Involvement of rat cytochrome 1A1 in the biotransformation of kaempferol to quercetin: relevance to the genotoxicity of kaempferol

Kaempferol is a flavonoid widely distributed in edible plantsand has been shown to be genotoxic to V79 cells in the absenceof external metabolizing systems. The presence of an externalmetabolizing system, such as rat liver homogenates (S9 mix),leads to an increase in its genotoxicity, which is attributedto its biotransformation to the more genotoxic flavonoid quercetin,via the cytochrome P450 (CYP) mono-oxygenase system. In thepresent work we investigated the mechanisms of the genotoxicityof kaempferol further. Special attention has been given to therole of CYP in the genotoxicity of this flavonoid. We studiedthe induction of mutations in Salmonella typhimurium TA98 inthe presence and in the absence of S9 mix and the inductionof chromosomal aberrations (CAs) and micronuclei (MN) by kaempferolin V79 cells in the presence and in the absence of S9 mix. Toevaluate the role of different CYP in the biotransformationof kaempferol we studied the induction of CAs and MN in V79cells genetically engineered for the expression of rat CYP 1A1,1A2 and 2B1. In addition we performed CYP inhibition studiesusing the above-mentioned indicators and high performance liquidchromatography (HPLC) analysis. The results obtained in thiswork suggest that rat CYP 1A1 is, among the cytochromes studied,the one that plays the major role in the transformation of kaempferolinto quercetin. The relevance of these findings to the humansituation is discussed. ⁴To whom correspondence should be addressed     

Evidence that the [3H]thymidine-induced adaptive response of human lymphocytes to subsequent doses of X-rays involves the induction of a chromosomal repair mechanism

When human lymphocytes are treated with [³H]thymidine ([³H]dThd),the observed number of chromosomal aberrations induced by [³H]dThdand subsequent doses of X-rays is less than the number inducedby X-rays alone. Experiments in which cells were examined atvarious times after exposure to the X-rays showed that thisphenomenon, termed an adaptive response to the endogenous radiationfrom tritium, is not an artefact caused by radiation-inducedmitotic delays, which could result in the sampling of metaphasecells that were irradiated in different parts of the G₂ phaseof the cell cycle, where sensitivity to X-rays changes dramatically.Reconstruction experiments in which labelled female cells wereco-cultured with unlabelled male cells now show that labelledand unlabelled cells progress to metaphase equally, and thereforethat the adaptive response is not the result of selection againsta radiosensitive population of cells that have incorporated[³H]dThd. Measurements of chromosomal aberrations induced inthe labelled female cells and unlabelled male cells that hadbeen co-cultured show that the adaptive response is restrictedto those cells exposed to radiation from the incorporation of[³H]dThd and that diffusible factors are not involved. The resultsare consistent with the proposal that this adaptive responseis the result of the induction of a hitherto unknown chromosomalrepair mechanism. It has now been found that this repair mechanismis induced at levels of radiation from [³H]dThd that in themselvesare too low to induce any discernible chromosomal aberrationsand that its activity is dependent on the enzyme poly(ADP-ribose)polymerase, because 3-aminobenzamide, an inhibitor of poly(ADP-ribosyl)ation,prevents the adaptive response. ¹To whom correspondence should be addressed     

Caffeine post-treatment causes a shift in the chromosome aberration types induced by mitomycin C, suggesting a caffeinesensitive mechanism of DNA repair in G2

Human lymphocytes were exposed in G₁ to mitomycin C (2.5 µMfor 2 h) and harvested at 3-h intervals from 48 to 84 h afterstimulation. All cultures were also post-treated in G₂ withcaffeine (2mM). Different types of chromosomal aberrations werescored in the first division metaphases. Caffeine increasesall chromosome aberration types by promoting a premature mitosisof damaged cells. However, when the frequency of damaged cellsis not affected by the caffeine post-treatment, a reductionof the frequency of the exchange-type aberrations was shown.The possibility that caffeine interferes with some mechanismof G₂ repair is discussed. ¹To whom correspondence should be addressed     

Induction of SCE by indirect mutagens in cultured rat hepatoma cells and in Chinese hamster V79 cells co-cultivated with hepatocyte primary cultures

