Characteristics of revertants induced by EMS and u.v. light from a 6-thioguanine resistant HGPRT deficient V79 Chinese hamster cell line

A spontaneous TG^R HAT^S V79 mutant had an HGPRT specific activityof less than 5% that of the wild type. Only two spontaneousrevertants were recovered from more than 10⁸ mutant cells screened.However, HAT resistant clones were recovered, after EMS mutagenesisat a frequency of 4.5 x 10^–6 and at a lower frequencyfrom u.v. irradiated mutant cells. Of more than 400 clones tested,over 90% had regained a HAT^R TG^s HGPRT ⁺ revertant phenotype.Nine of these revertants that were further characterised hadHGPRT specific activities within the wild type range and showedwild type electrophoretic mobility. These data suggest thatthe original HGPRT^– phenotype was of a mutational ratherthan an adaptational origin.     

探讨18F-FDG SPECT/CT在胃癌疗效评价中的价值

 目的 探讨¹⁸F-FDG SPECT/CT（¹⁸F-FDG coincidence single photon emission computed tomography/multi-detector computed tomography,¹⁸F-FDG SPECT/CT）所测定T/B值在胃癌临床疗效评价的价值。 方法 对31例未治疗的新发的原发性胃癌患者化疗前后进行MDCT增强扫描与 ¹⁸F-FDG SPECT/CT断层显像并测定T/B值。根据患者MDCT的疗效判定结果,初步探讨¹⁸F-FDG SPECT/CT的T/B值进行疗效评价的标准。 结果 ¹⁸F-FDG SPECT/CT中的T/B值下降率R_△T/B与肿瘤最大径之和的下降率R_△MD存在显著的直线相关(相关系数R_s=0.867, P<0.001);¹⁸F-FDG SPECT/CT中的R_△T/B预测胃癌疗效的ROC曲线图的面积为0.943,其准确率为94.3%。若分别设R_△T/B为35%、40%为基线,判定客观缓解OR（Objective remission,OR, CR+PR）的灵敏度分别为100%、85.7%,特异度94.1%、100%,阳性预测值93.3%、100%,阴性预测值100%、 89.5%;判定有效率RR的结果分别为48.4%、38.7%。 结论 胃癌T/B值的改变率进行疗效评价具有较高的准确性,我们推荐T/B值下降35%以上作为临床判定客观缓解、无缓解的分界点。     

Differential effects of complete and second-stage tumour promoters in normal but not transformed human and mouse keratinocytes

The complete tumour promoter phorbol, 12-myristate, 13-acetate(PMA) induces terminal differentiation in the majority of normalcultured human and mouse keratinocytes but a subpopulation existswhich is resistant to this effect (PMA^R). We have compared withPMA the effects of mezerein (Mez) and phorbol, 12-retinoate,13-acetate (PRA) on the ability of normal and transformed humanand mouse keratinocytes to terminally differentiate in an attemptto elucidate why the latter two compounds are inefficient completetumour promoters but are effective as second-stage promoterswhen given after PMA in the two-stage promotion regimen. BothPMA and Mez increased cornified envelope formation in a similarway in normal and transformed keratinocyte cultures inducinga 20- to 25-fold increase over the solvent controls in normalkeratinocytes but only a 2-fold increase in line SCC-27 (a cellline derived from a human squamous cell carcinoma). However,while quantitative dose response studies of the effect of phorbolesters on colony forming ability revealed a proportion of normalhuman and mouse keratinocytes which were resistant to PMA, nonormal keratinocytes were resistant to Mez or PRA. In contrast,cell lines derived from papillomas and squamous cell carcinomasshowed a resistant fraction of similar size with all three compounds.Furthermore, when Mez or PRA were mixed with PMA the survivalof line SCC-27 was the same as when the cultures were treatedwith the compounds individually indicating that the keratinocyteswhich were resistant to PRA or Mez were also the PMA^R subpopulation.A non-tumourigenic subclone of line SCC-12 (clone F.2), previouslyshown to possess all known properties of transformed keratinocytesexcept defective terminal differentiation in suspension cultureresponded to PMA and Mez in a similar way to normal keratinocytes,suggesting that resistance of the PMA^R subpopulation to second-stagepromoters requires the expression of a defect in the keratinocyteterminal differentiation programme.     

