Induction of translocations by cyclophosphamide in different germ cell stages of male mice: cytological characterization and transmission.

Cytological and fertility tests were performed in F1 male mice derived from different germ-cell stages of male parents treated with cyclophosphamide (350 mg/kg body weight). The objectives of the present experiment were: (I) to determine the sensitivity of the male germ-cell stages to the induction of translocations by the compound, and (2) to characterize translocation configurations in F1 and F2 males, in order to obtain information about the pattern of chromosome breakage induced and its transmission to subsequent generations. Of 508 F1 males studied, 39 were partially sterile. The group of males conceived 8-21 days after treatment contained by far the highest proportion of partially sterile animals (30%). It was also the only group in which totally sterile animals (11%) were found. Of 25 semisterile males from this group, 24 gave evidence of translocations when spermatocytes were scored at diakinesis. The translocation frequencies in F1 derived from treated spermatozoa and spermatocytes were 14 and 1%, respectively. No translocations were detected cytologically in 6 semisterile males derived from treated spermatogonial stages. These results indicate that spermatid stages are especially sensitive to the mutagenic action of cyclophosphamide. In 21 of the 31 semisterile translocation males (68%), the majority of the spermatocytes contained 18 bivalents plus a ring-of-four configuration, indicating that both breakpoints were relatively centrally located; and in several of these males, the frequency of cells with rings was close to 100%. In another 9 F1 males (29%) the predominant multivalent configuration was a chain-of-four, indicating one of the breakpoints to be relatively more terminally located; and in one male (3%), the majority of cells had two unequal bivalents, indicating both breakpoints to be fairly close to the ends of the chromosomes involved. Determination of centromere positions by the use of C-banding showed that chain-of-four configurations in any one male were predominantly of a given type..     

Heritable translocation test and dominant-lethal assay in mice with methyl methanesulfonate.

A dominant-lethal test and a heritable translocation test were performed with methyl methanesulphonate (MMS) at 40 mg/kg by treating the sensitive periods of post-meiotic spermatogenesis i.e. spermatozoa and spermatids. In the dominant-lethal test 25 to 60% dominant-lethal mutations were obtained depending on the mating intervals. In the heritable translocation test 11% sterile and partially sterile F1 males were observed in 250 offspring of the MMS group. All of the 14 partially sterile and 6 of the 14 sterile F1 males were demonstrated to be translocation carriers. Fertility of the partial steriles was about 40% of normal fertility. The translocation frequencies in the primary spermatocytes of the partially sterile F1 males varied between 2 and 99%. Transmission of partial sterility and translocations was confirmed in the F2 generation. There were no partially sterile or sterile males among the 245 controls.     

Effectiveness and efficiency of ethyl methanesulphonate and ethylene oxide for the induction of mutations in rice

A comparison of the mutagenic efficiency and effectiveness of ethyl methanesulphonate (EMS) and ethylene oxide (EO) on two different genotypes of rice showed that the mutagenic efficiency sharply decreased with increase in the concentration of EO whereas the efficiency of EMS increased with an increase in the concentration. The effectiveness was inversely proportional to dose of EO whereas it varied little with an increased dose of EMS.     

Thiono compounds. 4. In vitro mutagenic and antineoplastic activity of TEPA and thio-TEPA

Tris (1-aziridinyl) phosphine oxide (TEPA) and tris (1-aziridinyl) phosphine sulfide (thio-TEPA) induced base pair mutations in the Ames mutagenic assay. Thio-TEPA required metabolic activation while TEPA was active without metabolic activation. Growth of a human vaginal carcinoma (A431), a human breast carcinoma (MDAMB-231), and a human cervical carcinoma (HeLa) were inhibited in soft agar in vitro at concentrations which induced mutagenesis in the Ames Assay. A fourth line, JEG choriocarcinoma, was sensitive to the antigrowth properties of both drugs at concentrations below that which induced mutagenesis. These data suggest that as more antineoplastic agents become available, and as mean survival times increase, knowledge of the relative in vitro sensitivity of a patient's neoplasm to a specific antineoplastic drug (i.e., dose required for growth inhibition) as a function of its mutagenic index might be useful for prediction of clinical remission, as well as the risk of secondary neoplasm induction.Abbreviations TEPA tris (1-aziridinyl) phosphine oxide - thio-TEPA tris (1-aziridinyl) phosphine sulfide - MEM Minimal Essential Media This study was supported by PHS grant No. CA 30321 awarded by the National Cancer Institute (DHHS).     

