Increase in clonal variation in Chinese hamster ovary cells after treatment with mutagens

Clonal variation has been studied in CHO cells. The variant phenotype was an altered morphology of clones in agar: the parental CHO cells give rise to solid clumps of cells (wild-type colonies); occasionally, dispersed colonies arise, and the cells display an invasive growth in agar (INGA-type colonies). The frequency of this altered phenotype can be enhanced by treatment with a variety of mutagens (EMS, ENU, 4NQO, N-Ac-AAF, ultraviolet light, and X-irradiation). Enhancement was not due to a selective killing of wild-type cells or to a side-effect of cytotoxicity, which suggests that DNA damage is the cause of the altered phenotype. The INGA-trait breeds true, but most of the isolated clones have an inherent instability.     

Different effects of mutagens on sister chromatid exchange induction in three Chinese hamster cell lines

The sister chromatid exchange (SCE) induction of mutagens with different mechanisms of action was comparatively investigated on permanent cell lines of the Chinese hamster (CHO, V-79, and DON) with and without exogenous metabolic activation and with the use of various experimental protocols. CHO and V-79 cells were treated with ethylmethanesulfonate (EMS), a direct mutagen; with the two indirect mutagens cyclophosphamide (CP) and benzo[a]pyrene (BP); as well as with the radical former hydrogen peroxide (H2O2) and hydroxyurea (HU), an inhibitor of DNA synthesis. Aside from an increased basal SCE level and a higher bromodeoxyuridine (BrdUrd) sensitivity, there was no decisive difference between CHO and V-79 cells. However, there was a distinct relationship between SCE induction and the experimental protocol used, which was most pronounced after HU treatment. Neither cell line was able to metabolize the indirect mutagen BP. Only in CHO cells did CP lead to increased SCE frequencies. However, in all cases, the simultaneous application of S9 mix produced a distinct SCE induction. In contrast, BP caused SCE induction in DON cells, whereas CP was not metabolized. The reason for these findings must obviously be sought in the metabolization of CP and BP via different monooxygenase systems, whose activity can differ in these permanent cell lines. One notable finding was that the number of SCE induced by H2O2 could be distinctly reduced by the simultaneous application of S9 mix. This effect can be explained by the fact that S9 mix contains H2O2-degrading enzymes. The results indicate that closely related cell lines differ in their capability for inducing SCE and that investigations of SCE inductions performed on only one cell line do not necessarily produce a representative response.     

Activity of mutagens in the fungus Gibberella fujikuroi

The efficacy of a number of mutagens was examined for Gibberella fujikuroi. Nitrosoguanidine (MNNG) was the most effective mutagen inducing mainly morphological and pigment mutants. X-ray produced small mutagenic effect. After UV-irradiation pigment mutants developed for the most part and a combination of UV- and x-ray-irradiation mainly influenced the growth rate. A combination of UV-irradiation with MNNG elicited the highest number of mutants with increased GA₃ production.     

New derivatives of 2-carboxychromanone-4 as potential antiinflammatory drugs. I. Cis-and trans-beta-2,4-dihydroxybenzoylacrylic acids and their derivatives.

Proof is presented that the product of the reaction of resorcin and maleic anhydride in the presence of aluminum chloride in ethylene chloride solutions is cis-beta-2,4-dihydroxybenzoylacrylic acid (I). Cis and trans acids (I and II) were isomerized to 2-carboxy-7-hydroxychromanone-4(III). Reactions of acetylation of compounds I, II and III were studied.     

Higher molecular weight insulin precursors as autoantigens in type I diabetes

Hypothetically, the formation of abnormal insulin precursors due to genetic and/or acquired disorders would result in autoantigens by virtue of the altered tertiary structure. These are unlikely to be accessible to the converting enzymes which in turn should produce extended exposure to the immune system. Subsequent humoral and cell-mediated immune response might thus initiate or aggravate the autoimmune destruction of B-cells in subjects genetically susceptible to Type I diabetes.     

Infection of chlorophyll-less protoplasts from etiolated Chinese cabbage hypocotyls by turnip yellow mosaic virus

Protoplasts were prepared from hypocotyls of dark-grown, 8-day-old Chinese cabbage seedlings. Pectinase and cellulase were used together in a one-step procedure. These hypocotyl protoplasts did not contain detectable amounts of chlorophyll. They could be infected in vitro with turnip yellow mosaic virus (TYMV). Virus replication was demonstrated by direct immunofluorescence, enzyme-linked immunosorbent assay, virus recovery by sucrose density gradient centrifugation, and infectivity tests on chinese cabbage plants. The chlorophyll-less hypocotyl protoplasts supported TYMV replication in the dark as well as in the light, while chlorophyll-containing leaf protoplasts allowed TYMV multiplication only in the light.     

