation of free radicals and the effectiveness of the defense systems against toxic radicals.     

氯化镉在Balb/c 3T3细胞转化过程中对细胞凋亡的影响

目的探讨氯化镉(CdCl2)在Balb/c 3T3细胞转化过程中对细胞凋亡的影响及其分子机制。方法N-甲基-N′-硝基-N-亚硝基胍(MNNG)启动后,CdCl2持续处理Balb/c 3T3细胞14 d,建立两阶段细胞转化模型。苔盼蓝排染法检测CdCl2的细胞毒性,AnnexinV-FITC/PI双染色结合流式细胞仪检测细胞凋亡率,小鼠毒理基因芯片检测CdCl2促癌作用过程中的基因表达变化。结果1 mg/L MNNG启动后,324μg/L CdCl2持续处理能诱导细胞转化灶的出现。CdCl2处理2 d后细胞存活率降低,凋亡率显著升高。同时基因表达谱分析筛选出的差异表达基因中,有11个基因的功能与细胞凋亡有关,10个基因的功能与细胞抗氧化机制有关。结论CdCl2在促癌过程能诱导Balb/c 3T3细胞凋亡,同时影响凋亡和抗氧化相关基因的表达。     

Dose-dependent effects of berberine on cell cycle pause and apoptosis in Balb/c 3T3 cells

In determining the morphological appearance of Balb/c 3T3 cells from berberine-treated (100 and 200 g/ml) cultures by light microscopy demonstrated that the high berberine concentration (200 g/ml) treatment was associated with the accumulation of numerous apoptotic cells, as identified by condensed nuclei and decrease in cell size. On the other hand, accumulation of cells in G₂/M phase instead of induction of apoptosis was observed after 48–72 h of 100 g/ml berberine treatment. Berberine was found mainly in cytoplasm during berberine-induced (100 g/ml) cell cycle G₂/M arrest, while it was highly concentrated in nuclei in the induction of apoptosis under high dose of berberine (200 g/ml) treatment. Further addition of berberine (100–200 g/ml) had little effect on the induction of apoptosis in the cells that had already been exposed to 100 g/ml of berberine for 48 h. Our results suggest that there may exist in Balb/c 3T3 cells an important threshold for regulation of cell cycle pause and induction of apoptosis, that is dose-dependent.     

岗田酸促癌作用过程中对Balb/c 3T3细胞凋亡的影响

目的初步探讨促癌物岗田酸(OA)在促进Balb/c 3T3细胞转化过程中对细胞凋亡的影响及其分子机制。方法以N-甲基-N′-硝基-N-亚硝基胍(MNNG)为启动剂,OA为促癌剂,建立Balb/c 3T3细胞转化模型。台盼蓝染色法检测OA的细胞毒性,AnnexinⅤ-FITC/PI双染色流式细胞术检测细胞凋亡率,小鼠毒理基因芯片分析OA促癌作用过程中的基因表达变化,同时采用荧光定量RT-PCR方法验证部分基因的表达情况。结果MNNG 1 mg.L-1启动后,7.8μg.L-1OA持续处理能促进Balb/c3T3细胞的转化。OA处理3,5和7 d后细胞存活率降低,同时细胞凋亡率显著升高;基因表达谱分析显示,Bnip3,Cycs和caspase3等细胞凋亡相关基因以及Gstp2,Txn1和Prdx1等抗氧化基因的表达发生明显改变。RT-PCR检测显示OA处理3,7 d时,SPP1和Txn1基因的表达变化与芯片检测结果相似。结论OA在促癌过程能诱导Balb/c 3T3细胞凋亡,影响线粒体凋亡通路相关基因和抗氧化基因的表达。     

