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H Vrieling

Publications and source records attributed to H Vrieling.

At least 37 records · Page 2Linked to original sources
PubMed1998

Enhanced UV-induced mutagenesis in the UV61 cell line, the Chinese hamster homologue of Cockayne's syndrome B, is associated with defective transcription coupled repair of cyclobutane pyrimidine dimers.

Cells from Cockayne's syndrome (CS) patients are hypersensitive to the cytotoxic effects of UV-irradiation and are defective in transcription coupled repair (TCR). We have examined the mutagenic consequences of impaired TCR in the Chinese hamster cell line UV61, the rodent homologue of CS complementation group B. Analysis of the two major UV-induced photolesions, cyclobutane pyrimidine dimers (CPD) and pyrimidine 6-4 pyrimidone photoproducts (6-4 PP), revealed that repair of CPD from the transcribed strand was strongly reduced in UV61 cells, but repair of 6-4 PP was indistinguishable from that in wild-type hamster cells. UV-induced mutation induction was enhanced in UV61 compared to that observed in repair proficient cells. The spectrum of UV-induced base substitutions in UV61 was clearly different from that observed in wild-type hamster cells and resembled the spectrum previously observed in nucleotide excision repair deficient hamster cells. In UV61 cells a strong strand bias for mutation induction was found; assuming that premutagenic lesions occur at dipyrimidine sequences, 76% of the mutations could be attributed to lesions in the transcribed strand. These data strongly favour the hypothesis that defective TCR of CPD is responsible for the enhanced UV-induced mutagenesis in UV61 cells.

Animals↗
PubMed1998

Elevated frequencies of benzo(a)pyrene-induced Hprt mutations in internal tissue of XPA-deficient mice.

Xeroderma pigmentosum (XP) patients are hypersensitive to sunlight and have a high predisposition to developing cancer. At the cellular level, XP patients are defective in nucleotide excision repair (NER). Recently, mice have been generated via gene targeting that are deficient in the expression of the XPA gene [A. de Vries et al., Nature (Lond.), 377: 169-173, 1995]. We have assessed the consequences of defective NER for mutagenesis in normal and XPA mice exposed to benzo(a)pyrene and 2-acetylaminofluorene. To study mutagenesis, mature T lymphocytes were isolated from the spleen and stimulated to proliferate in vitro to select for mutants at the endogenous Hprt locus. Background mutant frequencies in normal and XPA mice were very similar and not influenced by age. Single doses of benzo(a)pyrene administered i.p. resulted in a dose-dependent increase of the Hprt mutant frequency in normal mice. In addition, after chronic exposure to benzo(a)pyrene, Hprt mutants were readily detectable in XPA mice at an early onset of treatment but only at a later stage in normal mice. In contrast, chronic treatment of either normal or XPA mice with 2-acetylaminofluorene did not increase Hprt mutant frequency above the background frequency. This absence of significant induction of Hprt mutants can be entirely attributed to the low frequency of 2-acetylaminofluorene-induced DNA adducts in lymphoid tissue. These results provide the first direct evidence in mammals that deficient NER leads to enhanced mutagenesis in endogenous genes in internal tissue after exposure to relevant environmental mutagens, such as benzo(a)pyrene.

2-Acetylaminofluorene↗
PubMed1998

Impairment of nucleotide excision repair by apoptosis in UV-irradiated mouse cells.

We investigated the relationship between nucleotide excision repair (NER) activity and apoptosis in UV-irradiated cells. Mouse erythroleukemia (MEL) and lymphoma (GRSL) cells exhibited enhanced sensitivity to the cytotoxic effects of UV radiation compared to hamster cell lines, although normal UV-induced hprt mutation frequencies were found. Determination of UV-induced repair replication revealed a limited capacity of MEL and GRSL cells to perform NER consistent with poor removal of cyclobutane pyrimidine dimers and pyrimidine 6-4 pyrimidone photoproducts from transcriptionally active genes during the first 8 h after UV exposure. However, both cyclobutane pyrimidine dimers and pyrimidine 6-4 pyrimidone photoproducts appeared to be processed to almost normal level 24 h after UV treatment. In parallel, we observed that the UV-irradiated MEL and GRSL cells suffered from severe DNA fragmentation particularly 24 h after UV exposure. Taken together, these data indicate a reduced repair of UV-induced photolesions in apoptotic cells, already established at the early onset of apoptosis. To test whether inhibition of repair in cells was due to inactivation of NER or to apoptosis-induced chromatin degradation, we performed in vitro excision assays using extracts from UV-irradiated MEL cells. These experiments showed that the NER capacity during early apoptosis was intact, indicating that slow removal of UV-induced photolesions in apoptotic cells is due to substrate modification (presumably degradation of chromatin) rather than direct inhibition of factors involved in NER.