The continuous rat hepatoma cell line H4IIEC3/G^– and rathepatocyte primary cultures (hpc) were compared with regardto their capacity to metabolize structurally different promutagens.The sensitivities of both activation systems were evaluatedby comparing the induction of SCE in H4IIEC3/G^– cellsthemselves with that in V79 cells co-cultured with hpc. Of thesix chemicals tested, aflatoxui B₁ (AFB1), cyclo-phosphamide,dimethylnitrosamine and nitrosomorpholine (NM) were shown tobe inducers of SCE in H4HEC3/G^– cells as well as in V79cells with hepatocyte activation. 7,12-Dimethylbenzanthracenegave positive responses in hpc/V79 co-cultures but not in H4IIEC3/G^–cells whereas benzo[a]pyrene was negative in both systems. Theseresults suggest that H4IIEC3/G^– cells retain metabolicactivities to convert different indirect mutagens into theiractive forms and clearly indicate the presence of liver specificcytochrome P-450-dependent mono-oxygenases. However, freshlyisolated hepatocytes are more efficient in metabolizing thetest compounds. Although hpc provide only external activation,the V79 cells system appears to be more sensitive for the detectionof promutagens.     

Absence of genotoxic effects in cells exposed to four ketonucleoside derivatives

The relationship between the structure and genotoxic potentialof four new cytostatic compounds — the ketonucleosidesKN-35, KN-43, KN-44 and KN-3 — was investigated in short-termin vitro assays of hypoxanthine-guanine phosphoribosyl transferaselocus mutation in V79 cells, induction of chromosomal aberrationsand sister chromatid exchanges on human lymphocytes, inductionof chromosomal aberrations and micronuclei in V79 cells, andtransformation of Syrian hamster embryo cells. None of the ketonucleosidesinduced mutagenic effects in any of the assays. Their failureto exhibit significantly genotoxic activity may be ascribedto the probable absence of any reaction between these drugsand the cellular DNA, and indicates that they act by some othermechanism which probably differs from the one observed withalkylating or intercalating antitumoural agents. This suggeststhat the cytotoxic activity of ketonucleosides cannot be relatedto genotoxicity.     

The molecular nature of mutations induced by adriamycin at the hprt locus of V79 cells

Adriamycin (AM), a widely used chemotherapeutic drug, induceda broad spectrum of gene mutations at the hprt locus of V79cells. The frequency and distribution of AM-induced deletionswas analyzed with multiplex polymerase chain reaction in twoV79 cell lines, which differed considerably in their spontaneousdeletion frequency. Among AM-induced mutants, deletions predominatedin both cell lines. Apart from total deletions of the hprt gene,partial deletions were found which were distributed all overthe hprt gene with breakpoints in nearly all introns. Underthe same experimental conditions, chromosome aberrations wereinduced by AM which mainly represented chromatid-type aberrations.Neither the induction of gene mutations nor the induction ofchromosome aberrations was enhanced by the repair inhibitor3-aminobenzamide. These results are discussed in the contextwith our earlier findings on bleomycin-induced mutations andit is suggested that at least two mechanisms lead to the formationof gene deletions. One of them seems to be associated with amisrepair process of frank DNA double-strand breaks and relatedto chromosome aberrations while the other is not. ¹To whom correspondence should be addressed     

Mutagenicity of extracts of brown and black masheri, pyrolysed products of tobacco using short-term tests

Black and brown varieties of masheri, which are pyrolysed tobaccoproducts were analysed for their mutagenic potentials usinga battery of test systems. Both materials were found to be mutagenicin Salmonella typhimurium strain TA98 with metabolic activationand also in V79 Chinese Hamster cells producing 8-azaguanineresistant mutations. Both varieties were found to induce statisticallysignificant increases in micronuclei formation as compared tothose produced in the solvent controls. Both varieties inducedstructural chromosomal aberrations in bone marrow cells of mice.Our data suggest that masheri is a potent mutagen in a varietyof test systems and is likely to have a mutagenic potentialvis à vis humans. ¹To whom correspondence should be addressed     

Activation and 'deletion' of self-reactive mature and immature T cells during ontogeny of Mls-1a mice: implications for neonatal tolerance induction