磁性附着体中软磁合金引起小鼠成纤维细胞L929凋亡的研究

目的研究磁性附着体中软磁合金对小鼠成纤维细胞L929凋亡的影响,进而评价其生物相容性。方法提取含钛合金、软磁合金、钴铬合金的浸提液,分别以25%、50%、100%浓度处理L929细胞24 h,并测量细胞凋亡率(AP)。结果随着金属浸提液浓度的增加,L929细胞凋亡率亦增加,软磁合金与含钛合金及钴铬合金之间AP值的差异均无统计学意义(P>0.05)。结论本实验为磁性附着体的临床应用提供一定的生物学依据。磁性附着体中软磁合金的生物相容性要求合临床应用材料的要求,可以在临床推广。     

Bradykinin stimulates DNA synthesis in competent Balb/c 3T3 cells and enhances inositol phosphate formation induced by platelet-derived growth factor

Both platelet-derived growth factor (PDGF) and bradykinin were found to induce a growth response in Balb/c 3T3 cells. However, whereas PDGF brought about a five-fold increase in the incorporation of [3H]thymidine into DNA, the response to bradykinin was never more than 50%. When bradykinin was present simultaneously with sub-optimal concentrations of PDGF the response was about 15% greater than with PDGF alone. In contrast, if the cells were made competent by a 5 hr preincubation with PDGF which was then washed away, subsequent addition of bradykinin induced a more than two-fold increase in incorporation of [3H]thymidine into DNA compared with competent cells subsequently incubated with serum-free medium alone. Bradykinin also acted synergistically with insulin when the two agents were added simultaneously to competent cells. PDGF induced marked increases in the concentration of inositol phosphates at 30 min after stimulation, but by this time point any effect of bradykinin had disappeared. However, the simultaneous presence of PDGF and bradykinin induced increases at 30 min that were 50-100% greater than with PDGF alone. It is concluded that the pathways by which PDGF and bradykinin initiate a growth response in BALB/c 3T3 cells only partly overlap. Their actions on the synthesis of inositol phosphates exhibit distinctive temporal characteristics, but can be co-operative at 30 min and at earlier time intervals. This effect was found to be time-dependent, and developed over the first 5 min.     

雷帕霉素诱导Balb/c小鼠CD4~+ CD25~+ foxp3~+调节性T细胞增殖

目的探讨雷帕霉素对Balb/c小鼠CD4+ CD25+ foxp3+调节性T细胞的作用。方法取8wk龄的SPF级Balb/c小鼠30只,随机分为两组,实验组每只灌胃雷帕霉素0.4mg.d-1,对照组灌胃每天予等体积无菌水,共3wk。无菌条件下肝素抗凝心脏采血,分离脾脏,制备单细胞悬液。采用流式细胞仪检测小鼠外周血和脾细胞CD4+CD25+T细胞,实时定量PCR检测小鼠脾细胞foxp3 mRNA的表达。结果实验组小鼠外周血和脾细胞中CD4+CD25+T细胞的比例分别为(9.95±4.65)%和(24.13±10.06)%,对照组小鼠外周血和脾细胞中CD4+CD25+T细胞的比例分别为(5.01±1.49)%和(8.48±3.19)%,差异均有显著性(P<0.01)。实验组小鼠脾细胞foxp3 mRNA的表达水平明显高于对照组,是对照组的6.029倍,差异有显著性(P<0.01)。结论雷帕霉素能够明显诱导Balb/c小鼠体内CD4+CD25+T细胞的增殖,并能提高foxp3+ mRNA的表达。     

A proteomic approach to investigate AuNPs effects in Balb/3T3 cells

Although gold nanoparticles (AuNPs) are currently used in several industrial products and biomedical applications, information about their biological effects is very limited. Thus, it is becoming crucial to assess their safety and adequately investigate the complexity of cell–nanoparticles interactions. In this work, the Balb/3T3 mouse fibroblast cell line was selected as an in vitro model to study the effects of AuNPs. Alteration of cellular processes and biochemical pathways caused by AuNPs exposure was investigated by analysing the differentially expressed proteome. Of interest was the difference observed in the protein pattern expression of cells exposed to AuNPs. It was found that 88 and 83 proteins were de-regulated after exposure to 5 and 15 nm AuNPs, respectively. Analysis of the proteome revealed that AuNPs triggers several pathways related to cellular growth and proliferation, cell morphology, cell cycle regulation, cellular function and maintenance, oxidative stress, and inflammatory response. Moreover, SPR analysis showed an increase of ECM proteins biosynthesis in cells exposed to AuNPs. We observed by TEM analysis that NPs are internalized and confined mainly in autophagosomes. Endoplasmic reticulum stressed and modification at mitochondrial level occurred. This study aims to improve existing knowledge necessary for a correct assessment of the balance between AuNPs potential adverse and beneficial effects and might have important implications for biomedical applications (e.g. nanomedicine).     

