A comparison of alternative distributions of postimplantation death in the dominant lethal assay

The action of beta-aminoethylisothiouronium bromide hydrobromide (AET) and sodium fluoride (NaF) on the clastogenic activity of Trenimon, cyclophosphamide, and bleomycin was tested on cultures of human peripheral lymphocytes with and without the addition of rat liver S9 mix. In addition, the influence of both anticlastogens on the SCE-inducing activity of Trenimon and cyclophosphamide was examined under the same conditions. In the absence of S9 mix both substances displayed the known anticlastogenic action when TR was the standard clastogen but acted coclastogenically in the experiments with BM. Under the influence of rat-liver S9 mix this action on TR-induced chromosome damage was decreased and only a slight anticlastogenic effect was observed in the experiments with activated cyclophosphamide. S9-activated BM lost some of its strong chromosome-damaging effect and AET proved clearly anticlastogenic under these test conditions. AET displayed a slight decreasing effect on SCE induced by TR, but had no effect on CP-induced SCE. No anti-SCE effect at all was found in the experiments with NaF. Detailed analyses revealed different actions of both anticlastogens on the different types of structural chromosome damage.     

Comparative studies on the specificity of anticlastogenic action in human lymphocytes in culture

Summary Comparative studies on human lymphocyte cultures yielded a certain specificity of the anticlastogenic action of the SH compounds 1-cysteine, cysteamine, and -aminoethylisothiouronium (AET) as well as of the amide 1-asparagine and the amino acid 1-methionine. This specific anticlastogenic activity manifested itself in specific changes of the spectrum of aberration types induced by the clastogens and of the pattern of intercellular distribution of the induced aberrations. It was clearly dependent on the concentration of the anticlastogens but was also influenced by the used clastogen. The use of different culture media yielded some quantitative influences on the anticlastogenic activity, but fundamental changes in the spectrum of anticlastogenic action have not been observed except with cysteamine. The patterns of activity ascertained for the different anticlastogens specifically differed from those changes in the spectrum and pattern of distribution of aberrations induced by a mere reduction of the concentration for instance of Trenimon. Therefore a direct reaction between the protectors and the clastogen Trenimon as the cause of the observed anticlastogenic action was again excluded. The presented data are also discussed under the aspects of the hypotheses of aberration induction as well as of their importance for further antimutagen research.Some parts of this paper have been supported by grants of Deutsche Forschungsgemeinschaft.     

The activity of bleomycin and modifiers of its clastogenic effect on the interphase chromatin of indian muntjak fibroblasts

Premature chromosome condensation was induced in Indian muntjak fibroblasts after exposure of the cells to bleomycin. Further experiments were devoted to the interaction of anticlastogens and a repair inhibitor, streptovitacin A. Chromosomal aberrations due to bleomycin treatment were S -phase-independently visible in the GI and G2 phase of the cell cycle. For premature chromosome condensation experiments, a 100 fold lower concentration of the mutagen produced a similar extent of chromosome damage as in metaphase studies. Additional exposure to the anticlastogens -aminoethylisothiouronium or N-acetylcysteine revealed differences between corresponding interphase and metaphase effects and between different exposure conditions. Streptovitacin A, known as an inhibitor of protein synthesis, acted like an anticlastogen in the G2 phase of the cell cycle. Our studies show that the premature chromosome condensation technique offers various qualitative insights into primary processes of mutagenicity and antimutagenicity, but requires further improvement and careful choice of the cell system for study.Abbreviationd AET -aminoethylisothiouronium - BM bleomycin - CHO Chinese hamster ovary - DMSO dimethylsulfoxide - FBS fetal bovine serum - NAC N-acetylcysteine - PCC premature chromosome condensation prematurely condensed chromosomes - PEG polyethylene glycol - SA streptovitacin A     

The action of anticlastogens in human lymphocyte cultures and their modification by rat-liver S9 mix. II. Studies with vitamins C and E

