Cycloheximide genotoxicity in in vitro and in vivo test systems.

The aim of this study was to investigate if there was any genotoxic effect produced by the antibiotic cycloheximide, widely used as a fungicide in agriculture as well as in everyday laboratory practice. The battery of test systems included the bacterium Salmonella typhimurium (strains TA98 and TA100), the yeast Saccharomyces cerevisiae (D7), Allium cepa somatic cells and mouse bone marrow cells. This combination of test systems enabled us to establish possible effects caused by cycloheximide at different levels of the genome and to indicate a possible mechanism of action. The results obtained in experiments showed that cycloheximide did not induce frameshift or base-pair substitution mutations in S. typhimurium regardless of metabolic activation. In S. cerevisiae cycloheximide had only toxic effects but no increase of mitotic gene conversion was noticed under the conditions of the experiment. However, in A. cepa somatic cells as well as in mouse bone marrow cells cycloheximide showed its activity causing different genetic damages, e.g., chromosome breaks, mitotic disturbances and nuclear abnormalities.

Molecular analysis of mutations induced by chemical carcinogens in mammalian cells. II. The use of recombinant DNA vectors.

One approach to molecular and mechanistic studies of mutagenesis in mammalian cells is to introduce a mutational target gene into the cells as part of a shuttle vector which is capable of replication in both mammalian cells and bacteria. Following mutagenesis in the mammalian cell host, the shuttle vector sequences are recovered from the mammalian cells and introduced into bacteria, where large amounts of the mutant gene can be produced for sequence analysis. The variety of shuttle vector systems which have been developed for this purpose will be described. Shuttle vectors have been widely used for the molecular analysis of mutations induced by physical and chemical agents and to investigate the factors which modulate mutation fixation. The data regarding chemically induced mutational spectra will be reviewed with particular emphasis on the studies aimed to dissect the complex process which lead from DNA lesion to mutation.

Misincorporation rate and type on the leading and lagging strands of UV-damaged DNA.

We have examined the fidelity of replication of the leading and lagging strands of UV-irradiated DNA by using an EBV-derived shuttle vector system which contains as marker gene for mutation analysis the bacterial gpt gene in both orientations relative to the EBV oriP. Human cells stably transformed with this vector were UV irradiated and gpt mutation rate and type were analysed. An increased mutagenicity associated with UV irradiation was observed, but the average error frequency was unaffected by the direction of replication of the target gene. Some variability by position and sequence context of leading and lagging strand errors was detected, suggesting that the different architecture of the replication complex for the two strands might, to some extent, affect mutation spectra. The comparable fidelity of translesion replication on the leading and lagging strands is in agreement with the current model for eukaryotic replication that postulates the simultaneous synthesis of both strands by a DNA polymerase with a proof-reading exonuclease.

Fidelity of replication of the leading and the lagging DNA strands opposite N-methyl-N-nitrosourea-induced DNA damage in human cells.

Semi-conservative replication of double-stranded DNA in eukaryotic cells is an asymmetric process involving leading and lagging strand synthesis and different DNA polymerases. We report a study to analyze the effect of these asymmetries when the replication machinery encounters alkylation-induced DNA adducts. The model system is an EBV-derived shuttle vector which replicates in synchrony with the host human cells and carries as marker gene the bacterial gpt gene. A preferential distribution of N-methyl-N-nitrosourea (MNU)-induced mutations in the non transcribed DNA strand of the shuttle vector pF1-EBV was previously reported. The hypermutated strand was the leading strand. To test whether the different fidelity of DNA polymerases synthesizing the leading and the lagging strands might contribute to MNU-induced mutation distribution the mutagenesis study was repeated on the shuttle vector pTF-EBV which contains the gpt gene in the inverted orientation. We show that the base substitution error rates on an alkylated substrate are similar for the replication of the leading and lagging strands. Moreover, we present evidence that the fidelity of replication opposite O6-methylguanine adducts of both the leading and lagging strands is not affected by the 3' flanking base. The preferential targeting of mutations after replication of alkylated DNA is mainly driven by the base at the 5' side of the G residues.

Strand bias of ultraviolet light-induced mutations in a transcriptionally active gene in human cells.

Ultraviolet (UV)-induced repair and mutational spectra were analyzed in an inducible marker gene, the metallothionein-l/guamine-xanthine phosphoribosyl transferase (gpt) fusion gene, carried by an Epstein-Barr virus-derived shuttle vector episomically maintained in human cells. The repair rate of UV photodimers from the shuttle-vector molecules was typical of transcriptionally active sequences, 70% of the dimers being removed within 8 h after irradiation. The spectrum obtained under basal gene transcription was compared with that obtained under induced transcription. In both cases, base substitutions at dipyrimidine sequences predominated. Multiple mutations and deletions probably due to recombinational events induced by UV damage were also observed. Most of the UV-mutated dipyrimidine sites were located in the transcribed strand and were independent of the transcriptional activity of the target gene. In contrast, the distribution of mutations throughout the coding region of the gpt gene was affected by transcription, with a preferential clustering of mutations occurring in the 3' half of the gene after transcription induction. The strand bias observed in the UV spectra most likely reflects selection for nonfunctional gpt protein.