Microsomal activation of 1- and 3-nitrobenzo[a]pyrene to mutagens in Chinese hamster ovary cells

1- and 3-nitrobenzo[a]pyrene (1- and 3-nitro-BaP) are environmentalpollutants and are S9-mediated mutagens in the Chinese hamsterovary (CHO) cell/hypoxanthine-guanine phosphoribosyl transferaseassay. In this study, we examined the pathways leading to themutagenic activation of these compounds in CHO cells. The microsomalmetabolites of 1-and 3-nitro-BaP, the 1- and 3-nitro-BaP trans-7,8-dihydrodiols-7, 8-dihydrodiols (trans-7, 8-dihydrodiols) andthe 1-and 3-nitro-BaP trans-9, 10-dihydrodiols, were isolatedand tested for mutagenicity. At the concentrations assayed,both trans-9, 10-dihydrodiols were non-mutagenic with and withoutS9 activation. In contrast, the trans-7, 8-dihydrodiols of 1-and 3-nitro-BaP were direct-acting mutagens and these responseswere similar in magnitude to the S9-mediated mutagenicitiesof the parent nitro-BaPs. S9 increased the mutagenic responsesof the trans-7, 8-dihydrodiols{small tilde} 20-fold. Inhibitionof epoxide hydrolase decreased the S9-mediated mutagenicityof 1-nitro-BaP by half, but doubled the S9-mediated mutagenicityof 3-nitro-BaP. These results suggest that in CHO cells: (i)the major route of mutagenic activation of 1- and 3-nitro-BaPinvolves S9-generated derivatives of the trans-7, 8-dihydrodiols,e.g. bay-region diol epoxides; (ii) reactive nitroarene oxidesmay conribute to mutation induction by 3-nitro-BaP; and (iii)metabolic routes involving trans-9, 10-di-hydrodiol formationresult in detoxification.     

Effect of turmeric on urinary mutagens in smokers.

Curcumin, the active principle of turmeric, is known to act as an anti-oxidant, anti-mutagen and anti-carcinogen in experimental animals. In the present study, anti-mutagenic effects of turmeric were assessed in 16 chronic smokers. It was observed that turmeric, given in doses of 1.5 g/day for 30 days, significantly reduced the urinary excretion of mutagens in smokers. In contrast, in six non-smokers, who served as control, there was no change in the urinary excretion of mutagens after 30 days. Turmeric had no significant effect on serum aspartate aminotransferase and alanine aminotransferase, blood glucose, creatinine and lipid profile. These results indicate that dietary turmeric is an effective anti-mutagen and it may be useful in chemoprevention.     

Generation of antigen-specific CD4+ T cell lines from naive precursors

The conditions required for sensitizing naive T cells to nominal antigen are poorly understood. In this report we describe an in vitro system for generating antigen-specific CD4⁺ T cells from previously unprimed individuals. Freshly isolated CD4⁺ T cells were cultured with keyhole limpet hemocyanin (KLH), sperm whale myoglobin (SWM), or human immunodeficiency virus (HIV) gp 160, antigens to which most persons have not been sensitized, in the presence of either dendritic cells (DC) or macrophages (MΦ). In short-term (< 8 days) cultures, CD4⁺ T cells or their CD4⁺, CD45RA (naive) subpopulation mounted significant proliferative responses to KLH, SWM, and HIV gp160, but only if the antigens were presented by DC. In contrast, CD4⁺, CD45RO (memory) T cells responded poorly to these antigens, although they responded vigorously to tetanus toxoid, a recall antigen, presented by either DC or MΦ. KLH- and SWM-specific CD4⁺ T cell lines were established from the starting population that had been sensitized in vitro, following repeated stimulation with antigen and MΦ in medium supplemented with interleukin-2 and interleukin-4. Despite the continued presence of these cytokines during T cell expansion, the expanded lines retained their ability to respond to the priming antigen in the absence of exogenous cytokines. When the CD45RA and CD45RO subpopulations were sensitized and expanded separately, the CD45RA cells alone gave rise to antigen-specific T cell lines, while the CD45RO cells proliferated nonspecifically. These results demonstrate that human naive CD4⁺ T cells can be sensitized in vitro to nominal antigens presented by DC and that the sensitized cells can be expanded into long-term lines that retain their antigen specificity.     

Evidence for TYMV-induced RNA and DNA synthesis in the nuclear fraction from infected Chinese cabbage leaves

Radioactively labeled nucleic acids prepared from the nuclear fraction from Chinese cabbage leaf tissue infected with turnip yellow mosaic virus (TYMV) contained two components not present in equivalent material from healthy leaves. One of these components had the properties expected for a virus-specific double-stranded RNA; the other contained DNA, with properties expected for DNA-RNA hybrids. In leaf tissue supplied with ³H-thymidine this DNA reached maximal labeling much more rapidly than the rest of the DNA.     

Social skills as precursors of cannabis use in young adolescents: a TRAILS study

Social skills (cooperation, assertion, and self-control) were assessed by teachers for a longitudinal cohort of (pre)adolescents, with measurements at average ages 11.1 (baseline) and 16.3 years (follow-up). Prospective associations with participants' self-reported use of cannabis, (age of) onset of cannabis use, and frequency of use at follow-up were examined using multinomial logistic regression analyses. Teacher-reported social skills predicted different aspects of cannabis use independent of better known factors such as presence of externalizing behavior and use of other substances. The direction of associations depended on the type of social skill. Good cooperation skills during early adolescence were associated with a reduced risk of lifetime cannabis use and a reduced risk of using cannabis on a regular basis. On the other hand, assertion at age 11 increased the risk of lifetime cannabis use and of using cannabis on an experimental basis.