The action of vitamins C (VC) and E (VE) on the clastogenic activity of trenimon (TR), cyclophosphamide (CP) and bleomycin (BM) was tested on cultures of human peripheral blood lymphocytes with and without addition of rat-liver S9 mix. In addition, the influence of both anticlastogens on the SCE-inducing activity of TR and CP was examined under the same conditions. A distinct dose-dependent anticlastogenic effect of VC was detected in the action of long-term treatment (24 h) with TR, if the vitamin was added to the cultures simultaneously with or before the clastogen. In the short-term tests (2 or 3 h clastogen treatment ending 23 h or 21 h before harvesting) simultaneous addition of both vitamins did reduce the chromosome-damaging action of TR whether S9 mix was present or absent. While VC also decreased the frequency of chromosome damage induced by S9-mix-activated CP, VE was inactive under the same conditions. Neither vitamin significantly affected the chromosome-breaking activity of BM if S9 mix was absent, but they increased the clastogenicity of BM metabolized by S9 mix. In contrast to their anticlastogenic efficacy neither of the vitamins displayed any significant anti-SCE effect, nor were they active in affecting the inhibition of cell proliferation caused by TR or CP.     

2,3,5-Tris-ethylenimino-1,4-benzoquinone (Trenimon): some chemical and biological properties

The three aziridine rings of 2,3,5-tris-ethylenimino-1,4-benzoquinone (Trenimon) were found to be active alkylating centres in the presence of acid catalyst, but differed in reactivity among themselves. At a given pH Trenimon was readily reduced by a number of agents including cysteine, which probably formed a substitution derivative. The cysteine derivative possessed three alkylating groups which were more reactive than those of pure Trenimon. Trenimon was soluble in fat solvents but the reduction and substitution derivatives were not. Trenimon was bound to hemoglobin and to albumins, probably by a substitution reaction between a sulfhydryl group of the protein and the 6-carbon atom of the drug. Binding to globulin occurred only following reduction of the protein. Trenimon was readily taken up by L5178Y lymphoma cells in suspension culture and was highly toxic but the cysteine derivative was not readily taken up and was less toxic than Trenimon by three orders of magnitude. The implications of the solubility and other physical properties of the alkylating agents are discussed, and evidence is presented that the biologically effective concentration of a drug is that which binds to the cell surface.     

The clastogenic effect of 5-methoxypsoralen plus UV-A in human lymphocytes in vitro and its modification by the anticlastogen β-aminoethylisothiouronium

Summary The treatment of human lymphocytes in vitro with 5-methoxypsoralen (5-MOP) plus UV-A induces a dose-dependent increase in the SCE rate and in structural chromosome aberrations. We carried out tests to see whether the clastogenic effect of 5-MOP plus UV-A could be reduced by the anticlastogen -aminoethylisothiouronium (AET). The occurrence of a protective effect proved to be dependent upon the conditions of treatment. When AET was present over a long period of time (22h) in cultures with 5-MOP, the number of breaks was reduced compared with such cultures without AET (reduction factor 0.5–0.6). On the other hand, a short period of action by AET (1.5 h) in the presence of 5-MOP produced no reduction of breaks. Posttreatment with AET (20 h) yielded an obvious protective effect (reduction factor 0.2–0.4). The possible mechanisms of the protective effect of AET are discussed.     

Protective effect of cysteine against X-ray- and bleomycin-induced chromosomal aberrations and cell cycle delay