Mutagenesis and lethality following S phase irradiation of xeroderma pigmentosum and normal human diploid fibroblasts with ultraviolet light

The mutagenic and lethal effects of u.v. light exposure in theDNA synthetic phase of the cell cycle were determined in xerodermapigmentosum complementation group A (XP-A), hereditary adenomatosisof the colon and rectum (ACR), and a normal, foreskin derivedcell strain (AG1522). For AG1522, an increased sensitivity tothe cytotoxic effects of u.v. light (survival curve D₀ = 3.2J/m²) was observed as compared to previous findings for confluent,non-proliferating cultures (D₀ = 4.2 J/m²). XP-A fibroblastswere markedly hypersensitive (D₀ = 0.5 J/m²) and ACR fibroblastsexhibited an intermediate response (D₀ = 2.0 J/m²). The mutagenicresponse of ACR fibroblasts, however, was similar to normalfibroblasts. A threshold of 1.5–2 J/m² was observed foru.v. induced mutagenesis in normal and ACR fibroblasts. XP fibroblasts,on the other hand, were strikingly hypermutable and demonstratedlittle or no threshold. When S phase mutagenesis was consideredas a function of survival level rather than u.v. light dose,XP fibroblasts remained significantly hypermutable as comparedwith normal fibroblasts at all survival levels. Previous mutagenesisresults with confluent, nonproliferating cultures of XP andnormal fibroblasts were reanalyzed as a function of cytotoxicity;XP hypermutability at all survival levels was also observed.     

Comparison of dose-effect relationships of carcinogens following low-dose chronic exposure and high-dose single injection: an analysis by the median-effect principle

Experimental dose-effect relationships of carcinogens followingeither acute (single dose) or chronic (time to tumor) exposureappears to conform with the median-effect principle (Chou, J.Theor. Biol. 59, 253–276, 1976) of the mass-action law:f_a/ (1 – f_a) = (D/D_m)^m, where D is dose or cumulativedose, D_m is the D required for the median-effect, m is the Hill-typecoefficient, and f_a is the fraction that is affected by D. Theparameters m and D_m are the basic characteristics for each carcinogenat specified experimental conditions. A plot of y = log [(f_a)-1-^–1]–1]^–1vs x = log (D) gives the slope, m, and the intercept, log D_m,at y = 0. Using previously reported data, it is shown that dose-effectrelationships of carcinogens obtained from various experimentaldesigns (e.g., mode of exposure, route of administration, ageat beginning of exposure to carcinogen, type of tumor produced,and strain and sex of animal used) can be normalized and compareddirectly on the same gauge and thus their consistency with themass-law principle can be clearly demonstrated. The analysissuggests that chemical carcinogens, like non-carcinogenic chemicals,exert their effects according to the principle of the mass-actionlaw. It also suggests that the interaction of the ultimate carcinogensand the probable targets is a multi-event or a slow-transitionprocess (i.e., m>1). The analytical procedure described byf_a = [1 + (D_m/D)^m]^–1 provides a simplified general methodfor assessment of low-dose risk of carcinogens.     

188 Re 标记DTPA-DG对肺癌A549 细胞增殖的影响

 目的 研究不同浓度¹⁸⁸ Re-DTPA-DG（^188Re标记二乙三胺五乙酸-葡糖胺）对人肺癌细胞A549增殖的影响，探讨其作用机制。方法 将人肺癌细胞A549分成3组，实验组（¹⁸⁸ Re-DTPA-DG组）、对照组（¹⁸⁸ Reoi淋洗液、缓冲液及等量的DTPA—DG）及生理盐水组（含生理盐水及等量缓冲液），前两组均设三种不同浓度，148、296、444MBq／L。观察细胞形态，采用四氮唑盐比色法（MTT法）评价¹⁸⁸Re-DTPA-DG对癌细胞A549增殖的影响。结果¹⁸⁸Re-DTPA-DG导致的细胞损伤比^188Re04-明显；随着药物浓度增加^{188 Re-DTPA-DG}导致的损伤越显著。结论¹⁸⁸ Re-DTPA-DG对肿瘤细胞增殖有较明显的抑制效应，其机制可能是由于¹⁸⁸ Re-DTPA-DG进入到细胞内，其辐射效应更能导致肺癌细胞损伤。     

Mutagenesis in Escherichia coli K-12 mutants defective in superoxide dismutase or catalase