We assessed the kinetics of V_ß6⁺ T cell eliminationin the lymph nodes and thymus during Mls-1^a mouse ontogeny.Our results suggest that induction of tolerance to Mls-1^a antigensinvolves mechanisms other than clonal deletion of immature Tcells in the thymus. Mature CD4⁺CD8^– (CD4SP) T cells wereaffected by Mls-1^a antigens earlier than immature thymocytepopulations. Up to 2 weeks after birth, reduced frequenciesof V_ß6⁺ T cells were detected only in CD4SP cellsfrom the thymus and lymph nodes, and generation of CD4SP cellsin the thymus was blocked at least 1 week earlier than thatof their CD4⁺CD8^loTCR^hl immature precursors. The number of V_ß6⁺CD4SPT cells increased during the first 2 weeks of life and remainedconstant thereafter. We thus found no evidence of deletion ofmature V_ß6⁺CD4SP T cells, as the reduced frequenciesin adult mice can be attributed to the dilution of previouslygenerated cells in lymphoid organs of growing mice, which increasein cellulartty after birth. V_ß6⁺CD4⁺ T cells wereactivated in vivo shortly after birth, as shown by a selectiveincrease in IL-2 receptor a chain expression in the thymus andlymph nodes from day 0 to day 2 after birth. It is thereforelikely that endogenous expression of Mls-1^a antigen shortlyafter birth activates V_ß6⁺CD4SP T cells and rendersthem anergic. This process of tolerance induction may precedethe clonal deletion of immature T cells in the thymus, describedin the adult mouse.     

Effects of non-MHC background genes on the induction of CD4+ T cells that prevent rejection of a highly immunogenic tumor, FBL-3

This study showed that non-MHC genes common to (DBA/2 H-2^d)and (DBA/1 H-2^q) gave rise to suppressor T (T_a) cells in thehybrid F₁ mice between C57BL/6 (B6) strain in the antl-FBL-3tumor responses. FBL-3, a Friend virus-induced tumor cell lineof B6 mouse origin, is highly immunogenic as shown by findingsthat syngenelc and hybrid F₁ mice with several other inbredstrains rejected up to 3 x 10⁷ tumor cells inoculated s.c. andgenerated potent CTL responses after mixed lymphocyte tumorcell culture. In contrast to these mice, (B6 x DBA/2) and (B6x DBA/1)F1 mice did not reject the tumor as the tumor dosesincreased. Progressive tumor growth in these F₁ mice was blockedby an I.p. Injection of cyclophosphamlde (250 mg/kg) on day10, but not on day 5, after tumor cell inoculation. Antl-CD4(GK1.5) mAb exerted similar therapeutic effects against tumorwhen given twice, between day 0 and 10, whereas the additionalinjection of antl-CD8 mAb enhanced the tumor growth in micethat otherwise rejected the tumor. Thus, In the response of(B6 x DBA/2)F, mice to FBL-3 tumor cells, CD4⁺ T₈ seemed todown-regulate the immunologically mediated regression of thetumor produced by CD8⁺ CTL. This was evidenced by limiting dilutionculture analyses, which showed that the frequency of an FBL-3-speclflcCTL precursor in the (B6 x DBA/2)F1 mice that rejected the tumorwith antl-CD4 mAb was 7- to 9-fold higher than that in micein which the tumor regressed spontaneously. That more than onegene was involved in suppressor T cell induction was shown bythe tumor growth pattern in (B6 x DBA/2)F₁ x B6 backcross andB6D2F2 mice.     

Anergy in vivo: down-regulation of antigen-specific CD4+ Th1 but not Th2 cytokine responses