An optimised data analysis for the Balb/c 3T3 cell transformation assay and its application to metal compounds

The Balb/c3T3 cell transformation assay (CTA) is an available in vitro system to detect the carcinogenic potential of chemicals. Currently, the European Centre for the Validation of Alternative Methods (ECVAM) is validating this test, assessing its reliability and relevance. Its endpoint is the formation of type III foci, which is, when using clone A31-1-1, a very rare event that usually does not occur at all for negative controls. The carcinogenic potential of a compound tested is assessed by comparing the number of foci in treated and untreated cells. The objective of the present work is to optimise the data analysis for this endpoint by applying the most commonly used approach by a t-test and the Fisher's exact test as an alternative approach. For this purpose selected metal compounds classified as carcinogenic (NaAsO2, CdCl2, cisPt), as suspected carcinogenic (C6H5)4AsCl, CH3HgCl), or as compounds without evidence of carcinogenic properties in humans ((NH4)2PtCl6, NaVO3) as well as a non-carcinogenic (AgNO3) were analysed. Our evaluation revealed that the t-test approach, which assumes normality of data, is not appropriate. The results demonstrated that the statistical analysis by Fisher's exact test, better reflecting the data properties, greatly facilitates the interpretation of Balb/c3T3 CTA data regarding carcinogenic potential.     

Cellular distribution and degradation of cobalt ferrite nanoparticles in Balb/3T3 mouse fibroblasts

The effect of the concentration of cobalt ferrite (CoFe₂O₄) nanoparticles (NPs) on their intracellular location and distribution has been explored by synchrotron radiation X-ray and fluorescence microscopy (SR-XRF) monitoring the evolution of NPs elemental composition as well. In cells exposed to low concentrations of CoFe₂O₄ NPs, the NPs preferentially segregate in the perinuclear region preserving their initial chemical content. At concentrations exceeding 500 μM the XRF spectra indicate the presence of Co and Fe also in the nuclear region, accompanied by sensible changes in the cellular morphology. The increase of the Co/Fe ratio measured in the nuclear compartment indicates that above certain concentrations the CoFe₂O₄ NPs intracellular distribution could be accompanied by biodegradation resulting in Co accumulation in the nucleus.     

Cadmium affects genes involved in growth regulation during two-stage transformation of Balb/3T3 cells

Cadmium (Cd), a carcinogenic metal in human and rodents, has been shown to transform cells in vitro. However, the carcinogenic mechanisms of Cd as a mutagen and/or promoter are not well clarified. We already reported that CdCl2 in a range of 1.5 approximately 360 ng/ml enhanced transformation of Balb/3T3 A31 cells induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG, 0.1 microg/ml) in a dose-dependent manner (Fang et al., Toxicol. In Vitro 15(3) (2001a) 51-7). In previous study, we observed that Cd stimulated cell proliferation on MNNG-initiated cells through inactivation of p53 and p27 and increase of proliferating cell nuclear antigen (PCNA) expression after 24 h treatment (Fang et al., Toxicology 163 (2001b) 175-84). The aim of this study is to further elucidate the long-term effect of Cd in terms of cell cycle control gene expressions during the promotion stage of in vitro two-stage transformation. For the purpose, we determined the expression levels of the genes involved in growth regulation, such as p53, p27, c-myc, mdm2, cyclins D1 and B1, CDK4, and PCNA in the cells treated with Cd for 14 days after MNNG-initiation. In MNNG+CdCl2 group, cells apparently expressed cellular tumor antigen p53 mRNA, but did not express the wild-type p53 protein; the protein and mRNA levels of p27 were reduced apparently in the cells of MNNG+CdCl2 group compared to the cells of control and MNNG group. In addition, the protein levels of cyclin D1, CDK4, PCNA, c-myc, and mdm2, and cyclin B1 mRNA level were higher in MNNG+CdCl2 group than control and MNNG group. Together with previous data (Fang et al., Toxicology 163 (2001b) 175-84), our results indicated that during the transformation process of MNNG-treated cells, Cd may activate oncogenes such as c-myc, mdm2, and cellular tumor antigen p53, inhibit the tumor suppressor genes such as wild-type p53 and p27, and consequently accelerate the proliferation of initiated cells. This work firstly demonstrates that Cd affects the genes involved in growth regulation on initiated cells during the promotion stage of in vitro cell transformation.     