Reactions of ethyleneimine with guanosine and deoxyguanosine

Ethyleneimine was reacted with guanosine in aqueous medium and the products were purified by Sephadex G-10 and high-performance liquid chromatography (HPLC). Two products were identified by UV-spectroscopy: imidazole-ring opened 7-alkylguanosine and 1-alkylguanosine, accounting for 80% and 14% of all adduct radioactivity, respectively. When the incubation with ethyleneimine and guanosine or deoxyguanosine was carried out at pH 6.0 for 1 h intact 7-alkylation products were detected. The half-life of imidazole ring opening of 7-alkylguanosine was 11, 5 and 2.8 min at pH-values 7.0, 7.7 and 8.0, respectively, as measured fluorometrically at 25 degrees C. The respective half-life of alkylated deoxyguanosine was 21 min at pH 7.7. The half-life of depurination of alkylated deoxyguanosine was 42 min at pH 6.0 and 25 degrees C.     

Relative mutagenicity of antineoplastic drugs and other alkylating agents in V79 chinese hamster cells,independence of cytotoxic and mutagenic responses

The mutagenic and cytotoxic effects of 4 antineoplastic drugs, vinblastine, vincristine, adriamycin and nitrogen mustard and of several monofunctional alkylating agents have been assayed in V79 Chinese hamster cells. Vincristine, vinblastine and nitrogen mustard did not significantly increase the frequency of TG^RHGPRT^? mutants but were all highly cytotoxic. Adriamycin and the monofunctional alkylating agents were all significantly mutagenic even at the lowest doses tested (approx. 70 % survival level). Induced mutant frequency increased linearly with increasing dose whereas dose-response curves for cytotoxicity for these effective mutagens invariably showed a shoulder followed by an exponential decline. At equitoxic doses the relative mutagenic effectiveness was MNU ENU EMS MMS ? DMS. MNU was approx. 20 times more effective than MMS and DMS.Measurement of the total amount of alkylation and the relative amounts of reaction with individual DNA bases at approx. equitoxic doses of MNU and DMS indicated a significantly higher O⁶/N⁷ ratio after MNU (0.15) than after DMS (0.005). However, approx. equal numbers of mutants/10⁵ cells/μM O⁶-Meguanine were induced by these 2 agents. These results support previous conclusions, that mutagenic and cytotoxic responses are independent in V79 cells.     

Repair processes and the response of dividing and non-dividing cells to methyl methanesulphonate and dimethyl sulphate.

A method for producing a viable non-dividing population of Chinese hamster V79 cells in suspension is described and the characteristics of the population outlined. The stationary population is more sensitive to methylating agents than a similar but exponentially growing population, the increased sensitivity arising from the loss of the shoulder from the survival curve. The extent of reaction of the agent with cellular macromolecules is similar in both cases. The repair capabilities of the two populations was examined. Non-semiconservative DNA repair synthesis occurs whether the cells are in a growth or no-growth condition when insulted. Repair of single-strand breaks, which arise following methylation, also proceeds up to the size of the replicon. The relationship of this stationary population to other no-growth conditions and its utility as a model for carcinogenesis studies is discussed.     