Efficient immunologic tolerance, defined as antlgen-speclflcunresponslveness, can be peripherally induced by the l.v. Injectionof syngenelc splenocytes coupled with antigen using ethylenecarbodilmlde (ECDI). We have previously reported that unresponslvenessinduced via l.v. Injection of syngenelc splenocytes coupledwith intact, UV-lnactlvated Theiler's murine encephalomyelitisvirus (TMEV-SP) resulted in ‘split tolerance’. Bothvtrus-speclflc delayed-type hypersensltlvlty and lgG2a levelswere inhibited, whereas lgG1 levels were increased when comparedwith sham tolerized controls. In the present report we demonstratethat tolerance induced by l.v. Injection of TMEV-coupled splenocytesresulted in antigen-specific inhibition of T cell proliferation,as well as IL-2 and IFN- production in response to both wholeTMEV and the immunodomlnant viral epitope. Additionally, toleranceinduction resulted in abrogation of T_h1 -derived [IL-2, IFN-and LT/tumor necrosis factor-ß (TNF-ß)]cytokine mRNA expression in response to In vitro stimulationwith UV-inactlvated TMEV as determined by reverse transcrlptasepolymerase chain reaction. In contrast, expression of T_h2-derived(IL-4, IL-6 and IL-10) cytokine mRNA was not affected in tolerizedmice. Tolerance functioned directly at the level of CD4⁺ T_h1cells at both the induction and effector limbs as depletionof CD8⁺ T cells both prior to in vivo tolerizatlon or in vitroculture had no effect on inhibition of T_h1-specific responses.The mechanism of In vivo tolerance induction appeared to beanergy of CD4⁺ T_h1 cells since IL-2, IFN- and LT/TNF-ßmRNA expression as well as virus-specific prollferatlve responsescould be restored by addition of rlL-2 to In vitro culturesof tolerant, CD4⁺ T_h1 populations. These results suggest thatin vivo ‘split tolerance’ Induced by l.v. Injectionof ECDI-flxed, antigen-coupled splenocytes involves anergy ofTMEV-speclflc, CD4⁺ T_h1 lymphocytes and concomitant primingof T_h2 cells. The induction of antlgen-speclflc, in vivo anergyhas important implications in the design of therapeutic strategiesfor immunopathologic diseases mediated by T_h1 lymphocytes, especiallyT cell-mediated autoimmune disorders.     

The sensitivity of IL-4 production for cAMP inducers is lost in CD4+ T cells from aged mice

CD4⁺ T cell clones have been demonstrated to display a differentialsensitivity for the induction of cAMP. In the present studywe investigated whether the differential sensitivity of CD4⁺T cell clones for cAMP inducers is also applicable to freshlyisolated phenotypically and functionally distinct CD4⁺ T cellsubsets that develop naturally in aging mice. Our results showthat the concanavalin A induced and anti-CD3 induced proliferativeresponse of CD4⁺ T cells from young mice is more sensitive forprostaglandin E₂ (PGE₂) and forskolin than that of their agedcounterparts, although the IL-2 production by these cells wasequally sensitive. In contrast, only a slight or no inhibitoryeffect of these cAMP inducers was found when the cells werestimulated with the combination of phorbol myristate acetateand lonomycln. In contrast to the findings obtained with T_n2clones, IL-4 production by freshly isolated CD4⁺ T cells wasinhibited by the cAMP inducers, whereas exogenous IL-2 had norestorative effect. However, the IL-4 production by CD4⁺ T cellsfrom aged mice was less sensitive than the IL-4 production byCD4⁺ T cells from young mice, although CD4⁺ T cells from agedmice showed significantly higher levels of intracellular cAMPin response to PGE₂. These higher levels of cAMP were relatedto the increased fraction of memory cells in aged mice: theMel-14^– Pgp-1⁺⁺ CD4⁺ T cells responded with at least 2-foldhigher levels of intracellular cAMP than the naive cells inyoung as well as in aged mice. Although memory CD4⁺ T cellsfrom young as well as aged mice responded vigorously to PGE₂by an enhancement of intracellular cAMP, only the IL-4 productionby cells from young mice was significantly inhibited. Therefore,it is not likely that the induction of cAMP is a major eventin the skewing of a primary response towards a T_h2 type of response.     