Escherichia coli K-12 strains with diminished levels of superoxidedismutase (SOD) due to inactivation of the sodA, sodB or sodAsodB genes were constructed in order to quantify the role ofO^–₂. in mutagenesis. Mutagenesis was monitored by selectingforward mutations to L-arabinose resistance (Ara^R). No sodAsodB mutant inability to grow in aerobic minimal medium wasfound, in contrast to that previously reported for a differentE.coli wild-type genetic background. The role of SOD for copingwith the damaging effects of superoxide became evident afterthe increase in intracellular O^–₂. flux by growing cellsunder hyperoxygenation, but particularly by using redox cyclingcompounds such as plumbagin, paraquat and menadione. Bacteriacompletely devoid of SOD activity showed very high levels ofAra^R-induced mutants at doses that were non-mutagenic for theSOD-proficient parental or the sodA or sodB single mutants.The mutagenicity of nifurtimox and quercetin were studied tofurther compare the responses of the SOD-deficient bacteriato those of their SOD-proficient counterparts. The relativeimportance of SOD and catalase for coping with the damagingeffects of O^–₂. and H₂O₂ was quantified by comparing SOD-deficientbacteria with isogenic catalase-deficient cells (a katG katEdouble mutant). The mutagenicities of plumbagin and menadionewere much higher in SOD-deficient than in catalase-deficientbacteria, in agreement with the role of the O^–₂. radicalin the so-called metal-catalyzed Haber—Weiss reaction.The relevance of catalase in protecting against the damagingeffects of H₂O₂ was evident from the hypersensitivity of thekatG katE double mutant to the mutagenic and lethal effectsof this oxidizing agent. It is concluded that the Ara mutagenicityassay combined with depletion in specific antioxidative enzymescould be a tool in establishing the extent to which DNA damageby oxygen radicals is relevant to mutagenesis.     

Limited sampling models for topotecan pharmacokinetics

BACKGROUND: Limited sampling models for the estimation of the topotecanArea Under the concentration versus time Curve (AUC) and itslactone ring opened form (AUC Tm), from one or more plasma concentrationdeterminations, are desired for further population-kinetic studies. PATIENTS AND METHODS: The models were developed and validated using 34 pharmacokineticcurves in 19 patients who participated in a phase I study. RESULTS: A single point model was selected as optimal: AUC (µmol/L. min) = 499(min) . C_2h(µmol/L) + 0.85(m²/mg . µmol/L. min) . dose(mg/m²), and for topotecan-metabolite (Tm), AUCTm(µmol/L.min) = 55.1 (min) . CTm_2h (µmol/L) / -0.011(m²/mg. µmol/L . min) . dose (mg/m²), where C_2h is the plasmaconcentration (µmol/L) of topotecan at 2 h after the endof a 30-min infusion, and CTm_2h the concentration of the openedform at the same time point. The models are valid for dosagesranging from 0.5 to 1.5 mg/ m²/day and proved to be unbiased(MPE% = –1.8% and –9.3%, respectively) and precise(RMSE% – 17.9% and 22.7%, respectively). From the predictedAUCs, the clearance (Cl = dose (µmol)/AUC(µmol/L. min)) could also reliably be predicted, as well as the totalAUC (AUC + AUC Tm) (RMSE% = 17.1% and MPE% = –0.02%).Half-life values could not be predicted with acceptable precisionand accuracy. CONCLUSION: The limited sampling models presented may be useful for futurestudies focused on pharmacokinetic/ pharmacodynamic relationshipsof topotecan in large populations. AUC, limited sampling model, pharmacokinetics, topotecan     

Persistence of benzo[a]pyrene and 7,8-dihydro-7,8-dihydroxybenzo[a]pyrene in Fischer 344 rats: time distribution of total metabolites in blood, urine and feces