Relationship between experimental results in mammals and man: II. Cytogenetic analysis of bone-marrow cells after treatment of cytembena and cyclophosphamide-cytembena combination

Cytogenetic analysis and the micronucleus test of bone-marrow cells was used to study the possible extrapolation of results from experimental animals to man.Cytembena was given i.p. in doses of 5, 10, 20, 40 and 80 mg/kg body wt. to Wistar rats in doses of 20, 40 and 80 mg/kg body wt. to ICR mice an dto Chinese hamsters. Five patients with various types of malignancy, so far medically untreated, received 20 mg Cytembena/kg body wt i.v.A combination of Cytembena and cylophosphamide was applied i.p. in single equal doses 1 : 1 of 5, 10, 20, and 40 mg/kg body wt to ICR mice, Chinese hamsters and Wistar rats. Patients were given i.v. 20 mg Cytembena and 20 mg cyclophosphamide/kg body wt.Bone-marrow cells were examined 24 h after the administration.The frequency of abnormal metaphases and chromosomal breaks after Cytembena treatment was low; nonetheless, the indicated dose-effect relationship was found in all the rodents used. The frequency of chromosomal breaks was 2–3 times higher in rodents in comparison with man, after treatment with a dose of 20 mg Cytembena/kg body wt.Highest frequencies of induced aberrations were found in mice. The rodents appeared to be 3–4 times more sensitive to the induction of chromosomal breaks and abnormal metaphases than man, after a dose of 20 mg Cytembena and 20 mg cyclophosphamide/kg body wt.     

Sister chromatid exchanges induced by cyclophosphamide in V79 cells cultured in diffusion chambers in mice

Chinese hamster cells V79 were cultured in diffusion chambers (DC) and implanted into mice. An exponential growth was observed from the 2nd to 4th day after implantation. The maximum growth was reached on the 6th day. After that, cell growth and viable cell counts decreased. Three days after implantation of DC with V79 cells, the hosts received 6 hourly injections of 0.2 ml of 5-bromodeoxyuridine (BUdR) solution at concentrations of 0.125 to 1.0 x 10(-2) M. DC were removed for chromosome and sister-chromatid exchanges (SCE) analyses 24 h after the first BUdR injection. The frequency of metaphases with differentially stained chromatids, with aberrations, and the number of SCE per cell increased with BUdR dose. The frequency of metaphases with differentially stained chromatids was also positively correlated with the duration of BUdR exposure or the number of hourly injections of BUdR-solution. The effects of cyclophosphamide (CY) in V79 cells in DC in mice were studied. Injections of CY at 2.5, 5, 10 and 15 microgram per gram of body weight to the hosts caused an increase in the number of SCE per cell in a linear manner. The results from this study indicate that V79 cells cultured in DC in mice may provide a potential test system for mutagenicity.     

An autoradiographic study of unscheduled DNA synthesis in the germ cells of male mice treated with X-rays and methyl methanesulfonate

Unscheduled DNA synthesis (UDS) in the germ cells of male mice after in vivo treatment with X-rays or methyl methanesulfonate (MMS) was assayed by use of a quantitative autoradiographic procedure. MMS induced UDS in meiotic through type III elongating spermatid stages, whereas X-rays induced UDS in meiotic through round spermatid stages. No UDS was detected in the most mature spermatid stages present in the testis with either MMS or X-rays. Taking into account differences in DNA content of the various germ-cell stages studied, we concluded that X-rays induced a maximum UDS response in spermatocytes at diakinesis--metaphase I. The level of UDS induced by MMS was about the same in all the stages capable of repair. Chromosome damage and UDS were measured simultaneously in the same spermatocytes at diakinesis 90 min after X-irradiation or MMS treatment. The level of UDS in most of the X-irradiated cells paralleled the extent of chromosome damage induced. A statistical analysis of these results revealed a positive correlation. As expected, MMS induced no chromosome aberrations above control levels. Therefore no correlation was determined between UDS and chromosome damage in this case. The distribution of UDS over the chromosomes treated at diakinesis with MMS or X-rays was studied. It was found that UDS occurred in clusters in the irradiated cells, whereas it was uniformly distributed in the MMS-treated cells.     