Development of short-term mutagenicity test systems in vitro: metabolic activation of indirectly acting mutagens by three immortal rat hepatocyte lines

The metabolic capacity to activate the indirectly acting promutagensaflatoxin B₁, cyclophosphamide, benzo[a]pyrene, 7,12-dimethylbenz[a]anthraceneand dimethylnitrosamine into DNA-reactive metabolites was investigatedin three immortalized rat hepatocyte cell lines (NRL cl-B, NRLcl-C and ARL) by analysing chromosome aberrations and sisterchromatid exchange (SCE). In all three cell lines a significantclastogenic and SCE inducing response was observed after exposureto each test compound. Furthermore, activities of the two enzymesaryl hydrocarbon hydroxylase and aldrinepoxidase, which playmajor roles in the cytochrome P450-dependent metabolism, couldbe determined in all cell lines. In contrast to the hepatocytelines in V79 Chinese hamster cells which were used as a referencecell line without any cytochrome P450 metabolizing capacity,no arylhydrocarbon hydroxylase or aldrinepoxidase activitieswere detected. A cytogenetic response to the test compoundswas only observed in the presence of the exogenous activatingsystem S9 mix. Due to the wide efficient and stable spectrumof their metabolizing capacities the tested rat hepatocyte linesoffer promising perspectives as alternative assay systems forthe detection of indirectly acting mutagens. ²To whom correspondence should be addressed     

Effect of 1 Gy X-rays in G1 phase on activation of cell cycle checkpoints in human lymphocytes: role played by chromosomal aberrations

The effect of X-irradiation (1 Gy) during G₁ on the transitionfrom G₁ to S and on the length of G₂ over cell cycles subsequentto irradiation was studied in human lymphocytes from six differentdonors. After irradiation a delay was observed in the onsetof S phase, as was an extension of the G₂ phase lasting throughoutthe three to four subsequent cell divisions. The extension ofG₂ and of the cell cycle as a whole is partly related to thepresence of chromosome aberrations in the cell. This is demonstratedby: (i) the presence of a larger number of chromosome aberrationsin M₁ cells corresponding to sampling times longer after thatof irradiation; (ii) the presence of a larger number of chromosomeaberrations in cells with a longer G₂. The most significantchromosome aberrations in this respect are isochromatid fragments.Lastly, we observed that irradiation during G₁ activates anothercheckpoint governing the way mitosis proceeds. This takes theform of an extension of metaphase; in this case also, in somecells, activation of a possible checkpoint during pre-anaphaseseems to be related to the presence of aberrations. ¹To whom correspondence should be addressed. Tel: +39 6 49912471; Fax: + 39 6 4456866; Email: olivien{at}axcasp.caspur.it     

Aneuploidy induction by benzo[a]pyrene and polyploidy induction by 7,12-dimethylbenz[a]anthracene in Chinese hamster cell lines V79-MZ and V79

We found that different ploidy effects were induced in fourChinese hamster-derived cell lines (V79-MZ, V79, CHL and CHO-K1)treated through two cell cycles with polycyclic aromatic hydrocarbonsin the absence of a metabolic activation system. 5-Bromodeoxyuridinewas used to investigate cell cycle delay and sister chromatidexchanges (SCE) induced by the chemicals. Benzo[a]pyrene (BP)induced aneuploidy at 2.5–10 µg/ml in V79-MZ cells.7,12-Dimethylbenz[a]anthracene (DMBA) induced polyploidy at3.125–6.25 and 6.25–12.5 µg/ml in V79-MZ andV79 cells respectively. Higher concentrations caused cell cycledelay and, therefore, did not affect ploidy. BP and DMBA didnot induce a significant increase in SCE frequency at the abovedoses. 3-Methylcholanthrene tested up to its solubility limit(10 µg/ml) did not induce numerical aberrations in anycell line. The clastogen mitomycin C, tested up to 0.01 µg/ml,did not produce numerical aberrations but did significantlyincrease SCE frequency in all cell lines. The spindle poisoncolchicine, tested up to 0.1 µg/ml, induced ploidy changesin the four cell lines that showed different sensitivities.Four cell lines showed no arylhydrocarbon hydroxylase activity,and V79-MZ, but not the other cells lines, showed high glutathioneS-transferase activity. Aneuploidy induction by BP and polyploidyinduction by DMBA in the absence of S9 mix in vitro have notbeen described before, and the finding might be due to the effecton tubulin. Due to their specificity and high sensitivity, theV79-MZ and V79 cell lines might be good systems for detectinganeuploidogens ³To whom correspondence should be addressed     

