The effect of folate deficiency on the hprt mutational spectrum in Chinese hamster ovary cells treated with monofunctional alkylating agents.

Folic acid deficiency acts synergistically with alkylating agents to increase DNA strand breaks and mutant frequency at the hprt locus in Chinese hamster ovary (CHO) cells. To elucidate the mechanism of this synergy, molecular analyses of hprt mutants were performed. Recently, our laboratory showed that folate deficiency increased the percentage of clones with intragenic deletions after exposure to ethyl methanesulfonate (EMS) but not N-nitroso-N-ethylurea (ENU) compared to clones recovered from folate replete medium. This report describes molecular analyses of the 37 hprt mutant clones obtained that did not contain deletions. Folate deficient cells treated with EMS had a high frequency of G>A transitions at non-CpG sites on the non-transcribed strand, particularly when these bases were flanked on both sides by G:C base pairs. Thirty-three percent of these mutations were in the run of six G's in exon 3. EMS-treated folate replete cells had a slightly (but not significantly) lower percentage of G>A transitions, and the same sequence specificity. Treatment of folate deficient CHO cells with ENU resulted in predominantly T>A transversions and C>T transitions relative to the non-transcribed strand. These findings suggest a model to explain the synergy between folate deficiency and alkylating agents: (1) folate deficiency causes extensive uracil incorporation into DNA; (2) greatly increased utilization of base excision repair to remove uracil and to correct alkylator damage leads to error-prone DNA repair. In the case of EMS, this results in more intragenic deletions and G:C to A:T mutations due to impaired ligation of single-strand breaks generated during base excision repair and a decreased capacity to remove O6-ethylguanine. In the case of ENU additional T>A transversions and C>T transitions are seen, perhaps due to mis-pairing of O2-ethylpyrimidines. Correction of folate deficiency may reduce the frequency of these types of genetic damage during alkylator therapy.     

The influence of the nucleotide excision-repair system on mutagenesis in Salmonella typhimurium LT2 after exposure to low doses of monofunctional alkylating agents.

The role of nucleotide excision repair in the mutagenicity of the monofunctional alkylating agents N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), methyl methanesulfonate (MMS), N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG), and N-ethyl-N-nitrosourea (ENU) in Salmonella typhimurium was examined. The mutagenic potential of the mutagenic agents used increased in the following order: MMS less than ENU less than ENNG less than MNNG. The results obtained confirm the involvement of nucleotide excision repair in the removal of mutagenic lesions from the DNA of S. typhimurium cells exposed to high doses of methylating as well as ethylating agents. At the low doses of all the alkylating agents used, the nucleotide excision repair-proficient strain was mutagenized more efficiently than the uvrB mutant. This phenomenon, a consequence of competition between nucleotide excision-repair enzymes and constitutive O6-methylguanine-DNA methyltransferase, is discussed.     

Evaluation of genotoxicity of clofazimine, an antileprosy drug, in mice in vivo. II. Micronucleus test in bone marrow and hepatocytes

The antileprosy drug, clofazimine, was tested for its possible genotoxicity using micronucleus (MN) tests in mice. A significantly higher incidence of MN in bone marrow erythrocytes, particularly in polychromatic erythrocytes, as well as in regenerated hepatocytes revealed a positive clastogenic effect of the drug. The drug also had a marked antimitotic effect as indicated by a negative correlation with the dose.     

Evaluation of genotoxicity of clofazimine, an antileprosy drug, in mice in vivo. I. Chromosome analysis in bone marrow and spermatocytes

Clofazimine, an antileprosy drug, was tested for its cytogenetic effect in mouse bone marrow and testis. Bone marrow metaphase analysis in adults treated directly for different periods (1, 2 and 4 weeks, 40 mg/kg/day) and with different doses (4, 20 and 40 mg/kg/day for 7 days) as well as in young animals exposed through lactation for different periods (2, 3, and 4 weeks) revealed significant increases in chromosomal aberrations over the controls. Analysis of diakinesis-metaphase I stages also exhibited a significantly elevated incidence of chromosome aberrations over controls after treatment for different periods. On the basis of the present result the drug may be considered a potential clastogen in mice.     

