Molecular toxicology最新文献

{"title":"Limitations of the fluorescent probe viability assay.","authors":"E J Massaro,&nbsp;K H Elstein,&nbsp;R M Zucker,&nbsp;K W Bair","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>In vitro assessment of the efficacy/capacity of toxicants (e.g., cancer chemotherapeutic agents, environmental pollutants, etc.) to damage/kill cells and/or inhibit growth (cell duplication) requires accurate measurement of target cell viability as a function of exposure. Rapid measurement of viability, such as can be achieved employing fluorescent probes of metabolic function in combination with instrumental analysis, is highly desirable. However, we observe that exposure to chemicals (of unrelated type) complicates the interpretation of viability data and, in the case of perturbed cells, questions the validity of viability growth assays based on intrinsic enzyme activity. Viability commonly is determined flow cytometrically (FCM) by the carboxyfluorescein diacetate (CFDA)/propidium iodide (PI) assay. Nonfluorescent CFDA is taken up by diffusion and converted via cytoplasmic esterase-catalyzed hydrolysis to carboxyfluorescein (CF), a negatively charged fluorescent molecule that is retained (incompletely) by the cell. As such, if CF fluorescence intensity is a relative measure of enzyme activity, it also can be considered an index of cellular vigor (metabolic rate). It is generally accepted that the viable cell excludes both basic dyes, such as PI, and acidic dyes, such as trypan blue, and uptake is indicative of irreversible cellular injury presaging cell death. We observe that, following incubation for 4 h with 0.5-1.0 microM tributyltin (TBT), a potent environmental toxicant, murine erythroleukemic cells (MELC) exhibit enhanced (supranormal) CF fluorescence compared to control cells. Apparent cell volume (ACV) is unaltered, and because such cells exclude PI, they are considered viable in terms of the CFDA/PI assay. However, rate of growth (increase in cell number over 48 h) is depressed, suggesting that supranormal CF fluorescence, even in the absence of PI uptake, is indicative of cellular perturbation. In effect, although CF fluorescence is the product of an enzyme-catalyzed reaction and, therefore, an indicator of vital function (enzyme activity), it apparently is not a reliable index of cellular vigor. At higher TBT concentrations (greater than 1.0, but less than 50.0 microM), the cells exhibit both increased CF fluorescence and PI fluorescence and are growth inhibited. MELC exposed to the cancer chemotherapeutic agents adriamycin, m-AMSA, or crisnatol (Burroughs Wellcome 770U82) also exhibit increased cellular CF fluorescence. However, rate of growth is decreased and ACV increased. The latter, measured either as a function of electrical resistance (Coulter volume) or by the FCM parameter axial light loss could account for the increase in mean CF fluorescence.(ABSTRACT TRUNCATED AT 400 WORDS)</p>","PeriodicalId":77750,"journal":{"name":"Molecular toxicology","volume":"2 4","pages":"271-84"},"PeriodicalIF":0.0,"publicationDate":"1989-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"13635807","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Xenobiotic regulation of glutathione S-transferase Ya gene expression.","authors":"K E Paulson","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>The gene for the rat GST Ya subunit has been examined in detail as a model for understanding the molecular mechanisms of inducibility by xenobiotics and their tissue-specific regulation. The focus of this article is to describe our current understanding of these mechanisms. The discussion will begin with the classification of the types of inducing agents. These pioneering studies suggested that there were multiple mechanisms for the inducibility of GSTs. In fact, the analysis of GST Ya gene expression has identified two different upstream activating elements and putative protein factors through which different classes of inducers act. Finally, the position-specific and tissue-specific regulation of the GST Ya gene will be discussed.