Comparative studies of synthesis, phosphorylation, DNA binding and proteolytic characteristics of a novel protein during phases of the mouse spleen cell cycle.

1. Cultured mouse spleen cells were exposed to the mitogen Concanavalin A followed by isoproterenol, and nuclei were electronically sorted from seven partitions of the cell cycle. 2. Several nuclear proteins, including stress proteins, which were cell-cycle-stage specific, were elicited by isoproterenol as determined by micro-electrophoresis and fluorography. 3. Two novel S-phase proteins (X0 and X') demonstrated differing synthesis and phosphorylation patterns during the cell-cycle phases. 4. X' showed DNA binding characteristics and proteolytic properties (hydrolyzing X0 or beta-galactosidase); both proteins were cell-cycle regulated.

Nitrobenzo[a]pyrene-induced DNA amplification in SV40-transformed Chinese hamster embryo cells.

Nitrobenzo[a]pyrenes (NBaPs) are ubiquitous environmental pollutants that produce mutations in Salmonella typhimurium and Chinese hamster ovary cells. In this study, 1-, 3-, and 6-NBaP induced amplification of SV40 DNA sequences in an SV40-transformed Chinese hamster embryo cell line which is sensitive to DNA amplification by various known carcinogens. Of the three isomers, 3-NBaP produced the highest level of gene amplification, which was 4.8 relative to untreated controls at a dose of 5 micrograms/ml. Considering the relationship between gene amplification and tumorigenesis, it seems prudent to carry out a more exhaustive analysis of the carcinogenic potential of these agents.

Synthesis and biochemical characteristics of nucleoproteins following a toxic dose of retinoic acid in cycling and differentiating HL-60 cells.

Exposure of HL-60 cells to 2 microM retinoic acid (RA), twice the dose necessary for differentiation, initiated rapid synthesis (2 h) of the nuclear stress proteins (SPs) e.g., 90, 70c, 70x, 22 (Mr x 10(-3)) during the G0 + G1 phase of the cell cycle as observed by polyacrylamide gel electrophoresis (PAGE). Synthesis of SPs was cell cycle correlated and not dependent on time-after-dosing, and all labeling disappeared from these proteins within 48 h of RA exposure. Stress proteins were not elicited with a 1 microM dose or less of retinoic acid. Non-stress nuclear proteins revealed changes in synthesis levels (e.g., actin, lamins, tubulins) which were cell cycle related and temporally associated with dosing. A major non-stress nuclear protein (Mr 120,000) which possessed an affinity for actin in binding assays, was cell cycle related in control cells, and was suppressed in synthesis in cells exposed to 2 microM retinoic acid. Two additional nuclear non-SPs 51 and 55 (Mr x 10(-3)) covalently bound the isotope [3H]retinoic acid, and their incorporation was cell cycle correlated during early periods of RA exposure. Except for the induction of SPs, the autoradiographs of nuclear proteins of RA dosed HL-60 cells, showed more quantitative than qualitative changes.

The induction of aneuploidy by 3-nitrobenzo[a]pyrene in Chinese hamster ovary cells.

Nitrobenzo[a]pyrenes are found in urban air particulates and particulates from diesel exhaust, gasoline engines and wood burning stoves. Following exposure of Chinese hamster ovary cells (CHO-K1-BH4) to 3-nitrobenzo[a]pyrene (3-NB[a]P), cells with multiple nuclei and/or nuclei with multiple lobes were observed. When CHO cells were treated with 5 micrograms/ml 3-NB[a]P for 5 or 20 h, aneuploidy was noted in these cells at 24-96 h post-exposure. The addition of N6, O2'-dibutyryl adenosine 3':5'-cyclic monophosphate to 3-NB[a]P-exposed CHO cell cultures appeared to reduce the amount of aneuploidy in treated cultures. Structure--activity studies showed that 1-NB[a]P was a much less effective inducer of aneuploidy than 3-NB[a]P and 6-NB[a]P was ineffective. 1-, 3- and 6-nitrosobenzo[a]pyrenes were not effective inducers of aneuploidy in CHO cells, and aneuploidy was not observed in cultures treated with 3-NB[a]P in the presence of S9 activation. It appears that the parent 3-NB[a]P is responsible for producing aneuploidy in CHO cells.

Effect of interleukin-2 on cell proliferation, sister-chromatid exchange induction, and nuclear stress protein phosphorylation in PHA-stimulated Fischer 344 rat spleen lymphocytes: modulation by 2-mercaptoethanol.