A comparison of the rates of elimination of [³H]benzo[a] pyrene(BaP) and 7,8-dihydro-7,8-diol-[³H]benzo[a]pyrene (BPD), aftersubcutaneous injection into Fischer 344 rats, shows they areboth eliminated at about the same rates and with the same patternover at least 7 days post-exposure. The end-rate of combinedurinary and fecal excretion was {small tilde}40 nmol/day. About20% of the injected BaP and {small tilde}3% of the injectedBPD remained at the site of injection for at least 9 days. Theremainder was distributed throughout the animal. If the rateof excretion continued at the observed steady-state rates, theBaP and BPD could persist for up to 40 days for each milligramof injected substance. The concentration of excretion productswere highest during day 1 and day 2 following exposure, decreasedexponentially to a concentration of {small tilde}0.5 µM(mixed metabolites) by day 5 following exposure, and then continuedto be excreted at that rate. Feces contained the highest totalamounts of radioactivity, which were {small tilde}2- to 4-foldhigher than the amounts in urine and {small tilde}15- to 50-foldhigher than in total blood. The conversion of organic ³H to³H₂O during the experimental period indicates.that whole-bodyphenol(quinone) formation was significant for BaP metabolism,but was much less for BPD metabolism. When BaP was injected,both blood and urine contained water-soluble, volatile tritiumcounts (³H₂O). Injection of BPD resulted in volatile ³H₂O inurine but not in blood. The persistence of BaP and BPD metabolitesin skin, blood, urine and feces compartments indicates thereis a substantial reservoir of the chemical(s) that could beused to replenish repaired or discarded DNA adducts.     

Role of reactive oxygen in tumor promotion: implication of superoxide anion in promotion of neoplastic transformation in JB-6 cells by TPA

The role of reactive oxygen (RO) in the promotion of neoplastictransformation of JB6 mouse epidermal cells by 12-O-tetradecanoylphorbol-13-acetate(TPA) was investigated using inhibitors of RO itself or RO generatingsystems of seven different types. Bovine erythrocyte CuZn superoxidedismutase (SOD) maximally decreased anchorage-independent (AI)colony induction by TPA in semi-solid agar in a dose-dependentmanner to 10% of TPA control level. The inhibitory effect wasspecifically on induction of transformation, not expressionof transformation. Copper (II) (3,5-diisopropylsalicylic acid)₂,which exhibits biomimetic SOD activity, was also effective.Two enzyme eliminators of H₂O₂, catalase and glutathione peroxidase,failed to prevent TPA-promotion. Among three hydroxyl radicalscavengers, D-mannitol and Na-benzoate were moderately activebut tetramethylurea did not specifically inhibit AI colony inductionby TPA. A quencher of singlet oxygen, 1,4-diazobicyclo-[2,2,2]octanewas also inactive. Antioxidants blocked AI transformation byTPA moderately (n-propyl gallate and tannic acid) or weakly(BHA). BHT did not specifically inhibit promotion of transformation.The effects of three inhibitors of the arachidonic acid cascadewere examined. NDGA and quercetin (lipoxygenase inhibitors)were moderately active but indomethacin (cyclooxygenase inhibitor)was much less active. Based on these results, we suggest thatsuperoxide anion (O₂^–) is required for promotion of transformationby TPA. H₂O₂ and ¹O₂ appear not to be required. Hydroxyl radicalsand lipid peroxides, possibly associated with O₂^– actionor formed in the course of oxidative metabolism of arachidonicacid also appear to be required but to a lesser extent. Productsof the lipoxygenase pathway of arachidonic acid metabolism butnot the cycloxygenase pathway may be important in promotionof transformation by TPA in JB6 mouse epidermal cells. The epidermalcells themselves can be both the source of and the target ofthe reacive oxygen in promotion.     

Fetal mouse susceptibility to transplacental lung and liver carcinogenesis by 3-methylcholanthrene: positive correlation with responsiveness to inducers of aromatic hydrocarbon metabolism

The role of metabolic activation of carcinogens in fetal tissueas a determinant of sensitivity in transplacental carcinogenesiswas investigated in a pharmacogenetic experiment utilizing backcrossesof C57BL/6 (Ah^bAh^b, responsive to induction of aromatic hydrocarbonmetabolism) and DBA/2 (Ah^dAh^d, non-responsive) mice. Responsive(C57BL/6 x DBA/2)F₁ and non-responsive DBA mothers, all carryingboth responsive (Ah^bAh^d) and non-responsive (Ah^dAh^d) fetuses,were given i.p. doses of the carcinogen 3-methylcholanthrene(MC) ranging from 5 to 175 mg/kg on gestation day 17. At 10months of age the metabolic phenotype of each offspring wasdetermined, and correlated with number of lung and liver tumors.Both male and female Ah^bAh^d (responsive) offspring in most dosegroups presented a consistent two- to three-fold higher incidenceof lung tumors than did non-responsive Ah^dAh^d littermates. Thedifference held for offspring of both (C57BL/6 x DBA)F₁ andDBA mothers and it was of statistical significance for one orboth sexes at most dosage levels. Hepatocellular tumors werealso significantly more frequent in responsive male Ah^bAh^d progenyof (C57BL/6 x DBA/2)F₁ mothers than in non-responsive Ah^dAh^dlittermates. Progeny of the DBA mothers exhibited significantlymore liver and lung tumors than did those of the (C57BL/6 xDBA/2)F₁ mothers receiving the same dose. These results suggestthat in this model system both maternal and fetal genotype forresponsiveness to induction of aromatic hydrocarbon metabolismare important factors modulating fetal carcinogenic risk.     