Cytogenetic effects induced by alkylated guanine in mammalian cells

Cytogenetic effects of 0⁶-ethylguanine (0⁶EtG) were investigatedin V79 Chinese hamster cells and human lymphocytes. The frequencyof micronucki and of polyploid cefls was increased in a dose-dependentmanner in V79 cells. Micronuclei were larger than those inducedby the alkylating agent diethylsulfate, suggesting that theyoriginated from chromosome loss rather than from chromosomebreakage. Chromosome displacement was also observed and furtherindicated damage to the spindle. In human lymphocytes, bothaneuploidy and polyploidy were induced. 0⁶EtG did not induceeither micronuclei or polyploidy in a V79 derivative, deficientin hypoxanthine-guanine phosphoribosyl transferase, suggestingthat conversion of the base to a nuckotide is necessary. Preliminarydata indicated that 7-ethylguanine was also able to induce aneuploidyand polyploidy in lymphocytes, although it was barely activein V79 cells. The hypothesis proposed is that alkylated nucleotidesmay behave as spindle poisons. As a consequence, treatment ofmammalian cells with alkylating agents may induce numericalchromosome aberrations via alkylation of guanine nucleotidepools, a pathway which has not so far been proposed.     

Comparison of S9 mix and hepatocytes as external metabolizing systems in mammalian cell cultures: cytogenetic effects of 7,12-dimethylbenzanthracene and aflatoxin B1

Two external metabolizing systems, S9 mix from Aroclor-induced rat livers and freshly isolated hepatocytes, were used for activation in cultures of human lymphocytes and V79 cells. 7, 12-dimethylbenzanthracene (DMBA) and aflatoxin B1 (AFB1) were employed as indirectly acting reference mutagens. Mutagenic effects were measured by induction of sister chromatid exchange (SCE). With DMBA, SCE-inducing effects were found to be quite similar after activation by S9 mix and activation by hepatocytes. In human lymphocytes nearly the same dose-effect relationships were found with both metabolizing systems; in V79 cells the hepatocyte-mediated induction of SCE was detectable at slightly lower concentrations than the S9-mediated SCE induction. In contrast with AFB1, S9 activation led to a stronger SCE induction than hepatocyte activation in both target cells. The induction of chromosomal aberrations by AFB1 after activation by the two metabolizing systems was also analysed in V79 cells. This experiment again revealed that AFB1 was more efficiently activated by S9 mix than by hepatocytes, and it appeared that AFB1 is a more potent inducer of chromosomal aberrations than of SCE. The different activation capacities of the two metabolizing systems for AFB1 may be due to the maintenance of inactivation mechanisms in hepatocytes or to the Aroclor induction of the S9 fraction. Our experiments have shown that the suitability of hepatocytes as an activation system is not restricted to microbial or eukaryotic point mutation assays, but that hepatocyte metabolism can also be successfully included in cytogenetic tests with short- and long-term cultures of mammalian target cells.     

血管生成素-1激活tyrosine kinase/PI3K增加血管内皮细胞［Mg2+］i

目的：探讨血管生成素-1（Ang-1）对人脐静脉内皮细胞（HUVECs）内游离镁离子浓度（［Mg²⁺］_i）的调节机制。方法:我们采用荧光指示剂mag-fura-2，运用PTi 阳离子测定系统动态测HUVECs的［Mg²⁺］_i。结果:Ang-1诱导的［Mg²⁺］_i增加与细胞外Mg2+浓度无关。Ang-1诱导的［Mg²⁺］_i增加与细胞内Ca2+浓度无关。经酪氨酸激酶阻断剂(tyrphostin A23 和 genistein), 磷脂酰3激酶阻断剂 (wortmannin和LY294002)预处理，均显著阻断Ang-1诱导的［Mg²⁺］_i增加。但经活化丝裂原激活激酶阻断剂(SB202190和PD98059) 预处理，不能阻断Ang-1诱导的［Mg²⁺］_i增加。结论:Ang-1通过酪氨酸激酶/磷脂酰3激酶信号传递途径使细胞内的Mg2+库释放Mg2+，从而增加HUVECs的［Mg²⁺］_i。     