</p>","PeriodicalId":77750,"journal":{"name":"Molecular toxicology","volume":"2 4","pages":"215-35"},"PeriodicalIF":0.0,"publicationDate":"1989-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"13635805","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Induction of a novel damage-specific DNA binding protein correlates with enhanced DNA repair in primate cells.","authors":"M. Protic, S. Hirschfeld, A. P. Tsang, M. Wagner, K. Dixon, A. Levine","doi":"10.1201/9781003075387-2","DOIUrl":"https://doi.org/10.1201/9781003075387-2","url":null,"abstract":"Pretreatment of mammalian cell with DNA-damaging agents, such as UV light or mitomycin C, but not the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA), results in the enhanced repair of subsequently transfected UV-damaged expression vectors. To determine the cellular factors that are responsible for this enhancement, we have used a modified gel retardation assay to detect the proteins that interact with damaged DNA. We have identified a constitutive DNA binding protein in extracts from primate cells that has a high affinity for UV-irradiated double-stranded DNA. Cells pretreated with UV light, mitomycin C, or aphidicolin, but not TPA or serum starvation, have higher levels of this damage-specific DNA binding (DDB) protein. These results suggest that the signal for induction of DDB protein can either be damage to the DNA or interference with cellular DNA replication. The induction of DDB protein varies among primate cells with different phenotypes: (1) virus-transformed repair-proficient cells have partially or fully lost the ability to induce DDB protein above constitutive levels; (2) primary cells from repair-deficient xeroderma pigmentosum (XP) group C, and transformed XP groups A and D, show constitutive DDB protein, but do not show induced levels of this protein 48 h after UV; and (3) primary and transformed repair-deficient cells from one XP E patient are lacking both the constitutive and the induced DDB activity. The correlation between the induction of the DDB protein and the enhanced repair of UV-damaged expression vectors implies the involvement of the DDB protein in this inducible cellular response.","PeriodicalId":77750,"journal":{"name":"Molecular toxicology","volume":"2 4 1","pages":"255-70"},"PeriodicalIF":0.0,"publicationDate":"1989-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"65953323","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Review: gene amplification--a cellular response to genotoxic stress.","authors":"C Lüke-Huhle","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>Recent years of cancer research have defined the role of key regulatory genes in oncogenesis. Oncogenes and suppressor genes are affected in the process of carcinogenesis either by mutations within the coding region, promoter mutations, or gene amplification. This review describes our studies on gene amplification in mammalian cells, with emphasis on the initiating events induced by carcinogenic chemicals and various types of radiation. The influence of genomic instability, cell dedifferentiation, and the malignant potential of a cell on their capacity to amplify genes is demonstrated by molecular biologic and cytogenetic studies on human and rodent cells. Cells that contain amplified DNA are at risk for chromosomal aberrations, sister chromatid exchanges, and rearrangements. Surviving cells show such cancer-prone genetic consequences.</p>","PeriodicalId":77750,"journal":{"name":"Molecular toxicology","volume":"2 4","pages":"237-53"},"PeriodicalIF":0.0,"publicationDate":"1989-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"13635806","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Induction of a novel damage-specific DNA binding protein correlates with enhanced DNA repair in primate cells.","authors":"M Protić,&nbsp;S Hirschfeld,&nbsp;A P Tsang,&nbsp;M Wagner,&nbsp;K Dixon,&nbsp;A S Levine","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>Pretreatment of mammalian cell with DNA-damaging agents, such as UV light or mitomycin C, but not the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA), results in the enhanced repair of subsequently transfected UV-damaged expression vectors. To determine the cellular factors that are responsible for this enhancement, we have used a modified gel retardation assay to detect the proteins that interact with damaged DNA. We have identified a constitutive DNA binding protein in extracts from primate cells that has a high affinity for UV-irradiated double-stranded DNA. Cells pretreated with UV light, mitomycin C, or aphidicolin, but not TPA or serum starvation, have higher levels of this damage-specific DNA binding (DDB) protein. These results suggest that the signal for induction of DDB protein can either be damage to the DNA or interference with cellular DNA replication. The induction of DDB protein varies among primate cells with different phenotypes: (1) virus-transformed repair-proficient cells have partially or fully lost the ability to induce DDB protein above constitutive levels; (2) primary cells from repair-deficient xeroderma pigmentosum (XP) group C, and transformed XP groups A and D, show constitutive DDB protein, but do not show induced levels of this protein 48 h after UV; and (3) primary and transformed repair-deficient cells from one XP E patient are lacking both the constitutive and the induced DDB activity. The correlation between the induction of the DDB protein and the enhanced repair of UV-damaged expression vectors implies the involvement of the DDB protein in this inducible cellular response.