镁锌合金对成骨细胞增殖的影响

目的研究镁锌合金对前成骨细胞MC3T3-E1细胞增殖的影响,探讨镁锌合金成为一种新型骨科内植物材料的可行性。方法在镁锌合金金属片上接种前成骨细胞MC3T3-E1,采用MTT法及碱性磷酸酶（ALP）检测试剂盒等方法检测MC3T3-E1细胞在材料表面的增殖活性,用左旋聚乳酸对照组。结果培养第1,3天后,镁锌合金表面成骨细胞增殖与对照组没有差异（P〉0.05）,第5、7、9后,镁锌合金表面成骨细胞增殖明显高于对照组（P〈0.01）。结论镁锌合金能够促进MC3T3-E1细胞的增殖,可以初步认为具有良好的生物相容性。     

Effect of indapamide on the proliferation of Balb/C 3T3 cells induced by platelet-derived growth factor.

The effects of indapamide (a nonthiazide antihypertensive diuretic) on the growth promoting activity of serum, platelet-derived growth factor (PDGF), fibroblast growth factor (FGF) or Ca3(PO4)2 on Balb/C 3T3 cells were studied. Indapamide inhibited the growth promoting activity of serum, but furosemide (a nonthiazide antihypertensive diuretic) had no such inhibitory effect. Indapamide inhibited the growth promoting activity of PDGF, but not that of FGF and Ca3(PO4)2. The present experiments demonstrate that indapamide selectively inhibits the growth promoting activity of PDGF.     

应用优化的3T3细胞中性红摄取试验评价6种化妆品的光毒性

目的 建立一种针对化妆品的体外光毒性检测方法.方法 选取6批市售化妆品样品,通过摸索样品的细胞毒性,确定光毒浓度区间,设立样品加阳性对照的参比物,再进行光毒性评价,通过Phototox软件分析结果.结果 6种市售化妆品光刺激因子≤2,表明均无潜在光毒性.建立适合于检测化妆品的样品阳性对照,且与阳性对照结果均表现强光毒性...     

Involvement of reactive oxygen species, protein kinase C, and tyrosine kinase in prostaglandin E2 production in Balb/c 3T3 mouse fibroblast cells by quinolone phototoxicity