A method to quantitate the relative initiating and promoting potencies of hepatocarcinogenic agents in their dose-response relationships to altered hepatic foci

The relative response to various initiating doses of diethylnitrosamine(DEN) and dimethylbenz[a]anthracene of the induction of numbersand size (vol. % of liver) of altered hepatic foci (AHF) inlivers of adult female rats of the Sprague-Dawley and Fischer344 (F-344) strains was studied by methods of quantitative stereologyin the presence and absence of the promoting agent, phenobarbital(PB, 0.05% in the diet). In all cases, a relatively linear responsewith dose, even at the lowest doses employed, was obtained exceptfor the numbers of AHF at the highest dose of DEN (30 mg/kg),which was not significantly different from that at a dose of10 mg/kg in F-344 female rats. Similar dose-response data wereobtained at various doses of two promoting agents effectivein hepatocarcinogenesis, PB and 2,3,7,8-tetrachloro-dibenzo-p-dioxin(TCDD), in livers of F-344 female rats following initiationwith DEN (10 mg/kg) 24 h post-70% hepatectomy. The responseto these agents exhibited threshold levels below which no increasein number or vol. % of liver of AHF was noted in comparisonwith that in livers of animals not treated with the promotingagents. At several subthreshold doses of both PB and TCDD aninhibition of AHF formation and growth (measured as vol. % ofliver) was observed. Based on quantitative stereologic calculations,parameters for the estimation for the relative potency of chemicalsas initiating or promoting agents have been established. Theseare defined as: initiation index = no. of foci induced x liver^–1x [mmol/kg body wt]^–1 and promotion index = V_f/V_c x mmol^–1x weeks^–1 where V_f is the total volume fraction (%) occupiedby AHF in the livers of rats treated with the test agent andV_c is the total volume of AHF in control animals which haveonly been initiated. These parameters were calculated for anumber of agents based on data published in the literature andfrom those reported herein. Neither parameter varied significantlywith the dose of the initiating agent based on the data in thispaper. The range of promotion indices extended over more thaneight orders of magnitude, whereas that of initiation indiceswas much less variable. Such parameters may be useful as quantitativeestimates of the potency of hepatocarcinogenic agents, suchvalues having potential application to risk estimations.     

Deuterium isotope effect on metabolism of N-nitrosodimethylamine in vivo in rat

The maximal rates of metabolic oxidation of N-nitrosodimethylamine(NDMA) and N-nitrosodimethylamine-d₆ (NDMA-d₆) in viva (V^H andV^D, respectively) have been measured by following ¹⁴CO₂ exhalationin rats after intraperitoneal injection of the two ¹⁴C-labelledcarcinogens at high doses (20 or 40 mg/kg). Complete deuterationof NDMA reduced only slightly the maximal rate of metabolismwhen the two substrates were administered separately (V^H/V^D1.2). However, much larger(4-fold) deuterium isotope effectswere observed when mixtures of NDMA with NDMA-d₆ were injected.These results are tentatively interpreted as evidence that C-Hbond cleavage is not a rate-limiting feature of overall metabolism,but that the complex between NDMA and the principal enzyme(s)metabolizing it in vivo freely equilibrates with unbound substrate.Single, large, intrapentoneal doses of NDMA and NDMA-d₆ produceda similar alkylation of rat liver DNA and also of kidney DNA.However, a small oral dose (54 µg/kg) of NDMA-d₆ produced1/3 less alkylation of liver DNA and 3 times as much alkylalionof kidney DNA as did an equimolar dose of NDMA. The reductionin alkylation of liver DNA correlates well with, and possiblyexplains, the decreased ability of NDMA-d₆ to induce liver tumorsin rats. The associated increase in the alkylation of kidneyDNA suggests that this change is due to a decrease in the amountof nitrosamine removed from the portal blood on the first passthrough the liver.     