Animals↗
PubMed1998

Methyl methanesulfonate-induced hprt mutation spectra in the Chinese hamster cell line CHO9 and its xrcc1-deficient derivative EM-C11.

The Chinese hamster cell mutant EM-C11, which is hypersensitive to the cell killing effects of alkylating agents compared to its parental line CHO9, has been used to study the impact of base excision repair on the mutagenic effects of DNA methylation damage. This cell line has a defect in the xrcc1 gene. XRCC1 can interact with DNA polymerase-beta, thereby suppressing strand displacement, and DNA ligase III, both of which have been implicated in base excision repair. XRCC1 may, therefore, allow efficient ligation of single-strand breaks generated during base excision repair. Both EM-C11 and CHO9 cells were treated with methyl methanesulfonate (MMS), a DNA-methylating agent reacting predominantly with nitrogen atoms generating adducts which are substrates for the base excision repair pathway. EM-C11 cells are much more sensitive to the cytotoxic effects of MMS than CHO9: for EM-C11, the dose of MMS inducing 10% survival is 6-fold lower than that for CHO9. In contrast, mutation induction at the hprt locus following MMS is similar in EM-C11 and CHO9. Molecular analysis of hprt gene mutations showed that although the largest class of hprt mutations, both in EM-C11 and CHO9 cells, consisted of GC > AT transitions, most likely caused by O6-methylguanine, the size of this class was smaller in EM-C11. The fraction of deletion mutants in EM-C11, however, was twice as large as that found in CHO9 cells. These results suggest that reduced ligation efficiency of single-strand breaks generated during base excision repair, as result of a defect in XRCC1, may lead to the formation of deletions.

Animals↗
PubMed1998

Strand-specific mutation induction by 1,2-dibromomethane at the hypoxanthine-guanine phosphoribosyltransferase locus of Chinese hamster ovary cells.

The nature of mutations induced by 1,2-dibromoethane (DBE) at the hprt (hypoxanthine-guanine phosphoribosyl-transferase) gene was analysed in Chinese hamster ovary (CHO-9) cells. Molecular characterization of 36 hprt mutants at the cDNA level yielded 19 GC-->AT transitions, two AT-->CG transversions, three frameshift mutations, two identical small deletions and 10 exon deletions. Further analysis of the deletion mutants by amplification of specific exons from genomic DNA showed two more GC-->AT transitions at splice sites and an approximately 70 bp deletion. Assuming that the S-[2-(N7-guanyl)ethyl]glutathione adduct is responsible for the GC-->AT transitions, 90% of the affected guanines were located in the non-transcribed strand of the hprt gene, suggesting a strand bias in repair of this adduct. Nearest neighbour analysis of induced GC-->AT transitions indicates a preference for a 5'-PyPuG DNA sequence, i.e. 15/21 mutated guanines were located in either a TGG or a CAG DNA sequence. These molecular data on DBE-induced mutations showed similar features as data from a study by Graves et al. (Mutagenesis, 11, 229-233, 1996) in which they analyzed 13 hprt mutants induced by DBE in CHO-K1 cells. Six of the seven GC-->AT mutations were on positions mutated more than once among the 36 hprt mutants in the present study. The combined findings suggest that some positions seem to be hot spots for DBE-induced mutations. Concerning the relevance of these in vitro studies for germ cell mutagenesis the conclusion may be that these data lend further support to the view that mutation spectra derived from in vitro systems have little predictive value for the nature of mutations induced in post-meiotic germ cells in vivo, as demonstrated for other alkylating agents in both Drosophila and mice.

Alkylation↗
PubMed1997

The design of a new mutation model for active genes: expression of the Escherichia coli lac operon in mammalian cells.