</p>","PeriodicalId":77750,"journal":{"name":"Molecular toxicology","volume":"2 4","pages":"255-70"},"PeriodicalIF":0.0,"publicationDate":"1989-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"13664194","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Characteristics of lead adaptation in a rat kidney cell line. II. Effect on DNA synthesis, protein synthesis, and gene expression.","authors":"B Hitzfeld,&nbsp;D M Taylor","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>The effects of adaptation of normal rat kidney cells (NRK 52-E) to growth in 5 or 10 microM lead nitrate on the rates of DNA synthesis and on the rate and pattern of protein synthesis was studied. The rate of [3H]thymidine incorporation into DNA was increased in normal cells, but remained unchanged in one lead-adapted cell line (only 5 microM NRK studied). Increased rates of [3H]leucine incorporation into nonadapted NRK cells were found only at times up to 3 h; in contrast, the lead-adapted cells showed such increases only at longer times. The most pronounced differences between normal and lead-adapted cells were found with lead concentrations of 10 or 50 microM lead nitrate. Lead-adapted control cells incorporated 170% of the [3H]leucine taken up by nonadapted cells. In both adapted and nonadapted cells the pattern of synthesis of specific proteins showed varied and dose-dependent differences between the three cell sublines examined. The changed sensitivity of both DNA and protein synthesis following lead exposure appears to be a potent parameter in the development of resistance, perhaps through the development of specific lead-binding proteins.</p>","PeriodicalId":77750,"journal":{"name":"Molecular toxicology","volume":"2 3","pages":"163-75"},"PeriodicalIF":0.0,"publicationDate":"1989-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"13632285","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Plasmid-aided insertion of MMTV-LTR and ras DNAs to NIH 3T3 fibroblast cells makes them responsive to 2,3,7,8-TCDD causing overexpression of p21ras and down-regulation of EGF receptor.","authors":"J Jankun,&nbsp;F Matsumura,&nbsp;H Kaneko,&nbsp;J E Trosko,&nbsp;A Pellicer,&nbsp;A H Greenberg","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>TCDD administered to NIH 3T3 fibroblast cells transfected with a plasmid containing MMTV-LTR and mouse ras DNAs caused an increased level of p21ras protein and down-regulation of EGF receptor. This effect occurred only in the cells with introduced N-ras or Ha-ras under transcriptional control of glucocorticoid-sensitive MMTV-LTR but not ones without these DNAs. The MMTV-LTR ras-incorporated cells treated with either dexamethasone or TCDD grew in soft agar to form colonies (anchorage independent growth), while nontreated cells did not, indicating profound cellular changes due to activation of N-ras by these two agents.</p>","PeriodicalId":77750,"journal":{"name":"Molecular toxicology","volume":"2 3","pages":"177-86"},"PeriodicalIF":0.0,"publicationDate":"1989-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"13705541","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Characteristics of lead adaptation in a rat kidney cell line. I. Uptake and subcellular and subnuclear distribution of lead.","authors":"B Hitzfeld,&nbsp;D M Taylor","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>Two sublines of normal rat kidney cell (NRK 52-E) that are resistant to 5 and 10 microM lead nitrate have been developed and characterized. The cellular and nuclear uptake of 210Pb, as well as the subnuclear distribution, was unchanged by lead adaptation: it remained at values of 2.3-6.6 pmole 210Pb/microgram protein/h. Also no difference in the slope of the curve of the uptake rate of the three cell types could be detected. Studies of the nuclear uptake of 210Pb showed that in both normal and lead-adapted cells the major lead fraction was associated with the chromatin and mostly (63-67%) with the nuclear proteins. Electron microscopic studies demonstrated that particulate lead was taken up in all lead-exposed cells. It was concluded that development of resistance to lead does not arise from a reduced rate of cellular uptake, from increased excretion, or from changes in nuclear uptake or in the subnuclear distribution within the cells.