Effects of chemical and physical mutagens on the frequency of a large genetic duplication in Salmonella typhimurium. II. Stimulation of duplication-loss from merodiploids.

Strains of Salmonella typhimurium which contain a duplication of approximately 30% of the genome may be obtained by a simple selective procedure. These strains are highly unstable, losing the duplication when grown on non-selective medium. In this paper we report that treatment of merodiploid bacteria with mutagenic agents stimulates the rate at which haploid segregants are obtained from merodiploid strains. The mutagens which have been tested for this effect are X-rays, ultraviolet light (UV), ethyl methanesulfonate (EMS), and the azaacridine half-mustard ICR-372.     

The role of DNA polymerases in DNA repair in Escherichia coli: DNA polymerase I-dependent repair following chemical alkylation of permeabilized bacteria.

Many mutagens and carcinogens damage DNA and elicit repair synthesis in cells. In the present study we report that alkylation of the DNA of Escherichia coli that have been made permeable to nucleotides by toluene treatment results in the expression of a DNA polymerase I-directed repair synthesis. The advantage of the system described here is that it permits measurement of only DNA polymerase I-directed repair synthesis and serves as a simple, rapid method for determining the ability of a given chemical to elicit “excision-repair” in bacteria.DNA ligation is intentionally prevented in our system by addition of the inhibitor nicotinamide mononucleotide. In the absence of DNA ligase activity, nick translation is extensive and an “exaggerated” repair synthesis occurs. This amplification of repair synthesis is unique for DNA polymerase I since it is not observed in mutant cells deficient in this polymerase. DNA ligase apparently controls the extent of nucleotide replacement by this repair enzyme through its ability to rejoin “nicks” thereby terminating the DNA elongation process.The nitrosoamides N-methyl-N-nitrosourea and N-ethyl-N-nitrosourea, as well as the nitrosoamidines N-methyl-N′-nitro-N-nitrosoguanidine and N-ethyl-N′-nitro-N-nitrosoguanidine, elicit DNA polymerase I-directed repair synthesis. Methyl methanesulphonate is especially potent in this regard, while its ethyl derivative, ethyl methanesulphonate, is a poor inducer of DNA polymerase I activity in permeabilized cells.     

Cloning and sequence analysis of genes involved in erythromycin biosynthesis in Saccharopolyspora erythraea: sequence similarities between EryG and a family of S-adenosylmethionine-dependent methyltransferases.

Summary Treatment of tomato seeds with ethyl methanesulphonate (EMS) followed by allyl alcohol selection of M₂ seeds has led to the identification of one plant (B15-1) heterozygous for an alcohol dehydrogenase (Adh) null mutation. Genetic analysis and expression studies indicated that the mutation corresponded to the structural gene of the Adh-1 locus on chromosome 4. Homozygous Adh-1 null mutants lacked ADH-1 activity in both pollen and seeds. Using an antiserum directed against ADH from Arabidopsis thaliana, which crossreacts with ADH-1 and ADH-2 proteins from tomato, no ADH-1 protein was detected in seeds of the null mutant. Northern blot analysis showed that Adh-1 mRNA was synthesized at wild-type levels in immature seeds of the null mutant, but dropped to 25% in mature seeds. Expression of the Adh-2 gene on chromosome 6 was unaffected. The potential use of the Adh-1 null mutant in selecting rare transposon insertion mutations in a cross with mutable Adh-1 ⁺ tomato lines is discussed.     