The protective effect of cysteine was studied in muntjac and human lymphocytes in vitro scoring chromosomal aberrations in harlequin stained first cycle metaphases, induced by X-irradiation at G₀. Its protective efficiency was also studied against the radiomimetic clastogen, bleomycin, in muntjac cells. 30 μg and 1 mg/ml of cysteine were given prior to 2, 3, and 4 Gy, and 2 mg/ml prior to only 4 Gy. 30 μg cysteine protected only against deletions in 4 Gy-treated cells while 1 mg protected against deletions by all three doses of X-rays. However, rearrangements were not reduced significantly in any of these, probably due to their low frequency. But when cysteine was increased to 2 mg, both types of aberrations were reduced significantly. This shows that a sufficient number of aberrations and an optimum concentration of the protector are essential for eliciting the best protective effect. This conclusion is further supported by the results of 2 mg cysteine treatment in human lymphocytes which yielded higher frequencies of rearrangements with 2 and 3 Gy X-rays than 4 Gy in muntjac, but had a relatively lower frequency of deletions. Thus the most abundant categories of aberration, i.e., deletions in muntjac and exchanges in humans, were reduced significantly by 2 mg cysteine, associated with a prominent reduction in the frequency of aberrant metaphases. Therefore, the differential protection observed with a low concentration of the protector and an insufficient yield of aberrations induced only indicates protection provided to the most frequent type of aberration by a protector when present in lower concentration.

Cysteine pretreatment yielded weak protection against the effects of bleomycin, but posttreatment caused a mild potentiation of the clastogenic effect of BLM without altering the cell cycle kinetics. In this context, an action of cysteine as a reducing agent on BLM is suggested. Although cysteine alone caused severe retardation of the cell cycle, when given prior to X-irradiation, not only its delaying effect was not observed, but also it reduced the X-ray induced cell cycle delay. This might be due to the oxidation of cysteine by its radical scavenging action.     

Evaluation of the co-mutagenicity of ethanol and delta 9-tetrahydrocannabinol with Trenimon.

The mutagenic potential of chronic treatments of male CF-1 mice with ethanol and delta 9-tetrahydrocannibinol (THC), and their comutagenic potential with a known mutagenic agent, Trenimon, were examined. This was accomplished by measuring the frequency of dominant lethal mutations arising from mating of treated males with nontreated females. Adult male mice were treated with 5% (v/v) ethanol as part of a liquid diet (28% ethanol-derived calories) for five weeks; 10 mg/kg body weight (p.o.) THC every two days for five weeks; a single injection of Trenimon (0.125 mg/kg, i.p.) on day 28 of diet treatment; and all combinations of treatments. The control group was pair-fed a liquid diet in which isocaloric sucrose replaced ethanol; these males were also given sesame oil (vehicle for THC) and saline (vehicle for Trenimon) on the same schedule as that for the treated males. Neither body weights nor hematocrits were adversely affected by any treatment. Both ethanol and Trenimon treatments resulted in a small (8-9%; p less than 0.05) decrease in testicular weight. The effect of combined treatment with ethanol and Trenimon was roughly additive. Treatment with THC had no effect on testicular weight. Seminal vesicle weights were not affected by any treatment. Treatments were without significant effect on fertility, as measured by the frequency of males producing pregnancies. Ethanol and Trenimon treatments produced approximately 3- and 7-fold increases, respectively in the frequencies of preimplantational loss over that seen for the control group (7.3%), resulting in significant ethanol and Trenimon effects (p less than 0.001). No interactive effects of ethanol and Trenimon treatments were noted. Frequencies of dead fetuses per pregnancy in the ethanol- and Trenimon-treated groups were increased approximately 2.5- and 4-fold, respectively, over the control value of approximately 16%. However, the effect of combined treatments was not greater than that due to Trenimon alone, resulting in Trenimon and ethanol effects (p less than 0.001) and ethanol-Trenimon interaction (p less than 0.001). The calculated mutation index resulting from each treatment yielded significant (p less than 0.001) ethanol- and Trenimon-induced effects. In contrast to effects of ethanol and Trenimon treatments, THC, given alone, or in combination with ethanol and/or Trenimon, had no effect on either preimplantational loss, fetal mortality or the resulting mutation index. The data suggest that chronic ethanol treatment, at levels resulting in minimal fertility impairment, increases the frequency of dominant lethal mutations. In contrast, chronic treatment with THC, as administered in the present study, appears to be without effect.(ABSTRACT TRUNCATED AT 400 WORDS)     

Comparative studies on the influence of radioprotectors and amino acids on the chromosome-damaging activity of 8-hydroxyquinoline sulfate in human lymphocytes in vitro

