Effects of spermine on formation of HGPRT− mutants induced by ethylmethanesulfonate,methylmethanesulfonate, and mitomycin C in V79 chinese hamster cells

Spermine is a polyamine found in bacteria, animal, and plant tissues. It is involved in a variety of biological processes, and its interaction with DMA stabilizes the secondary structure of the double helix. Spermine is one of the first reported antimutagens, reducing the mutation rate in several prokaryotic test systems, while in eukaryotic organisms conflicting results have been obtained. In light of the significant antimutagenic effect of spermine, it is important to evaluate its activity in mammalian cells in culture. The present study was undertaken to evaluate the ability of spermine to suppress the level of HGPRT mutants induced by ethylmethanesulfonate, methylmethanesulfonate, and mitomycin C. Spermine reduced the mutation frequency induced by ethylmethanesulfonate and methylmethanesulfonate but did not affect survival; with mitomycin C survival was reduced but mutation rate was not influenced.     

Human cytochrome P450 2E1 and sulfotransferase 1A1 coexpressed in Chinese hamster V79 cells enhance spontaneous mutagenesis

Genetic engineering of target cells for investigating the genotoxicity associated with specific xenobiotic‐metabolizing enzymes is useful for elucidating metabolic activation and inactivation processes. We constructed a V79‐derived cell line expressing both human cytochrome P450 (CYP) 2E1 and human sulfotransferase (SULT) 1A1. We previously reported that this cell line (V79‐hCYP2E1‐hSULT1A1) efficiently activates various important pro‐genotoxicants. Here we present data on the expression level and stability of the heterologous enzymes, measured by immunoblotting, enzyme activities, and mutagenic responses to CYP2E1‐ and SULT1A1‐dependent promutagens. Unexpectedly, these cells demonstrated greatly elevated spontaneous gene mutation frequencies (determined at the Hprt locus), and elevated frequencies of sister chromatid exchange, as compared with control V79 cells and V79‐derived lines engineered for other enzymes. Therefore, V79‐hCYP2E1‐hSULT1A1 cells require regular cleansing in aminopterin‐containing medium when used for Hprt gene mutation assays. In a 4‐week time course without such selection, V79‐hCYP2E1‐hSULT1A1 demonstrated a progressive increase in the spontaneous mutant frequency from 2.9 to 155 × 10^?6. This phenomenon was moderately, strongly, and completely prohibited in the presence of CYP2E1 inhibitor 1‐aminobenzotriazole, SULT1A1 inhibitor pentachlorophenol and both in combination, respectively. This protection indicates that the enhanced spontaneous mutagenicity involves the activity of the expressed enzymes rather than being caused by an accidental genetic alteration that might have occurred during transfection. We postulate that human CYP2E1 and SULT1A1 activate an endogenous cellular molecule or a medium component to become mutagenic. It will be challenging to identify this compound and to see whether it is involved in spontaneous mutagenesis and carcinogenesis in vivo. Environ. Mol. Mutagen., 2010. © 2009 Wiley‐Liss, Inc.     

Genotoxicity of 1‐methylpyrene and 1‐hydroxymethylpyrene in chinese hamster V79‐derived cells expressing both human CYP2E1 and SULT1A1