The effect of interleukin-2 (IL-2) on cell proliferation, sister-chromatid exchange (SCE) frequency, and the phosphorylation of nuclear stress proteins was evaluated in phytohemagglutinin (PHA)-stimulated spleen lymphocytes isolated from Fischer 344 rats. In addition, the ability of 2-mercaptoethanol (2-ME) to modulate the induction of these biological responses was characterized. Cell proliferation, as measured by the mitotic index, increased significantly (P less than .003) from a range of 3-4% in PHA-stimulated cultures to a range of 8-11% in PHA-stimulated cultures exposed to IL-2. The average generation time (AGT) did not respond to IL-2 in a concentration-dependent manner and decreased significantly (P less than .05) when 20 microM 2-ME was included with IL-2 in the culture medium. The number of SCE increased significantly (P less than .004) from control frequencies, which ranged from 13.1 to 15.6 SCE per cell, to frequencies of 18.5 to 21.5 SCE per cell as the concentration of IL-2 in the culture medium increased to 50 half-maximal units per ml. A reduction in SCE frequency was observed when cells were cultured with 20 microM 2-ME and IL-2 compared to IL-2 alone. Three nuclear proteins, with relative molecular masses of approximately 13,000-18,000, 20,000, and 80,000, were phosphorylated in IL-2-exposed G1-phase nuclei. Elicitation of these nuclear proteins in IL-2-exposed cells was not affected by exposure to 2-ME.

Evaluation of the genotoxicity of gentian violet in bacterial and mammalian cell systems.

Previous studies indicate that gentian violet (GV), a triphenylmethane dye used in agriculture and human medicine, is a clastogen in vitro and a carcinogen in chronically exposed mice and rats. Data on its genotoxic activity, however, have been incomplete and partly contradictory. Mutagenesis and DNA damage experiments were conducted to re-evaluate the genotoxic potential of GV in both bacterial and mammalian cell systems. GV was mutagenic in Salmonella typhimurium tester strains TA97 and TA104, but there was little mutagenic activity detected in strains TA98 and TA100. A rat liver homogenate fraction (S9) tended to increase mutagenicity. The major microsomal metabolites of GV, pentamethylpararosaniline and N,N,N',N'-tetramethylpararosaniline were less mutagenic in TA97 and TA104, while N,N,N',N"-tetramethylpararosaniline was a weak mutagen in Salmonella. GV was not mutagenic in Chinese hamster ovary (CHO) cell strain CHO-K1-BH4, and was a questionable mutagen in CHO-AS52 cells. While GV produced DNA damage as measured by sedimentation of nucleotids derived from B6C3F1 mouse lymphocytes treated in vitro, no damage was found in lymphocytes isolated from mice dosed with GV. GV was also a weak producer of gene amplification in an SV40-transformed Chinese hamster cell line. The results indicate that GV is a point mutagen in bacteria; however, since similar exposure conditions produced weak mutagenic activity in mammalian cells, GV may be carcinogenic by virtue of its clastogenic activity.

Comparison of sister chromatid exchanges in spleen and thymus lymphocytes from AKR, C57BL/6N x DBA/2J F1, and CBA mice following in vivo exposure to N-methyl-N-nitrosourea.

The induction of sister chromatid exchange (SCE) by N-methyl-N-nitrosourea (MNU) was studied in spleen and thymus lymphocytes from AKR mice, which are highly susceptible to MNU-produced thymomas, CBA mice, which are much less sensitive to induction of thymomas by MNU, and C57BL/6N x DBA/2J F1 mice. MNU produced dose-related increases in SCE in concanavalin A (Con A)- and lipopolysaccharide-stimulated spleen lymphocytes and Con A-stimulated thymus lymphocytes from each mouse strain at 1 and 24 h posttreatment. MNU-induced SCE were generally higher in Con A-stimulated spleen lymphocytes compared to lipopolysaccharide-stimulated spleen lymphocytes and Con A-stimulated thymus lymphocytes from each mouse strain. On the whole, MNU-produced SCE were lower in AKR and CBA spleens than in C57BL/6N x DBA/2J F1 spleens. In addition, MNU-induced SCE levels in thymus lymphocytes from all three strains of mice were, for the most part, similar. These results indicate that if differences in MNU-induced genotoxicity in AKR, CBA, and C57BL/6N x DBA/2J F1 thymus lymphocytes exist, these differences cannot be ascertained by use of the in vivo/in vitro SCE assay.

Triethylene melamine-induced sister-chromatid exchange in murine lymphocytes exposed in vivo.

The induction of sister-chromatid exchange (SCE) by triethylene melamine (TEM), a known animal carcinogen, was investigated in an in vivo exposure/in vitro culture murine lymphocyte assay. Dose-related increases in SCE were observed in B6D2F1 mice following a single i.p. injection of 0.5, 1 or 2 mg/kg TEM. SCE frequencies remained elevated over baseline levels at 24 h post exposure. It is hoped that studies of this nature can determine whether the in vivo/in vitro murine lymphocyte SCE assay is useful for predicting the carcinogenic potential of an agent.

Inhibition of 7,12-dimethylbenz[a]anthracene-induced genotoxicity in Chinese hamster ovary cells by retinol and retinoic acid.