The influence of the radioprotectors cystaamine and aminoethylisothiouronium (AET) as well as the amino acids l-cysteine, l-alanine, l-arginine, l-asparagine, l-glutamic acid, l-histidine and l-methionine on the cytogenetic action of 8-hydroxyquinoline sulphate (8-HCHS) was tested in human lymphocyte cultures in vitro. An excess of l-cysteine, cysteamine, and l-asparagine, when added to the cultures simulataneously with 8-HCHS, distinctly reduced the chromosome-damaging effect of this agent. l-Glutamic acid and AET exerted a protective activity to a lesser extent. l-Methionine only displayed some effect in reducing the relatively rare isochromatid aberrations induced by 8-HCHS. The other amino acids had no effect on the chromosome-damaging action of this substance.The dose dependence of the protective activity as well as the degree of effectivity and the spectrum of action of the different protectors are compared. The possible mechanisms of action are discussed.     

Inhibition of a mouse hepatoma by the alkylating agent trenimon linked to immunoglobulins

Summary Trenimon was conjugated in active alkylating form to rabbit anti-mouse H6 hepatoma globulin (AHG) with retention of antibody activity. H6 hepatoma-inoculated mice were given various combinations of conjugates, free Trenimon, and unconjugated immunoglobulins in daily injections for 5 days. Linkage of Trenimon to immunoglobulins reduced systemic toxicity of the drug, with comparative retention of its antitumor activity. The antitumor action of Trenimon was potentiated by AHG irrespective of whether the drug was directly linked to AHG or free AHG was administered along with Trenimon linked to normal rabbit globulin (NRG).In vitro, Trenimon bound to AHG was less inhibitory to hepatoma cells than free Trenimon, but more inhibitory than Trenimon-NRG conjugates. There was no significant endocytosis of conjugates by the hepatoma cells. This suggests that unlike free Trenimon, the target molecules of Trenimon-immunoglobulin conjugates are not intracellular DNA but are located on the surface of the hepatoma cells.     

Induction of micronucleated erythrocytes by MEA,AET, WR-2721 and X-rays

The induction of micronucleated polychromatic erythrocytes (MNPCEs) was assessed in the bone marrow of adult male Swiss mice treated with MEA (cysteamine HCl), AET (2-aminoethylisothiouronium Br.HBr), or WR-2721 (S-2-(3-aminopropylamino)ethyl phosphorothioic acid), at a dose of 200 mg/kg body weight, and/or exposed to 6 Gy X-rays. MEA, AET, or WR-2721 was given alone or 15 min prior to X-ray exposure, and the frequency of MNPCEs was determined 24 h after the aminothiol treatment and X-irradiation of mice. A genotoxic effect was shown for MEA, AET, WR-2721, and X-rays, as well as a protective effect of the aminothiols against X-ray-induced genotoxicity in the mouse erythropoietic system. The aminothiol drugs given alone, without subsequent X-irradiation, elevated the frequency of MNPCEs, and WR-2721 appeared to be less toxic than AET and MEA. After exposure of mice to X-rays, the number of MNPCEs was distinctly increased. MEA, AET, or WR-2721 administration prior to X-irradiation resulted in a reduction of the X-ray-induced elevation of the frequency of micronuclei, but a stronger radioprotective effect was obtained following WR-2721 and AET treatment than after MEA application. So, the genotoxic and radioprotective effect of the aminothiols was dependent on the compound applied.     