1‐Methylpyrene (1‐MP) is a widespread pollutant that is carcinogenic in animals following metabolic activation. Previous studies have shown that benzylic hydroxylation of 1‐MP, catalyzed by multiple CYP isoforms, gives rise to 1‐hydroxymethylpyrene (1‐HMP), which becomes bioreactive following further metabolism by various sulfotransferase (SULT) isoforms. However, the mutagenic and chromosome damaging effects of 1‐MP and 1‐HMP in mammalian cells have not been investigated. In this study a Chinese hamster V79‐derived cell line expressing both human CYP2E1 and human SULT1A1 was used to investigate the ability of 1‐MP and 1‐HMP to induce cytotoxicity (using the CCK‐8 assay), micronuclei and Hprt gene mutations. The role of each enzyme was investigated through co‐exposure in the presence of an enzyme inhibitor. We found that at concentrations of 0.5–4 μM and 5–20 μM, under conditions where no reduction in cell viability/growth occurred, 1‐HMP and 1‐MP induced micronuclei in V79‐hCYP2E1‐hSULT1A1 cells in a concentration‐dependent manner; however, both compounds were inactive in V79 cells. Similarly, they both caused an increase in Hprt mutant frequency in V79‐hCYP2E1‐hSULT1A1 cells in these concentration ranges, with 1‐MP impairing cell viability/growth at 10 μM and above in the mutagenicity assay. The compounds were again both inactive in V79 cells. The effects of 1‐HMP in V79‐hCYP2E1‐hSULT1A1 cells were blocked or reduced by addition of pentachlorophenol (PCP), a SULT1 inhibitor; the genotoxicity of 1‐MP was significantly reduced by either 1‐aminobenotrazole, a CYP2E1 inhibitor, or PCP. The results suggest that human CYP2E1 and SULT1A1 cooperate to activate 1‐MP and cause genotoxicity in mammalian cells. Environ. Mol. Mutagen. 56:404–411, 2015. © 2014 Wiley Periodicals, Inc.     

Evaluation of a new procedure for the flow cytometric analysis of in vitro,chemically induced micronuclei in V79 cells

Measurement of the frequency of micronuclei induced in cells by ionizing radiation or by chemical treatment is widely used to analyze cytogenetic damage. The microscopic scoring of micronuclei is a tedious and time-consuming procedure. Therefore, attempts have been made to automate micronuclei scoring by means of image analysis or flow cytometry. A new procedure for the flow cytometric analysis of chemically induced micronuclei in V79 Chinese hamster cells has been established in our laboratory. Debris was separated from micronuclei by means of a new gating procedure using area and width fluorescence of the stained suspension of micronuclei and nuclei. In order to test the sensitivity and specificity of this improved method of flow cytometric analysis, five well-known mutagenic compounds were tested. With the new technique, the frequency of micronuclei measured and analyzed corresponded well with results obtained by conventional microscopy. In addition, a large series of negative compounds, and weak, middle, and strong micronuclei inducers, were tested in order to establish criteria for discrimination between genotoxic and nongenotoxic compounds by flow cytometry. This new procedure for flow cytometric detection of micronuclei represents a quick, reliable, and relatively simple method for in vitro micronucleus testing. Environ. Mol. Mutagen. 32: 387–396, 1998 © 1998 Wiley-Liss, Inc.     

Molecular and cellular influences of butylated hydroxyanisole on Chinese hamster V79 cells treated with N-methyl-N'-nitro-N-nitrosoguanidine: antimutagenicity of butylated hydroxyanisole

The antioxidant butylated hydroxyanisole (BHA) is a rodent carcinogen that also reduces the mutagenicity and carcinogenicity of other agents. In this study, we have evaluated possible mechanisms for the antimutagenicity of BHA by investigating its effects on N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-treated Chinese hamster V79 cells. Mutant frequency was determined using the hprt/V79 assay, while plating efficiency was used to measure cytotoxicity, and apoptosis was measured by flow immunofluorocytometry. In addition, DNA strand breaks and the kinetics of strand-break rejoining were investigated by the alkaline elution of DNA and by single-cell gel electrophoresis (SCGE). Although the higher concentration of BHA (0.5 mM) increased the cytotoxicity of MNNG and the lower concentration of BHA (0.25 mM) did not change it, both concentrations were antimutagenic in MNNG-treated cells, with the greater effect occurring at the lower BHA concentration. Neither BHA nor MNNG nor BHA + MNNG increased the level of apoptotic nuclei, and BHA did not change the level of MNNG-induced DNA strand breaks, though it did inhibit their rejoining. Determination of O(6)-methylguanine-DNA-methyltransferase (MGMT) activity confirmed that V79 cells do not synthesize active MGMT protein; MGMT activity was also undetectable after MNNG and BHA + MNNG treatment. The ability of BHA to reduce the level of MNNG-induced mutations did not correlate with cytotoxicity, induction of apoptosis, the level of DNA strand break induction, or MGMT activity. A modified SCGE assay showed that BHA significantly reduced the level of formamidopyrimidine-DNA-glycosylase + endonucleaseIII-sensitive sites, which at least partially are caused by oxidative DNA lesions. The results suggest that the protective effect of BHA on MNNG-induced mutagenicity is best explained by the antioxidative activity of BHA, which may scavenge free radicals that participate in MNNG-induced mutagenicity.     