The design of a novel transgenic mouse model is described that should allow analysis of mutations at a single cell level in all tissues of a model animal. The model is based on the correct regulation of the Escherichia coli lac operon in mammalian cells. Induction of a mutation in the lacI gene will result in the loss of transcriptional repression of the lacZ gene in mutated cells. Expression of beta-galactosidase can subsequently be detected at the single cell level. The model was first tested in vitro using transfection of mouse LTK- cells. LacZ expression was very heterogeneous in most of the stable transfectants and seemed to be subject to epigenetic inactivation. One clone (IIB1) was isolated that stably expressed lacZ in more than 99% of its cells. Subsequent introduction of the lacI gene into IIB1 cells resulted in correct transcriptional repression of the lacZ gene that could be alleviated by IPTG, an allosteric inducer of lacI repression. However, in time the extent of beta-galactosidase induction gradually declined suggesting that the prolonged repressed transcriptional state triggers epigenetic inactivation. Variegated expression of the lacZ gene was not confined to cultured cells since several transgenic lines also did not express the lacZ transgene. This study shows that while the susceptibility of the lacZ gene to inactivation processes poses a fundamental problem, correct regulation of the expression of a reporter gene by the lacI repressor protein is feasible in mammalian cells when assayed at the single cell level. Thus, the model can in principle be used for the detection of mutagenic events at the lacI locus. Targeting of the lacZ gene to an endogenous housekeeping gene might prevent epigenetic inactivation. Alternatively, with the use of another reporter gene in the mutation detection system the proposed transgenic mouse model could be realized.

Animals↗
PubMed1996

Induction of hprt gene mutations in splenic T-lymphocytes from the rat exposed in vivo to DNA methylating agents is correlated with formation of O6-methylguanine in bone marrow and not in the spleen.

The suitability of splenic T-lymphocytes as a substitute tissue for detection of genotoxic effects induced in vivo by chemical agents that are organ-specifically activated was tested in rats exposed to single doses at the potent lung-carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), acetoxymethylmethylnitrosamine (AMMN) or N-methyl-N-nitrosourea (MNU). NNK, AMMN and MNU methylate DNA most likely via the formation of a methanediazohydroxide ion that decomposes to a methyl diazonium ion. For all three agents, an increase in the levels of 06-methylguanine and 7-methylguanine in DNA of rat liver and lung was detected by reverse phase HPLC and electrochemical detection. Treatment with NNK did not result in the formation of O6-methylguanine and 7-methylguanine in DNA of bone marrow and spleen, corresponding with the absence of metabolic activation pathways for this compound in these tissues. For AMMN formation of both 06-methylguanine and 7-methylguanine was detectable in DNA of the spleen, whereas in DNA of bone marrow only very low frequencies of 7-methylguanine were found at a toxic dose. MNU induced O6-methylguanine and 7-methylguanine in both spleen and bone marrow. Using splenic T-lymphocytes from the rat no increase above control levels of the hprt mutant frequencies was found for NNK and AMMN for all exposure levels tested, 32 days after chemical exposure. For MNU a dose-dependent increase in hprt mutant frequency was found at exposure levels of 0.097 mmol/kg up to 0.582 mmol/kg. DNA sequence analysis was performed on PCR products of hprt cDNA from 39 MNU-induced 6-thioguanine-resistant T-lymphocyte clones. Single base pair substitutions were found in 25 of these mutants (64%), GC-->AT transitions being the predominant type of mutation (19 of 25; 76%). These mutations are probably caused by mispairing of 06-methylguanine with thymine during DNA replication. The results indicate that formation of mutagenic lesions in the spleen is not correlated with an enhanced frequency of 6-thioguanine-resistant splenic T-lymphocyte clones from rats, 32 days after exposure in vivo to DNA damaging agents. This suggests that mutation-fixation in T-lymphocytes does not occur in the spleen but at other sites in the body such as bone marrow, after which these mutated cells migrate to the spleen.

Alkylating Agents↗
PubMed1995

Marked differences in the role of O6-alkylguanine in hprt mutagenesis in T-lymphocytes of rats exposed in vivo to ethylmethanesulfonate, N-(2-hydroxyethyl)-N-nitrosourea, or N-ethyl-N-nitrosourea.

The role of DNA alkylation at the O6 position of guanine in the induction of gene mutations in vivo was studied in the hprt gene of rat T-lymphocytes from spleen exposed in vivo to the monofunctional ethylating agents ethylmethanesulfonate (EMS) and N-ethyl-N-nitrosourea (ENU), or the hydroxyethylating agent N-(2-hydroxyethyl)-N-nitrosourea (HOENU). All chemicals showed an exposure-dependent increase in hprt mutant frequency. HOENU and ENU, however, were much more mutagenic than EMS when compared at equimolar levels. DNA sequence analysis was performed on PCR products of hprt cDNA from 40 EMS-, 35 HOENU-, and 46 ENU-induced 6-thioguanine-resistant T-lymphocyte clones. Thirty EMS-induced mutants contained a single base pair substitution with GC to AT transitions being the predominant type of mutation (26 of 30) which are probably caused by mispairing of O6-ethylguanine with T during DNA replication. No strand specificity of mutated G's among GC to AT transitions was observed. Twenty-three HOENU- and 42 ENU-induced mutants contained a single base pair substitution. In contrast to EMS, GC to AT transitions were found at a low frequency, 4 of 23 for HOENU and 5 of 42 for ENU, indicating that O6-hydroxyethylguanine and O6-ethylguanine are less important in HOENU- and ENU-induced mutagenesis in vivo, respectively. Also here no strand bias for mutated G's was observed, although the number of this type of mutation was limited. The most frequently induced base pair alterations by HOENU and ENU were transversions at AT base pairs, 16 of 23 and 28 of 42, respectively, with AT to TA being the predominant type of mutation. In both ENU and HOENU mutational spectra, an extreme strand bias for mutated T's toward the nontranscribed strand was found. The results suggest that DNA damage induced in rat T-lymphocytes in vivo by HOENU and ENU is processed in similar ways.