In vitro binding of aflatoxin B1 and 2-acetylaminofluorene to rat, mouse and human hepatocyte DNA: the relationship of DNA binding to carcinogenicity

DNA binding levels were determined and compared in culturedhepatocytes from male and female rats as well as other animalspecies following exposure to aflatoxin B₁ (AFB₁) or 2-acetylaminofluorene(2-AAF). When human, rat (both male and female) and mouse hepatocytesin primary culture were exposed to 2.0 ? 10^–7 M [³H]AFB₁(sp. act. 2.63 µCi/nmol) for 24 h, male rat hepatocyteshad the highest degree of [³H]AFB₁-DNA binding (203 pmol/mgDNA) and human hepatocytes contained the next highest bindinglevel (42 pmol/mg DNA). Hepatocytes from female rats contained38 pmol/mg DNA while cultured mouse hepatocytes contained only1.4 pmol/mg DNA. When the same dose of [³H]AFB₁ was administeredto the cultured male rat hepatocytes at 24 h, 48 h, 72 h and1 week after seeding, and incubated for 24 h, the DNA bindinglevels were 189, 175, 76, 75 pmol/mg DNA respectively. In parallelexperiments to the cultured male rat hepatocytes above, theAFB₁-DNA binding levels in the cultured female hepatocytes were42, 41, 37 and 34 pmol/mg DNA respectively. Human, male andfemale rat hepatocytes in primary culture were exposed to 5.2? 10^–5 M 2-acetyl-amino [9–¹⁴C]fluorene (sp. act.0.0094 µCi/nmol) for 24 h. It was determined that malerat hepatocytes contained the highest amount of radioactivelylabeled 2-AAF bound to their DNA (1.57 nmol/mg DNA), femalerat hepatocytes contained 0.62 nmol/mg DNA and human hepatocytescontained 0.29 nmol/mg DNA. Results from our in vitro hepatocyteculture system correlate well with in vivo animal studies dealingwith species and sex differences in DNA binding and carcinogenicsusceptability. This indicates that hepatocytes in vitro maintainmany of the biological properties necessary for carcinogen responsesimilar to liver cells in vivo. In addition, comparison of genotoxiceffect in cultured hepatocytes from animals as well as humansmay be useful in evaluating carcinogenic potential of xenobioticsin human liver.     

Differences in the calcium-mediated regulation of gap junctional intercellular communication between a cell line consisting of initiated cells and a carcinoma-derived cell line

Differences in calcium-mediated regulation of gap junctionalintercellular communication (GJIC) between a cell line consistingof mouse epidermal initiated cells (3PC) and a mouse epidermalcarcinoma-derived cell line (CA3/7) were studied. Under lowextracellular calcium (Ca²⁺_e) conditions (0.05 mM) CA3/7 cellsshowed a low level of GJIC compared with 3PC cells. High Ca²⁺_e(1.20 mM) raised GJIC between CA3/7 cells to the GJIC levelof 3PC cells, which in turn remained unchanged under these conditions.Raising the free intracellular calcium concentration (Ca²⁺₁),using a calcium ionophore (ionomycin) or the Ca²⁺-ATPase inhibitorthapsigargin under low Ca²⁺_e conditions, did not affect theGJIC level between 3PC cells, and increased GJIC between CA3/7cells. Intracellular calcium chelation in 3PC cells under lowCa²⁺_e conditions by ethylene glycol-bis(ß-amino-ethylether) N, N, N', N'-tetra-acetic acid acetoxy-methyl ester (EGTA-AM)decreased GJIC in this cell line. High Ca²⁺_e conditions protectedboth cell lines from a decreased GJIC by EGTA-AM exposure. Inhibitionof calmodulin (CaM) by calmidazolium (CDZ) or N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide(W-7) under low Ca²⁺_e conditions, inhibited GJIC in 3PC cellsand increased GJIC in CA3/7 cells. Inhibition of Ca²⁺/CaM-dependentprotein kinase (Ca²⁺/CaM-PK) by 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine(ML-7) decreased GJIC in both cell lines. Western analysis showedthat Cx43 was more phosphorylated in both cell lines in concurrencewith different effects on the GJIC level. Under conditions inwhich GJIC was inhibited, a decreased immunostaining of Cx43on the plasma membrane was found. The level of immunostainingof the cell adhesion molecule E-cadherin on the plasma membranesof both cell types remained unchanged under conditions in whichGJIC was changed by modulaters of (Ca²⁺)₁, CaM activity, orthe Ca²⁺/CaM-PK activity. These results indicate that differencesexist between 3PC cells and CA3/7 cells in the GJIC regulationby intracellular calcium and calmodulin.     