First results of in vitro cultivation of stem cells from bone marrow and peripheral blood in an autologous test system of patients with hematological diseases

In 17 patients with haematological diseases an autologous test system was elaborated for determining the percentage of unipotent myeloic stem cells in the bone-marrow and peripheral blood and implemented by experiments. In comparing the results obtained by means of the traditional heterologous culture method, differences (diminutions, increase of aggregate numbers) could be found which allow certain conclusions to be drawn on the patient's real bone marrow function. For reasons of standardization, cultivation in the heterologous system cannot be abandoned. The investigations are continued.     

The association of nonsense mutation with exon-skipping in hprt mRNA of Chinese hamster ovary cells results from an artifact of RT-PCR.

RT-PCR of RNA from CHO cells with nonsense mutations in the hprt gene frequently detects minor hprt mRNA species lacking one or more exons. Many nonsense mutants also contain greatly reduced concentrations of the major, normally spliced hprt mRNA. In this study, we examined the hypothesis that exon-deleted mRNAs are normal constituents of CHO cells, but are not detected in wild-type parental cells and most missense mutants because their amplification is suppressed by relatively high concentrations of normally spliced hprt mRNA. A protocol designed to specifically detect exon-deleted mRNAs was conducted using RNA from parental cells and identified all the exon-deleted species typical of nonsense mutants. Quantitative analysis of parental cell RNA measured these exon-deleted mRNAs at < or = 0.7% of the abundance of the full-sized species. Nonsense and missense mutants had comparable amounts of exon-deleted mRNAs, which varied both above and below parental concentrations. The relative concentrations of particular exon-deleted species could be explained by the location of nonsense mutations remaining in the mRNA or by structural effects of mutations on splicing. Exon-deleted mRNAs were detected by RT-PCR when the concentration of the most abundant exon-deleted species was > or = 2% of the full-length mRNA. This occurred for mutants with nonsense mutations in internal exons. RT-PCR conditions were shown to suppress the amplification of exon-deleted species 40-fold when full-length mRNA was abundant, which occurred for parental lines and missense mutants. Our results verify that RT-PCR conditions can produce an artifactual association between nonsense mutation and exon-skipping when minor, exon-deleted mRNA is relatively enriched.     

Further evaluation of an in vivo micronucleus test on rat and mouse skin: results with five skin carcinogens.

In a previous paper, we presented a practical in vivo micronucleus (MN) test that used rat skin as the target organ. To evaluate the test, as well as to determine the reproducibility and applicability of the method to mice, we used it to test the effect of five skin carcinogens (N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 4-nitroquinoline 1-oxide (4NQO), 7,12-dimethylbenz[a]anthracene (DMBA), and benzo[a]pyrene (B[a]P)) on rat and mouse skin. All five compounds significantly and dose-dependently increased the MN frequencies in the basal cells of the chemical-treated skin. These results indicated the reproducibility of the test results and also the applicability of the test to mice as well as rats.     

The dicyemid Mesozoa as an integrated system for morphogenetic studies. I. Description, isolation and maintenance.

The morphological simplicity of the dicyemid Mesozoa is such as to allow mapping of enery individual cell during the development of the organisms. Individual cells are of a size amenable to micromanipulation and a number of potential morphogenetic makers is readily apparent. The possibility of raising the animals in vitro and obtaining developmental mutants makes these organisms excellent candidates for use as an integrated system to correlate cytodifferentiation and morphogenesis with genetic control. An axenic, nearly defined, medium in which the misozoans can be kept for over three months has been developed. Methods for isolating and maintaining the organisms are described.     