The effect of sodium fluoride on DNA synthesis, mitotic indices and chromosomal aberrations in human leukocytes treated with trenimon in vitro

Human leukocyte cultures were set up with Ham's F-10 medium and stimulated with PHA-M. Treatment of the cells in G₁ from 15–20 h with 0.5 × 10⁻⁶ M Trenimon resulted in a considerable cell cycle delay, as measured by [³H]-TdR autoradiography and determination of mitotic indices. Under these conditions only few cells incorporated the tracer at the same time as most cells did in untreated cultures. However, this did not lead to a mitotic activity at the same time as obtained in controls. Most of the treated cells started their DNA synthesis and mitotic activities with a delay of around 20 h, as compared with the controls. Continuous treatment of the cells with 10⁻³ M NaF had no effect on [³H]TdR labelling or mitotic indices in otherwise untreated cultures, but led to an impressive effect on DNA synthesis in Trenimon-treated cultures, without a considerable effect on the mitotic indices. This finding could beexplained as due to a lower alkylation in cellular DNA in the presence of NaF. More cells can start with their DNA synthesis, although they are, like Trenimontreated cultures, incapable of completing it normally. Analyses of the effect of NaF on chromosomes aberrations induced by Trenimon revealed that pre-, simultaneous and post-treatments significantly enhanced the frequency of undamaged mitoses. Continuos fluoride treatment also protected the cells from Trenimon-induced damage, but the effect was not significant, possibly because of heavily damaged mitoses which appeared under these conditions. We interpret our findings as an indication of a real anti-mutagenic activity of NaF.     

Chromosomal breakage,endomitosis, endoreduplication,and hypersensitivity toward radiomimetric and alkylating agents: A possible new autosomal recessive mutation in a girl with craniosynostosis and microcephaly

A high frequency of spontaneous chromosomal breakage, endomitosis, endoreduplication and hypersensitivity toward both the alkylating agent Trenimon and the radiomimetric drug bleomycin was observed in phytohemagglutinin-stimulated peripheral lymphocytes from a girl with craniosynostosis, microcephaly, ptosis, bird-like facies, and moderate mental retardation. We also observed abnormal chromosomal spiralization and some aspects of abnormal cellular division. Several fruitless attempts were made to establish a cell line. The parents were consanguineous, supporting the existence of a new, rare, autosomal, recessive condition in man. The mutation might involve a gene involved in DNA repair and/or regulation of the mitotic cycle.     

Chemical radioprotection of hemopoietic colony-forming cells: comparative effect of AET, anoxia, and urethan

The bone marrow colony-forming unit (CFU) technique of Till and McCulloch was employed to test the radioprotective effect of AET, anoxia, urethan on marrow cells irradiated in vivo. For AET and anoxia, a dose-reduction factor of 1.9 to 2.1 was found. Since the marrow cells were assayed for CFU content immediately after irradiation of the donor, the observed effect can be interpreted as a "true" radiation dose reduction. By contrast, urethan injection did not increase the survival of marrow CFU assayed immediately after whole-body x-irradiation. However, both urethan and AET afforded radioprotection of endogenous CFU content of spleen and bone marrow, but not of endogenous spleen colony count. It is concluded that the mechanism of radioprotection by urethan is fundamentally different from that of AET or anoxia.     

The rate of sister chromatid exchanges parallel to spontaneous chromosome breakage in Fanconi's anemia and to trenimon-induced aberrations in human lymphocytes and fibroblasts.

The incidence of structural chromosome aberrations and the rate of sister chromatid exchanges (SCE) was investigated in lymphocyte cultures from a patient with typical Fanconi's anemia and his parents. The rate of SCEs was found to be normal. In experiments with the alkylating agent Trenimon the SCE rates proved to be a sensitive indicator for the induction of structural aberrations: in presence of an induced aberration rate half as high as the spontaneous rate in the Fanconi's anemia case, the rate of SCEs was found to be quintupled. Dose-effect relationships for the induction of SCE rates by Trenimon were studied over a wide dose range in lymphocyte and fibroblast cultures. The results reflect the same difference in sensitivity earlier observed in the induction of structural chromosome aberrations, fibroblasts being far more sensitive.     