Fluoro-substituted N-nitrosamines. 4. Comparative genotoxic activities of N-nitrosodibutylamine and three fluorinated analogues in two bacterial systems

N-Nitrosodibutylamine (NDBA) and three fluorinated analogues(N-nitroso (4,4,4-trifluorobutyl)butyl-amine, F₃NDBA; N-nitrosobis(4,4,4-trifluorobutyl)amine, F₆NDBA; and N-nitrosobis (2,2,3,3,4,4,4-heptafluorobutyl) amine, F₁₄NDBA) were comparatively investigatedfor biological activity in two bacterial systems. Opposite ordersof magnitude were obtained for their potency in the two tests.For inducing his⁺ reversion in auxotrophic strains of Salmonellatyphimurium the sequence was F₃NDBA > F₆NDBA > NDBA andfor inducing lethal DNA damage in repair deficient strains ofEscherichia coli WP2 it was NDBA > F₆NDBA F₃NDBA. F₁₄NDBAwas not active in either test system.     

In vivo mutagenesis of the reporter plasmid pSP189 induced by exposure of host Ad293 cells to activated polymorphonuclear leukocytes

We measured the mutation frequency and spectrum induced by exposureof the mutation reporter plasmid _pSP189 in vivo to phorbol myristateacetate (PMA)-activ-ated human polymorphonuclear leukocytes(PMNs). The mutation frequency induced in the supF tRNA geneof _pSP189 transfected into human Ad293 cells by a 30 min exposureto 4X10⁶ activated PMNs/ml was 3- to 9-fold higher than thebackground mutation frequency of 0.1–1.8X10^-5. The enhancedmutation frequency caused by activated PMNs required replicationof the reporter plasmid in host Ad293 cells. Fifty five uniqueactivated PMN-associated mutants characterized by sequencingincluded base substitutions (55%) and deletions (45%), however,no small (1–3 bp) deletions were observed. Ninety fourpercent of point mutations occurred at C: G base pairs, withC: G     

Inductions of oxidative DNA damage and mesothelioma by crocidolite, with special reference to the presence of iron inside and outside of asbestos fiber

Inductions of oxidative DNA damage (oh⁸dG) in vitro and peritonealmesothelioma in rats (F344, female) were compared between crocidolite(CR) and de-ironized crocidolite [DCR, washed by HCl and ethylenediaminetetraacetic acid (EDTA)] to verify the hypothesis that reactiveoxygen species contribute to carcinogenesis, focusing on therole of iron present inside or outside of the CR. The yieldof oh⁸dG was 14.6 oh⁸dG/10⁵ in CR and 30.2 in DCR under simpleincubation with DNA. In the incubation systems added severalchemicals and H₂O₂, OCR induced higher levels of oh⁸dG thanCR. Especially, the addition of Fe₂O₃ and H₂O₂ to OCR increasedoh⁸dG in DNA depending on the Fe₂O₃ concentration, however,this tendency was not observed in the same system of CR. Surprisingly,7 out of 10 rats died within 2 days after the injection of 10mg of Fe₂O₃ following the DCR injection (5 mg/rat), showingnecroses of hepatocytes from the surface of each lobe whereCR and Fe₂O₃ particles had been deposited together. There wasno death in other groups of rats. One year after the i.p. injectionof CR (5 mg/rat, single injection), mesotheliomas were foundin all rats administered OCR and Fe (2 mg/rat, once a week,for 35 weeks), in 4 rats of OCR alone (n = 10), in 5 rats ofCR alone (n = 10) and in none of the rats administered Fe₂O₃alone (n = 10). Therefore, present results indicate that theinduction of oxidative DNA damage changed even when the sametype of asbestos was washed by chemical treatment, and Fe₂O₃promoted the development of mesothelioma which was induced byOCR.     