We examined the effect of an antioxidant and protein kinase inhibitors on prostaglandin E₂ (PGE₂) release from Balb/c 3T3 mouse fibroblast cells induced by quinolone phototoxicity. Simultaneous administration of sparfloxacin (SPFX) or lomefloxacin (LFLX) at 12.5 to 100 μ M and ultraviolet-A (UVA) irradiation for 10 min markedly elevated PGE₂ concentration in the incubation medium, whereas levofloxacin (LVFX) at concentrations up to 100 μ M and UVA irradiation did not increase PGE₂ concentration. Pretreatment with 100 μ M pyrrolidine dithiocarbamate (PDTC), an antioxidant, or 1 μ M calphostin C, a selective inhibitor of protein kinase C (PKC), completely inhibited the elevation of PGE₂ in the 24-h incubation medium; pre-treatment with 10 μ M H7, a cyclic nucleotide-dependent protein kinase, and PKC or 1 μ M herbimycin A, a tyrosine kinase inhibitor, inhibited the PGE₂ elevation by 29 to 39%. Conversely, 25 nM staurosporine significantly augmented the PGE₂ elevation by quinolones plus UVA. Interleukin-1β (IL-1β ) and tumor necrosis factor α (TNFα ) were not detected in the incubation medium of 3T3 cells after quinolone plus UVA, corresponding to the lack of effect of antibodies against IL-1α , IL-1β , and TNFα on PGE₂ release from 3T3 cells. These results suggest that PGE₂ production in 3T3 cells by quinolone phototoxicity is modulated by reactive oxygen species, PKC, and tyrosine kinase, but not by IL-1 or TNFα . Received: 23 September 1997 / Accepted: 1 December 1997     

Immunomodulatory effect of caffeic acid phenethyl ester in Balb/c mice

Caffeic acid phenethyl ester (CAPE), an the active component of propolis, is known to have anticarcinogenic, antiviral and various biological activities; however, the effect of CAPE on the immunomodulatory activity in vivo remains unknown. We have investigated the effect of CAPE on the immune system in female Balb/c mice. CAPE (0, 5, 10, 20 mg/kg) was given to mice orally for 14 days. Immunomodulatory activity was evaluated by assessment of body and organ weight, lymphocyte blastogenesis, plaque-forming cell (PFC) assay, lymphocyte subpopulation by flow cytometry and cytokine production. Even though the change of body weight was not observed in CAPE-administered group, thymus weight and/or cellularity of thymus and spleen are decreased at the all dose groups of CAPE (5, 10, 20 mg/kg). On the other hand, CAPE had no effect on B lymphocyte proliferation induced by lipopolysaccharide (LPS) but increased T lymphocyte blastogenesis induced by concanavalin A (Con A) at the dose of 20 mg/kg. In the case of lymphocyte subpopulation, the population of T and B cells was not changed but CD4(+) T cell subsets are significantly increased in exposure to CAPE. The antibody responses to T lymphocyte dependent antigen, sheep red blood cell and keyhole limpet hemocyanin (KLH) were increased more than 10 mg/kg in CAPE-treated group. Likewise, the cytokine, IL-2, IL-4 and IFN-gamma were significantly increased at the dose of 20 mg/kg CAPE group. These results suggest that CAPE could have immunomodulatory effects in vivo.     

2种光源对体外3T3细胞光毒性试验结果的比较

目的 比较2种光源对体外3T3细胞光毒性试验结果的影响。方法 参照化学品体外3T3中性红摄取光毒性试验指导原则（OECD 432）,采用模拟阳光光源和紫外光源对6种参考物质进行试验。结果 6种参考物质在2种光源照射后其光刺激因子和平均光效应均接近OECD 432中的参考值,各个物质在无光和有光下的IC₅₀值也相近。结论 2种光源对体外3T3细胞光毒性试验结果基本无影响。     

The Use of Uninduced Rat Livfr S-9 to Supplement BALB/3T3 Cells in the In Vitro Transformation Assay

ABSTRACT

Because of limited inherent capacity to metabolize chemicals to their reactive form, the BALB/3T3 transformaticn assay using clone A31–1–1 cells requires metabolic supplementation with rodent liver homogenate preparations (S-9). Activation by 8–9 is limited, however, by its cytotoxicity to the cells, thus necessitating a reduction in treatment time from the usual 24–72 to 1–4 hr. With cyclophosphamide (CP) as a test chemical, we were able to increase the treatment time to 24 hr by using lower concentrations of cofactors and S-9 prepared from the livers of untreated rats. Statjstically significant increases in transformed foci were induced in cultures treated with 50 or 100 vg CP/ml in the presence of 100 or 200 vg of uninduced rat liver S-9/ml and 76 or 380 μg each of NAPH, NADP, NADPE, and glucose-6-phosphate per ml of incubation medium.