Energy, range dynamics and global species richness patterns: reconciling mid-domain effects and environmental determinants of avian diversity

Spatial patterns of species richness follow climatic and environmental variation, but could reflect random dynamics of species ranges (the mid-domain effect, MDE). Using data on the global distribution of birds, we compared predictions based on energy availability (actual evapotranspiration, AET, the best single correlate of avian richness) with those of range dynamics models. MDE operating within the global terrestrial area provides a poor prediction of richness variation, but if it operates separately within traditional biogeographic realms, it explains more global variation in richness than AET. The best predictions, however, are given by a model of global range dynamics modulated by AET, such that the probability of a range spreading into an area is proportional to its AET. This model also accurately predicts the latitudinal variation in species richness and variation of species richness both within and between realms, thus representing a compelling mechanism for the major trends in global biodiversity.     

Role of the mismatch repair system and p53 in the clastogenicity and cytotoxicity induced by bleomycin

The mismatch repair (MMR) system and p53 protein play a pivotal role in maintaining genomic stability and modulate cell chemosensitivity. Aim of this study was to examine the effects of either MMR-deficiency or p53 inactivation, or both, on cellular responses to bleomycin. The MMR-deficient colon carcinoma cell line HCT116 and its MMR-proficient subline HCT116/3-6, both expressing wild-type p53, were transfected with an expression vector encoding a dominant-negative p53 mutant, or with the empty vector. Four transfected clones, having the following phenotypes, MMR-proficient/p53 wild-type, MMR-proficient/p53 mutant, MMR-deficient/p53 wild-type, MMR-deficient/p53 mutant, were subjected to treatment with bleomycin. Loss of MMR function alone was associated with increased resistance to apoptosis, chromosomal damage and inhibition of colony formation caused by bleomycin. Loss of p53 alone resulted in abrogation of G1 arrest and increased sensitivity to apoptosis and chromosomal damage induced by the drug, but did not affect clonogenic survival after bleomycin treatment. Disabling both p53 and MMR function led to abrogation of G1 arrest and to a moderate impairment of drug-induced apoptosis. Chromosomal damage was reduced in the MMR-deficient/p53 mutant clone with respect to the MMR-proficient/p53 wild-type one, when evaluated 48 h after bleomycin treatment, but was comparable in both clones 96 h after drug exposure. Clonogenic survival of the MMR-deficient/p53 mutant clone was similar to that of the MMR-deficient/p53 wild-type one. The effects of MMR-deficiency on cellular responses to bleomycin were confirmed using the MMR-proficient lymphoblastoid cell line TK6 and its MMR-deficient subline MT1, both expressing wild-type p53. In conclusion, our data show that loss of MMR and p53 function exerts opposite and independent effects on apoptosis and chromosomal damage induced by bleomycin. Moreover, inactivation of MMR confers resistance to the cytotoxic activity of the anticancer agent in cells expressing either wild-type or mutant p53.     

Die Schutzwirkung von Cysteamin und β-Aminoäthylisothiouronium (AET) gegen die chromosomenschädigende Aktivität von Trenimon® in menschlichen Lymphocyten in vitro

Zusammenfassung Die als Cytostatikum verwendete alkylierende Substanz Trenimon^® (2.3.5-Tris-äthylenimino-benzochinon-1,4) verursacht an menschlichen Chromosomen in hohem Maße sekundäre Strukturaberrationen. Durch Zugabe von Cysteamin und AET in Kulturen menschlicher Lymphocyten in vitro war es möglich, die aberrationsauslösende Wirkung des Trenimon^® zu reduzieren.Der Schutzeffekt beider Substanzen erstreckte sich auf verschiedene Aberrationsparameter wie Gaps, Brüche und Chromosomenumbaufiguren (Exchanges). Eine Abhängigkeit des Schutzeffektes von der Protektorkonzentration konnte für beide Substanzen nachgewiesen werden. Die cytostatische Wirkung des Trenimon^® wurde sowohl durch AET als auch Cysteamin nicht beeinträchtigt.

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The protective effect of cysteamine and -aminoethylisothiouronium (AET) on the chromosome-damaging activity of Trenimon^® Dose-effect-ratios

Summary The cytostatic alkylating substance Trenimon^® (2,3,5-tristhyleneimine-1,4-benzoquinone) is extremely detrimental to human chromosomes to a high degree. Addition of cysteamine or aminoethylisothiouronium (AET) made reduction of this aberration-inducing activity of Trenimon possible. The protective effect of both cysteamine and AET was demonstrated in different types of aberrations (gaps, breaks, and interchanges). It was dependent on the concentration of the protective agent in each case. The cytostatic activity of Trenimon^® was affected by neither AET nor cysteamine.