香加皮杠柳苷对MCF-7细胞周期及p21WAF1/CIP1表达的影响

 目的：研究香加皮杠柳苷（CPP）对人乳腺癌MCF-7细胞周期及p21^WAF1/CIP1表达的影响 ,探讨其抗肿瘤作用及作用机制。方法：采用MTT法检测不同浓度CPP（1.25、2.50、5.00、 10.00、20.00 ng/ml）作用不同时间（24、48、72 h）对MCF-7细胞的增殖抑制作用;应用流 式细胞术分析不同浓度CPP （2.50、5.00、10.00 ng/ml）分别作用于MCF-7细胞6、12、24、 48、72 h对肿瘤细胞周期的影响;并采用RT-PCR和免疫细胞化学技术检测细胞周期相关因子 p21^WAF1/CIP1的表达。结果：CPP能明显抑制MCF-7细胞的增殖,并呈浓度及时间依赖性,作用 于MCF-7细胞48 h的IC₅₀为（4.88±0.16）ng/ml。流式细胞术结果显示,CPP作用MCF-7细胞24 h时,G₀/G₁期细胞明显增多,而S期和G₂/M期细胞显著减少,与对照组相比差异有统计学意义 （P<0.05）,其中5.00 ng/ml组G₀/G₁期细胞由对照组的（49.33±3.25）%升高至（79.47± 2.40）%,S期和G₂/M期细胞由对照组的（28.47±1.59）%和（22.20±2.09）%分别下降至（10.13±3.26）%和（10.40±1.41）%。经CPP处理的MCF-7细胞中p21^WAF1/CIP1 mRNA的表达明显增强,p21^WAF1/CIP1/β-actin光吸度比值与对照组相比明显增高（P<0.05）。免疫细胞化学结果显示,CPP组MCF-7细胞中p21^WAF1/CIP1 蛋白的表达随作用浓度的增加而增强,其中10.00 ng/ml组肿瘤细胞p21^WAF1/CIP1的表达呈强阳性。结论：香加皮杠柳苷（CPP）具有显著的体外抗肿瘤作用,且有效剂量很小,其IC₅₀仅为（4.88±0.16）ng/ml。CPP可使MCF-7细胞发生G₀/G₁期阻滞,并可使细胞周期相关基因p21^WAF1/CIP1的mRNA及蛋白表达增强,提示阻滞细胞周期可能是CPP体外抗肿瘤的作用机制之一。     

A mammalian cell mutant with enhanced capacity to dissociate a bis-benzimidazole dye-DNA complex

The bis-benzimidazole dyes (specifically Hoechst 33258 and themore lipophilic derivative Hoechst 33342) are non-intercalatingAT base pair-specific ligands which bind to cellular DNA bynon-covalent association with the minor groove. The interactionof dye with cellular DNA is thought to be the principal pathwayfor the cytotoxic, mutagenic and DNA-damaging properties ofsuch agents. Upon binding and near UV light excitation, dyemolecules exhibit fluorescence enhancement such that dye/DNAassociation and dissociation in individual cells can be monitoredby flow cytometry. We have studied dye uptake and the DNA -dye dissociation characteristics of a Hoechst 33258-resistantmouse cell line (Hoe^R4l5) compared to the response of the parentalcell line Ltk^–. Hoe^R4l5 was found to show similar levelsof cross resistance ({small tilde} 10-fold) to Hoechst 33258and Hoechst 33342 comparedto parental responses except thatthe more lipophilic ligand was {small tilde}30-fold more toxic.Estimates of Hoechst 33342 uptake using flow cytometry or radiolabellingmethods indicated that resistance could not be attributed toreduced cellular uptake, low initial levels or different modesof DNA binding. Both cell lines showed similar initial levelsof dye-induced DNA strand-breakage. However, Hoechst 33342 resistancedid correlate with an enhanced capacity (10-fold) ofHoe^R4l5to remove dye from cellular DNA compared with the relativelylong retention (T_1/2 300 min) of ligand by the parental cellline. Our results are consistentwith the view that ligand persistencerather than indirect DNA damage is a more important factor inthe cytotoxicity of non-intercalating DNA-binding ligands. Amodel is presented of the cellular processes of DNA damage recognitionand surveillance for ligands which interact with the minor grooveof DNA.