Human CYP2E1-dependent mutagenicity of benzene and its hydroxylated metabolites in V79-derived cells: Suppression and enhancement by ethanol pretreatment

Benzene is a human carcinogen that requires metabolic activation. We previously observed that benzene and its hydroxylated metabolites induce micronuclei in mammalian cells expressing human CYP2E1. This study was initially aimed to study another endpoint, the induction of gene mutations by those compounds in the same cell models. A V79-derived cell line expressing human CYP2E1 and sulfotransferase (SULT) 1A1 (V79-hCYP2E1-hSULT1A1) pretreated with ethanol (a CYP2E1 stabilizer) was used in the hprt gene mutagenicity assay. Phenol, hydroquinone, catechol, and 1,2,4-trihydroxybenzene all induced gene mutations, while they were inactive, or only weakly positive (hydroquinone), in parental V79-Mz cells. Unexpectedly, benzene was non-mutagenic in both cell lines, but it became positive in V79-hCYP2E1-hSULT1A1 cells using regimes of short exposure/long recovery without ethanol pretreatment, for both gene mutations and micronuclei formation. In silico molecular simulation showed binding energies and positions favorable for each compound to be oxidized by human CYP2E1, benzene demonstrating the highest affinity. By tunnel analysis, ethanol binding did not limit benzene to pass tunnel S, which was specifically active for benzene. However, its end product, acetic acid, decreased the occurrence of tunnel S from 5.4 to 2.2% and extended the length of its bottleneck from 5.5 to 9.0 Å. With residual ethanol molecules still being present in CYP2E1 for a period of time after benzene exposure, the acetic acid formed could limit the entrance of benzene, thus inhibit its metabolic activation. In summary, ethanol may interfere with the activation of benzene to mutagenic metabolites, at least in cultured cells.     

Overexpression of heme oxygenase-1 (HO-1) in V79 cells results in increased resistance to hyperbaric oxygen (HBO)-induced DNA damage

Heme oxygenase (HO) catalyzes the rate-limiting step in the oxidative degradation of heme to biliverdin. The isoform HO-1 is inducible by a variety of agents causing oxidative stress and has been suggested to play an important role in cellular protection against oxidant-mediated cell damage. Using treatment of cell cultures with hyperbaric oxygen (HBO) as a model for oxidative stress, we have shown an induction of HO-1 in isolated human lymphocytes after a single HBO exposure and protection of these cells against DNA damage by subsequent oxidative stress. In contrast, V79 Chinese hamster cells showed neither a comparable adaptive protection nor an induction of HO-1 after HBO exposure, which makes this cell line an attractive model system for a further characterization of HO-1-mediated protection. In the present study, we investigated whether overexpression of HO-1 renders V79 cells more resistant to DNA damage induced by HBO. Transient transfection of V79 cells with a full-length human HO-1 cDNA resulted in a 2-3-fold increase in HO-1 protein levels. Comet assay experiments with and without FPG posttreatment for the determination of oxidative DNA base damage showed that HO-1 overexpressing V79 cells were significantly protected against oxidative DNA damage induced by a single HBO exposure. Furthermore, HO-1-transfected cells exhibited a clearly reduced induction of micronuclei after HBO treatment. Since the observed protective effects were abolished by cotreatment with the HO-1 inhibitor tin-mesoporphyrin, our study suggests that a low-level overexpression of HO-1 provides protection against oxidative DNA damage induced by HBO.     