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Herrn Professor Dr. G. Koch zum 60. Geburtstag gewidmet.

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Die Arbeit enthält die Ergebnisse einer Dissertation von R. Becher, die 1972 der Medizinischen Fakultät der Universität Erlangen vorgelegt wurde.     

Generation of ROS by CAY10598 leads to inactivation of STAT3 signaling and induction of apoptosis in human colon cancer HCT116 cells

Abstract

Prostaglandin E₂ (PGE₂) has been reported to play critical roles in cell fate decision by interacting with four types of prostanoid receptors such as EP1, EP2, EP3 and EP4. The present study was aimed at investigating the effect of the EP4-specific agonist CAY10598 in human colon cancer HCT116 cells. Our study revealed that treatment with CAY10598 significantly reduced the cell viability and induced apoptosis in HCT116 cells, as evidenced by the induction of p53 and Bax, release of cytochrome c, cleavage of caspase-9, -7, and -3, and PARP, and the inhibition of Bcl-2, Bcl-xL and survivin expression. Moreover, treatment with CAY10598 diminished the phosphorylation of JAK2, leading to the attenuation of STAT3 activation in HCT116 cells. CAY10598-induced apoptosis in cells which were transiently transfected with EP4 siRNA or treated with an EP4 antagonist prior to incubation with the compound remained unaffected, suggesting an EP4-independent mechanism of apoptosis induction by CAY10598. We found that treatment with CAY10598 generated reactive oxygen species (ROS) and pretreatment of cells with N-acetyl cysteine rescued cells from apoptosis by abrogating the inhibitory effect of CAY10598 on the activation of JAK2/STAT3 signaling. In conclusion, CAY10598 induced apoptosis in HCT116 cells in an EP4-independent manner, but through the generation of ROS and inactivation of JAK2/STAT3 signaling.     

Micronuclei frequency induced by bleomycin in human peripheral lymphocytes: correlating BLHX polymorphism with mutagen sensitivity

Mutagen sensitivity assay, by measuring chromosome damage induced by an in vitro treatment of peripheral lymphocytes with bleomycin, has been proposed as a biomarker for assessing cancer susceptibility. Recently, a single nucleotide polymorphism (SNP A1450G) of the gene for bleomycin hydrolase (BLHX), a specific neutral cysteine protease able to metabolise bleomycin, was proposed as a plausible candidate to variation in mutagen sensitivity. To shed more light on the effect of BLHX genotype on the expression of chromosome damage induced in vitro by bleomycin, we determined mutagen sensitivity for 45 non-smoker healthy volunteers. The level of bleomycin-induced chromosome damage was assessed as frequencies of micronuclei (MN) in cytokinesis-blocked lymphocytes. The subjects were genotyped for the BLHX gene, to determine the possible effect of this polymorphism on mutagen sensitivity. No difference in the spontaneous value of MN was detected between the homozygotes wild-type (A/A) and the carriers of variant alleles A/G heterozygotes or G/G homozygotes (MN/1000 binucleated (BN) cells: 6.69+/-2.53 and 6.37+/-4.87, respectively). A substantial effect of BLHX polymorphism in predetermining individual mutagen sensitivity status was observed: subjects with the BLHX A/A genotype displayed significantly lower mean levels of bleomycin-induced MN frequency than the carriers of A/G or G/G variant alleles combined (12.00+/-3.76 MN/1000 BN vs. 16.37+/-8.86 MN/1000 BN, respectively; P=0.029). The multiple regression analysis, including BLHX genotype and age, confirmed the significant effect of BLHX variant alleles (A/G, G/G) on the chromosome damage induced by bleomycin (P=0.01), whereas age correlated only with the spontaneous MN frequency.