雌激素对内皮素诱导心肌细胞肥大反应的影响及其机制

目的： 探讨雌激素（E₂）对内皮素-1（ET-1）诱导心肌细胞肥大反应的影响及其机制。方法： 以ET-1刺激体外培养的新生大鼠心肌细胞，建立心肌肥大细胞模型；观察E₂对ET-1诱导心肌细胞肥大反应的影响，并检测心肌细胞中ERK1/2活性的变化。结果： ET-1可使心肌细胞蛋白质含量、表面积和［³H］^-亮氨酸掺入显著增加，这些作用可被E₂明显抑制。E₂预处理抑制ET-1诱导的心肌细胞ERK1/2活性的增加。雌激素受体拮抗剂他莫昔芬（Ta）可抑制E₂对ET-1诱导的心肌细胞［³H］^-亮氨酸（［³H］^-Leu）掺入和ERK1/2活性的作用。MEK1/2抑制剂PD98059预处理使ET-1诱导的心肌细胞［³H］^-Leu掺入减少，使E2对ET-1诱导的心肌细胞肥大反应的抑制作用加强。结论： E₂通过雌激素受体抑制ET-1诱导的心肌细胞肥大反应，这一过程与ERK1/2信号转导途径有关。     

Induction of unresponsiveness to the superantigen staphylococcal enterotoxin B: cyclosporin A resistant split unresponsiveness unfolds in vivo without preceding clonal expansion

The continuous presence of antigen and powerful immune responses(exhaustive cell proliferation) of llgand reactive T cells arecurrently thought to condition clonal deletion and/or inductionof unresponsiveness to endogenous or exogenous superantigens(SAg). Here we report that in vivo induction of unresponsivenessto the SAg staphylococcal enterotoxin B (SEB) can be an immediateprocess. Within hours a large portion of ligand reactive V_ß8⁺T cells becomes clonally deleted by apoptosis. In parallel,the remaining V_ß8⁺ T cells are unresponsive to SEB,yet at the same time express functional IL-2 receptors (IL-2R)and thus are highly responsive to the growth promoting effectsof IL-2. In a subsequent step refractory IL-2R⁺V_ß8⁺T cells undergo a wave of cell proliferation for 48 h, presumablydriven by IL-2. Thereafter a large proportion of V_ß8⁺T cells succumb to apoptosis, the remaining cells display thehallmarks of split unresponsiveness, i.e.they display a selectivefallure to produce IL-2 upon SEB stimulation in vitrocombinedwith a preserved capability to express functional IL-2R. Earlydeletion and Induction of unresponsiveness to SEB are cyclosporinA (CsA) resistant, while clonal expansion with subsequent celldeletion is blocked by CsA, yet the development of split unresponsivenessis not impaired by CsA. The results suggest that IL-2 drivengrowth of refractory T cells may mimic powerful immune responsesof ligand reactive V_ß8⁺ T cells. Since unresponsivenessto SEB precedes in vivo expansion, the results as such questionthe concept of ‘exhaustive cell proliferation’ asa prerequisite for induction of unresponsiveness. In additionthey suggest that unresponsiveness (tolerance) can be inducedin the presence of CsA.     

Quercetin and the mutagenicity of wines

Various studies have shown the mutagenicity of red wine. Themajor mutagens identified in red wine have been flavonoids,i.e. rutin and its aglycone quercetin. Besides flavonoids, however,it has recently been reported that H₂O₂ may account for themutagenicity of red wine in the L-Arabinose resistance test.In the present study we report on the role of flavonoids inthe mutagenicity of red wine in the Ames assay. Different winesfrom Portugal and Spain have been tested after concentrationin XAD-2 columns in strains TA98 and TA104 of Salmonella typhimuriumconcurrently with the determination of the respective contentof quercetin by HPLC. A similar approach was used for pilotscale productions of red wines. In all cases quercetin couldbe demonstrated as the major mutagen in red wines. The levelsof quercetin in finished wines and during the wine-making processshowed a good fit with the levels of mutagenicity detected.Catalase had no effect whatsoever on the mutagenicity of winesin both TA98 and TA104. These results do not rule out a rolefor H₂O₂ in the mutagenicity of wines, detected in other geneticend-points, because H₂O₂ can be formed from the autooxidationof quercetin. ⁴To whom correspondence should be addressed