Normal acute and chronic inflammatory responses in sphingosine kinase 1 knockout mice

Sphingosine-1-phosophate, generated from the phosphorylation of sphingosine by sphingosine kinase enzymes, is suggested to function as an intracellular second messenger for inflammatory mediators, including formyl peptide, C5a, and Fc. More recently, a role for sphingosine kinases during inflammation has also been proposed. Here we show that sphingosine kinase 1 knockout mice exhibit normal inflammatory cell recruitment during thioglycollate-induced peritonitis and that sphingosine kinase 1-null neutrophils respond normally to formyl peptide. In the collagen-induced arthritis model of rheumatoid arthritis, sphingosine kinase 1 knockout mice developed arthritis with normal incidence and severity. Our findings show that sphingosine kinase 1 is dispensable for inflammatory responses and support the need for more extensive studies of sphingosine kinases in inflammation.     

Immunomodulatory effects of thymopentin under acute and chronic inflammations in mice

The effects of thymopentin, a synthetic analog of the active center of the thymus hormone thymopoietin, on the immune status of mice with two different models of inflammation induced by injection of lipopolysaccharide (LPS) from Gram-negative bacteria were studied. Acute inflammation was induced by a single injection of LPS in a dose of 250 μg/100 g of body weight, and chronic inflammation (sepsis) was modeled by daily injection of LPS for 11 days with a gradual increase in the dose range from 25 to 250 μg/100 g of body weight. Under acute inflammation, a preliminary injection of thymopentin did not induce any additional stimulation of cytokine production increased by LPS. On the contrary, whereas the chronic introduction of LPS was characterized by a depressed production of several cytokines, thymopentin produced an immunostimulating effect. Thus an increase in the production of nitric oxide, interferon-μ, and Hsp70 was demonstrated. In addition, a more effective restoration of the number of thymus cells, as well as an increase in macrophage tumor necrosis factor-α production were observed after cessation of LPS + hormone injections. The results show that preliminary application of thymopentin promotes the regulation of immune cell activity under acute and chronic inflammation.     

腺苷和一氧化氮在中枢神经系统相互作用及与癫痫相关性

腺苷和一氧化氮(Nitric oxide,NO)都是十分活跃的具有多种生物活性的内源性物质。近年来,关于腺苷和NO在周围组织和中枢神经系统中的相互作用被广泛关注。腺苷在中枢神经系统中广泛存在,可作为整合中枢兴奋和抑制性神经递质的调节因子;NO在中枢神经系统中具有广泛的生物学意义,既兼有第二信使和神经递质的性能,又是效应分子,参与多种生理功能,代谢衍生物有一定的中枢神经毒性。在中枢神经系统中,腺苷和NO之间可能有一定联系,本文综述了二者在中枢神经系统中的相互作用及其与癫痫的相关性,以期为中枢神经系统相关疾病的发病机制研究及防治方法提供新的思路。     

Unscheduled DNA synthesis in spermatogenic cells of mice treated in vivo with the indirect alkylating agents cyclophosphamide and mitomen

Cyclophosphamide (CPA) and mitomen (DMO) are chemical mutagens that require metabolic activation to produce their biological effect. We have used an in vivo UDS assay in various meiotic and postmeiotic germ-cell stages of male mice to study DNA repair after treatment with these chemicals. EMS, a compound requiring no metabolic activation, was also used for comparative purposes.CPA and DMO induced UDS in meiotic through early-to-midspermatid stages, but no UDS was detected in late spermatids and mature sperm. While EMS produced a maximum UDS response in the germ cells immediately after treatment, CPA and DMO did not produce a maximum response until ～0.5 to 1 h after injection. This delay is attributed to the time required for CPA and DMO to be enzymatically vonverted active alkylating metabolites.Unlike the results found with EMS, mutation frequencies (dominant lethals, translocations, specific-locus mutations) following CPA treatment are not noticeably reduced in germ-cell stages in which UDS occurred. In the case of DMO, mutations are induced only in mature spermatozoa, and these germ-cell stages represent only a fraction of those in which no UDS is detected. The results with CPA and DMO thus still leave unclear the relationship between DNA repair and the differential spermatogenic response of mice to genetic damage.