The effects of retinol and retinoic acid on cytotoxicity and mutation expression at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in Chinese hamster ovary (CHO) cells with or without exogenous metabolic activation were studied in the presence and absence of known chemical mutagens. Neither retinol nor retinoic acid induced mutants at the HGPRT locus of CHO cells at concentrations ranging from 1 microM to 50 microM without exogenous metabolic activation, and at concentrations ranging from 1 microM to 25 microM in the presence of Aroclor 1254-induced rat liver S9. Retinol and retinoic acid did not affect 100 micrograms/ml ethyl methanesulfonate-induced cytotoxicity or mutations at the HGPRT locus of CHO cells in the absence of exogenous metabolic activation at concentrations ranging from 1 microM to 25 microM. In contrast, retinol and retinoic acid inhibited 7,12-dimethylbenz[a]anthracene-induced cytotoxicity and mutation induction at the HGPRT locus of CHO cells when either uninduced Sprague-Dawley rat liver S9, Aroclor 1254-induced Sprague-Dawley rat liver S9 or co-cultivated primary Sprague-Dawley rat hepatocytes were used to provide metabolic activation. These data show that retinoids are capable of inhibiting mutation induction in mammalian cells in vitro by a chemical promutagen requiring metabolic activation to a reactive form, and suggest that such inhibition is due to an alteration of mutagen metabolism.

Mutagenicity of the mononitrobenzo[a]pyrenes in Chinese hamster ovary cells mediated by rat hepatocytes or liver S9.

Previous studies have shown that 1- and 3-nitrobenzo[a]pyrene (NBaP) were mutagenic in the Salmonella reversion assay without exogenous activation and that 1-, 3- and 6-NBaP were mutagenic in the presence of hepatocytes or liver homogenate (S9). In the present study, 1-, 3- and 6-NBaP were tested for mutagenicity in Chinese hamster ovary (CHO) cells under activation conditions similar to those used in the bacterial studies. None of the NBaPs was mutagenic without exogenous activation and none was mutagenically activated by hepatocytes from unpretreated rats or rats pretreated with Aroclor 1254 or 3-methylcholanthrene. Benzo-[a]pyrene (BaP), the parent compound, induced a strong mutagenic response under all hepatocyte mediation conditions. The NBaPs did produce positive mutagenic responses with S9 activation (3- = 1- greater than 6-NBaP), but these moderate responses were less than those of BaP. The difference between the bacterial and CHO results under the variety of activation conditions suggests the importance of the endogenous metabolism of the target cell as well as the source and the type of exogenous metabolic activation.

A rapid method for preparation of urinary bladder epithelium for flow cytometric analysis.

A rapid and reliable method is described for preparation of urinary bladder epithelium for flow cytometric analysis. This method has proven useful for measurement of cell cycle kinetics in bladder epithelium from normal fetal, neonatal and adult mice as well as animals that have been exposed to urinary bladder toxicants. A modified Vindelov 's propidium iodide stain was instilled directly into the urinary bladder through the urethra, allowing the propidium iodide to lyse the epithelial cells and stain the DNA in the liberated nuclei. The stained epithelial cell nuclei were then removed from the lumen and analyzed by flow cytometry without centrifugation or filtration techniques. This method allows examination of epithelial cell nuclei without contamination from other tissue elements due to the fact that the propidium iodide did not act beyond the epithelial basement membrane under our experimental conditions.

Methapyrilene is inactive in the hepatocyte-mediated Chinese hamster ovary/hypoxanthine-guanine phosphoribosyl transferase mutational assay.

Methapyrilene, tested at both non-hepatocytotoxic and hepatocytotoxic concentrations, failed to induce somatic mutations in the hepatocyte-mediated Chinese hamster ovary/hypoxanthine-guanine phosphoribosyl transferase (HGPRT) mutational assay. These data support the conclusion that methapyrilene induces the carcinogenesis process through a non-DNA damaging mechanism, suggested when this antihistamine previously failed to induce a genotoxic response in several in vitro test systems developed to detect carcinogens and mutagens.

Flow cytometric analysis of the effect of phenytoin and its major metabolite on mitogen stimulated mouse spleen cells.

The in vitro immunopharmacological effects of phenytoin (PHT) and its major metabolite 5-(4-hydroxyphenyl)-5-phenylhydantoin (HPPH) were investigated by flow cytometric analysis of the DNA content of mitogen stimulated mouse spleen cells. The qualitative effects of PHT and HPPH were similar in concanavalin A stimulated mouse spleen cells with both compounds causing an increase in the percentage of S phase cells. The data suggests that this effect is due to an augmentation of cell cycling as demonstrated by the significant increase in 4N cells in PHT treated cultures relative to control cultures following colcemid treatment. A PHT time course study revealed an increase in S phase cells and a subsequent increase in 4N cells. PHT had no significant effect on congenitally athymic nude mouse spleen cells stimulated with lipopolysaccharide (LPS) except at the highest concentration tested (80 micrograms/ml) where a depression of cell cycling was observed. HPPH caused a colcemid-like accumulation of 4N cells in the LPS stimulated nude mouse spleen cell cultures. PHT and HPPH were found to be effective in enhancing cell cycling in cultures containing a significant population of T-cells stimulated with a T-cell mitogen whereas an inhibitory effect was observed in cultures without T-cells stimulated with a B-cell mitogen. The capacity of PHT to enhance the mitogenic action of concanavalin A may relate to its capacity to induce immunologic abnormalities and lymphadenopathy in humans.