The circumfusion system for multipurpose culture chambers. I. Introduction to the mechanics, techniques, and basic results of a 12-chamber (in vitro) closed circulatory system

A self-contained mechanical system for circulating nutrient fluid through 12 tissue culture chambers is described in detail. This system utilizes nonperforated cellophane membranes in the chambers which separate the circulating nutrient from the tissue culture environments. The nutrient, therefore, is dialyzed through the cellophane of each chamber; some cell products are retained in the microenvironment between the closely apposed cellophane and cover slip, whereas the other cell products move from chamber to chamber in the circulating nutrient. The resultant environmental conditions directed by the circumfusion systems are highly favorable for maintaining the differentiation of chick embryo tissues over protracted periods; a number of micrographs are shown.     

The in vivo induction of sister chromatid exchanges in the bone marrow of the Chinese hamster. II.N-nitrosodiethylamine (DEN) and n-isopropyl-alpha-(2-methyl-hydrazino)-p-toluamide (Natulan), two carcinogenic compounds with specific mutagenicity problems.

N-Nitrosodiethylamine (DEN) and N-isopropyl-alpha-(2-methylhyadrazino)-p-toluamide (Natulan) were examined with the in vivo SCE method of Vogel and Bauknecht. Only Natulan showed a positive effect with a significant increase of induced SCE between 10 and 25 mg/kg b.w. The six-point curve of the dose effect was of the plateau type. With DEN only a slight increase with high doses could be obtained, which was not significant when 50 or 100 cells were counted. Compared with the results of other tests published, Natulan gives positive results preferentially with in vivo mammalian tests but not with microorganisms. On the other hand, DEN is inactive in vivo on the chromosomal level, but preferentially induces point mutations at the molecular level in microorganisms and Drosophila. It is recommended to include in a battery of true mutagenicity tests also cytogenetic tests (in vivo SCE test and micronucleus test).     

The effect of caffeine on cytotoxicity, mutagenesis, and sister-chromatid exchanges in Chinese hamster cells treated with dihydrodiol epoxide derivatives of benzo[a]pyrene

The effect of caffeine on Chinese hamster V79 cells after treatment with the highly mutagenic (+/-)-7 beta,8 alpha-dihydroxy-9 alpha, 10 alpha-7,8,9,10-tetrahydrobenzo[a]pyrene, and the weaker mutagen (+/-)-7 beta,8 alpha-dihydroxy-9 beta,10 beta-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene, B[a]P-deiol-epoxide II, was studied at both the biological and molecular levels. Caffeine, at nontoxic dose levels, caused a synergistic reduction in cell survival induced by both isomers and also inhibited DNA elongation as measured by alkaline sucrose-gradient analysis of nascent DNA. However, caffeine did not affect the induction of either ouabain-resistant mutants or sister-chromatid exchanges by either isomer. These results suggest that enhanced cell killing by caffeine in benzo[a]pyrene-diol-epoxide treated V79 cells may be related to caffeine's inhibitory effect on DNA elongation. However, inhibition of DNA elongation by caffeine did not influence the resulting induced levels of mutagenesis or sister-chromatid exchanges.     

Effects of 3-aminobenzamide on Chinese hamster cells treated with thymidine analogues and DNA-damaging agents. Chromosomal aberrations, mutations and cell-cycle progression

The nuclear enzyme, poly(ADP-ribose) synthetase is involved in the repair of damaged DNA. We report here the results obtained with 3-aminobenzamide (3AB), an inhibitor of this enzyme, on induced biological effects. 3AB increases the frequency of chromosomal aberrations induced by DMS, EMS, ENU, bleomycin and CldUrd. The magnitude of the effect is dependent on the type of chemical used, the combinations with DMS and EMS being the most potent ones. No potentiation was observed after treatment of cells with MMC. Mutation frequencies were determined on the HPRT locus and showed that 3AB did not increase the frequency of gene mutations induced by EMS, ENU and CldUrd. Cell-cycle progression is affected when cells are grown in medium containing CldUrd and 3AB, primarily when the inhibitor is present during the second cell cycle when substituted DNA becomes replicated. The extent of the effect depends on the amount of analogue incorporated and is independent of the presence of the analogue in the medium during the second cell cycle. Analysis of chromosomal aberrations in delayed G2 cells with the aid of the premature chromosome-condensation technique revealed numerous aberrations after incorporation of CldUrd and treatment with 3AB.