</p>","PeriodicalId":77750,"journal":{"name":"Molecular toxicology","volume":"2 3","pages":"151-62"},"PeriodicalIF":0.0,"publicationDate":"1989-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"13632284","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Covalent modification of hepatic microsomal lipids of rats by carbon tetrachloride.","authors":"B S Kaphalia,&nbsp;G A Ansari","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>The present study was undertaken to isolate and identify various lipids bound to 14C label during hepatic microsomal metabolism of 14CCl4 in vitro under anaerobic conditions and in vivo in rats. The two major radioactive fractions identified by thin-layer chromatography each for neutral lipids and phospholipids from in vitro and in vivo experiments corresponded to fatty acids and triglycerides and to phosphatidylcholine (PC) and phosphatidylethanolamine (PE), respectively. Approximately 89% of the radioactivity associated with phospholipids was found in PC and PE fractions. Hydrolysis of PC and PE with phospholipase A2 (EC 3.1.1.4) released about 50% of the total radioactivity as lipid moieties corresponding to fatty acids. The radioactive neutral lipids and the lipid moieties hydrolyzed from PC and PE were methylated with boron trifluoride in methanol. These methylated lipids were separated by reversed-phase high-performance liquid chromatography (HPLC), and the elution profiles of 14C label found for the lipids obtained from in vitro experiments were similar to those from in vivo. The major radioactive fractions eluted immediately after methyl oleate were identified as trichloromethyloctadecenoic and trichloromethyleicosatrienoic acid methyl esters by chemical ionization mass spectrometry. The mass spectral analysis of these fractions also indicated the formation of dichlorocarbene adduct of oleic acid. However, similar mass spectrometric detection of trichloromethylated lipids was not evident in neutral lipids and phospholipids isolated from in vivo studies. The 14C-labeled lipids eluted as a nonpolar fraction exhibited a high molecular weight containing more than three chlorines. Dimerization and cross-linking of trichloromethylated lipids based on HPLC and mass spectral analysis are also discussed in this paper.</p>","PeriodicalId":77750,"journal":{"name":"Molecular toxicology","volume":"2 3","pages":"199-213"},"PeriodicalIF":0.0,"publicationDate":"1989-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"13632288","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Inactivation of plasma alpha 1-proteinase inhibitor by acrolein: adduct formation with lysine and histidine residues.","authors":"J C Gan,&nbsp;G A Ansari","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>Four new peaks were observed upon amino acid analysis of alpha 1-proteinase inhibitor (alpha 1-PI), which was inactivated by acrolein under in vitro conditions. The first peak emerged just before ammonia, the second and third between ammonia and lysine, and the fourth between histidine and arginine. The new fourth peak was also observed when model compounds of lysine (N-acetyllysine or polylysine) were reacted with acrolein and subsequently processed for amino acid analysis. This new compound was purified by high-voltage paper electrophoresis and subjected to fast atom bombardment mass spectrometry, which showed a protonated molecule ion at m/z 203 [M + H]+ followed by m/z 186 [M + H+ - NH3]+. This compound was thus identified as 3-oxopropyllysine, a lysine adduct of acrolein. Similarly, when a model polypeptide of histidine, polyhistidine, was reacted with acrolein under the same conditions as alpha 1-PI, three new peaks (besides histidine) emerged from the column. Their elution times corresponded to the first three new peaks found in the hydrolysates of acrolein treated alpha 1-PI.</p>","PeriodicalId":77750,"journal":{"name":"Molecular toxicology","volume":"2 3","pages":"137-45"},"PeriodicalIF":0.0,"publicationDate":"1989-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"13665247","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}