Matrix metalloproteinases in acute inflammation: induction of MMP-3 and MMP-9 in fibroblasts and epithelial cells following exposure to pro-inflammatory mediators in vitro

Recent studies have demonstrated important pro-inflammatory roles for two matrix metalloproteinases (MMPs)-MMP-3 (stromelysin-1) and MMP-9 (gelatinase B)-in acute lung injury [Am. J. Respir. Cell Mol. Biol. 24 (2001) 1]. A role for MMP-3 in skin inflammation has also been demonstrated [Proc. Natl. Acad. Sci. U. S. A. 96 (1999) 6885]. While leukocytes (neutrophils and macrophages) are known to elaborate these tissue-destructive enzymes, parenchymal cells are also capable of synthesizing MMPs. In the present study, we examined the production of MMP-3 and MMP-9 by rodent lung fibroblasts, type II epithelial cells, and vascular endothelial cells. Dermal fibroblasts were also examined. Cells were examined under control conditions and in response to agonists that induce acute inflammatory tissue injury (IgG-containing immune complexes and lipopolysaccharide [LPS]). In the absence of stimulation, MMP-3 and MMP-9 were not detected or were present at low level. However, upon stimulation with either of the two pro-inflammatory agonists, production of both enzymes occurred in fibroblasts and epithelial cells (though not in endothelial cells). The observation that resident cells in the tissue parenchyma can elaborate MMPs in direct response to pro-inflammatory stimuli provides insight into possible mechanisms by which tissue damage occurs in acute inflammation.     

前列腺素E1联合碱性成纤维细胞生长因子干预人牙髓干细胞增殖和血管生成能力的变化

文题释义：前列腺素E1：是一种生物活性物质,广泛存在于体内各组织细胞,由BERGSRTOEM等于1960年首先分离获得,并详细研究了其分子结构,具有舒张血管、稳定细胞膜降低血小板黏附率、改善血液黏度及抗炎等作用,可促进骨髓间充质干细胞和神经干细胞的增殖、迁移,并促进细胞分泌血管内皮生长因子,进而促进血管生成。碱性成纤维细胞生长因子：是一种肝素黏合多肽,也是一种重要的潜在有丝分裂原,属于成纤维细胞生长因子家族,对细胞有丝分裂及分化具有很强的调节作用,特别是对中胚层和神经外胚层来源的细胞作用更强,可促进细胞生长,研究发现牙髓中的成纤维细胞可表达碱性成纤维细胞生长因子,在体外能明显促进人牙髓干细胞增殖。  摘要背景：前列腺素E1及碱性成纤维细胞生长因子可促使人牙髓干细胞增殖,但两者联合应用对人牙髓干细胞增殖和血管生成能力的影响还未见研究。目的：探究前列腺素E1联合碱性成纤维细胞生长因子对人牙髓干细胞增殖和血管生成能力的影响。方法：①体外分离培养人牙髓干细胞,经表面标志物检测鉴定后,分别以5,10,20,50,100 μg/L前列腺素E1及5,10,20,50,100 μg/L碱性成纤维细胞生长因子单独作用于体外培养的人牙髓干细胞,以未加药物处理的细胞作为空白对照组,采用CCK-8法分别在1,2,3 d检测人牙髓干细胞增殖情况,筛选最佳药物作用质量浓度和时间。②将体外培养的人牙髓干细胞分为4组：空白对照组、前列腺素E1组、碱性成纤维细胞生长因子组、前列腺素E1+碱性成纤维细胞生长因子组,采用CCK-8法检测人牙髓干细胞增殖情况,然后提取人牙髓干细胞条件培养基,以ELISA测定条件培养基中血管内皮生长因子、内皮抑素水平,以小管形成实验检测人牙髓干细胞条件培养基干预后人脐静脉血管内皮细胞的体外小管形成能力。结果与结论：①前列腺素E1、碱性成纤维细胞生长因子的最佳作用质量浓度均为20 μg/L,最佳作用时间为2 d;②与空白对照组相比,前列腺素E1组、碱性成纤维细胞生长因子组、前列腺素E1+碱性成纤维细胞生长因子组人牙髓干细胞相对增殖率、血管内皮生长因子水平、人脐静脉血管内皮细胞体外血管生成能力均显著升高(P < 0.05),内皮抑素水平显著降低(P < 0.05);前列腺素E1+碱性成纤维细胞生长因子组上述各指标均优于前列腺素E1组、碱性成纤维细胞生长因子组(P < 0.05);③结果表明,前列腺素E1联合碱性成纤维细胞生长因子可促进人牙髓干细胞增殖,增强人脐静脉血管内皮细胞体外小管形成能力。 ORCID: 0000-0002-7727-1883(项海东) 中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

17 Beta-estradiol and not genistein modulates lacI mutant frequency and types of mutation induced in the heart of ovariectomized big blue rats treated with 7, 12-dimethylbenz[a]anthracene

In industrialized countries, heart disease rates are higher among women after menopause. Recent studies indicate that consumption of phytoestorogens, e.g., isoflavones such as genistein (GE), may have potential cardiovascular health benefits; however, no studies have evaluated the effect of these agents on toxicant-induced damage in the heart. Since estrogen receptors are found in the heart, and GE mimics estrogenic effects, we have examined whether or not dietary GE or 17 beta-estradiol (E2) modulates the lacI mutant frequency (MF) in the heart of ovariectomized (OVX) Big Blue rats exposed to the model carcinogen 7,12-dimethylbenz[a]anthracene (DMBA). Groups of female rats were administered 80 mg/kg DMBA or vehicle by gavage and were chronically fed with diets containing 0, 250, or 1,000 microg/g GE or 5 microg/g E2. Sixteen weeks after carcinogen treatment, the animals were sacrificed and the hearts were removed and processed for determining the frequency and types of mutations in the heart tissue. GE and E2 supplementation alone resulted in nonsignificant increases in MF. The DMBA-induced lacI MF in the heart was sevenfold higher than the control (119.8 +/- 18.7 x 10(-6) vs. 17.4 +/- 3.2 x 10(-6); P < 0.001). GE in the diet had no significant effect on DMBA mutagenicity, while feeding E2 to DMBA-treated rats caused a significant reduction in the MF (119.8+/- 18.7 x 10(-6) vs. 61.4 +/- 13.5 x 10(-6); P < 0.017). DNA sequence analysis revealed that the majority of DMBA-induced mutations in rats fed control diet were A:T-->T:A (42%) and G:C-->T:A (19%) transversions, followed by G:C-->A:T (13%) and A:T-->G:C (8%) transitions. Feeding E2 altered the DMBA-induced mutational spectra by decreasing A:T-->T:A (23%) and G:C-->T:A (13%) transversions and increasing G:C-->A:T (24%) and A:T-->G:C (21%) transitions. Taken together, the results suggest that DMBA can induce gene mutations in heart tissue of OVX rats, and while dietary GE had little or no effect on DMBA-induced mutation, dietary E2 reduced the mutagenicity of DMBA.     

A novel model of drug hapten-induced hepatitis with increased mast cells in the BALB/c mouse

Clinical evidence suggests that idiosyncratic hepatitis following administration of halogenated volatile anesthetics is mediated by autoimmune responses. No murine model to study mechanisms of anesthetic-induced or any other form of drug-induced idiosyncratic hepatitis exists. Anesthetics are believed to trigger hepatitis by covalently linking a trifluoroacetyl (TFA) chloride hapten to hepatic proteins, forming haptenated self-proteins. To test this hypothesis, we developed a hapten-induced model of hepatitis by immunization with syngeneic S100 liver proteins covalently coupled to TFA (TFA-S100). We found that TFA-S100 induced hepatitis was more severe than disease induced by S100 plus adjuvants or by the adjuvant alone and was characterized by neutrophil, mast cell, and eosinophil infiltration. TFA-specific IgG1 antibodies directly correlated with hepatitis, whereas S100 autoantibodies did not. TNF-alpha, IL-1beta, and IL-6 released from splenocytes collected 2 weeks after TFA-S100 inoculation were increased resembling the elevated serum cytokines reported in patients with autoimmune hepatitis (AIH). Three weeks after inoculation, the peak of hepatitis, we noted decreased numbers of Kupffer cells and lower levels of IL-6 and IL-10 in the liver, cytokines produced by Kupffer cells. This is the first report, to our knowledge, of a hapten-induced model of hepatitis with immune and autoimmune features.