Adenine↗
PubMed1995

Mechanisms and biomarkers of genotoxicity. Molecular dosimetry of chemical mutagens.

The relevance of the use of DNA adduct frequencies as a parameter for the extent of mutation induction by monofunctional alkylating agents was investigated in cultured Chinese hamster cells and in rat skin fibroblasts treated in vivo with the test chemicals. The nature of the biologically significant DNA adducts was investigated by DNA sequence analysis of mutations induced at the hypoxanthine-guanine phophoribosyltransferase (hprt) gene. The results show that under conditions where O6-alkylguanine is a persistent DNA lesion more than 50% of the mutations are GC to AT transitions indicating that the frequency of O6-alkylguanine is a good parameter for mutation induction. However, in target cells which are able to remove alkyl groups from the O6 position of guanine, alkylating agents with a low nucleophilic selectivity (e.g. N-ethyl-N-nitrosourea (ENU) and N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG)) exert most of their mutagenic activity most likely via the induction of O2-ethylthymine.

Animals↗
PubMed1995

Detection of point mutations in T lymphocytes.

We exposed experimental animals to a series of alkylating agents that induced mutations at the X-linked hprt gene of T lymphocytes. We then isolated the mutant cells and analyzed the molecular nature of the mutations by amplification of hprt cDNA sequences with the use of reverse transcriptase PCR followed by DNA sequence analysis, and then correlated the mutational spectra obtained to the spectra of DNA adducts caused by the alkylating agents used. The nature of the base-pair changes causing the mutations was characteristic for the reaction pattern of the genotoxic agent with DNA. However, we also found a clear influence of DNA repair processes; i.e., in those cells that were able to remove certain types of DNA damage, the class of mutations expected from that type of damage was reduced.

Alkylating Agents↗
PubMed1994

Analysis of repair of cyclobutane pyrimidine dimers and pyrimidine 6-4 pyrimidone photoproducts in transcriptionally active and inactive genes in Chinese hamster cells.

Irradiation of cells with short wave ultraviolet light (UV-C) induces both cyclobutane pyrimidine dimers (CPD) as well as pyrimidine 6-4 pyrimidone photoproducts (6-4 PP). We have focused on the removal of both types of DNA photolesions from the transcriptionally active adenine phosphoribosyltransferase (APRT) and hypoxanthine-guanine phosphoribosyltransferase (HPRT) genes and the inactive c-mos gene. Induction levels of both CPD and 6-4 PP were similar for all three genes analyzed, with the induction of 6-4 PP being about 3-fold lower than of CPD. Repair of CPD was analyzed using the CPD-specific enzyme T4 endonuclease V; repair of 6-4 PP was examined employing Escherichia coli UvrABC excinuclease. Unlike the HPRT gene, in which CPD were removed selectively from the transcribed strand, both strands of the 16-kilobase fragment encompassing the 2.6-kilobase APRT gene were repaired efficiently. This suggests the existence of multiple transcription units in the APRT region including transcription units running in the opposite direction of the APRT gene. Only a marginal part of the CPD was removed from the inactive c-mos gene after 24 h. In all three genes investigated, 6-4 PP were repaired more rapidly than CPD and, as demonstrated for the HPRT and APRT genes, without strand specificity. The difference in the repair phenotype of CPD between the HPRT gene and the APRT gene coincides with differences between both genes with regard to the DNA strand distribution of previously published UV-induced mutations.

Adenine Phosphoribosyltransferase↗
PubMed1994

Differences in dorsal and ventral pigmentation result from regional expression of the mouse agouti gene.

The agouti coat color gene encodes a paracrine signaling molecule that controls the production of yellow and black pigment by melanocytes within hair follicles. Some agouti alleles affect the dorsum and ventrum independently, which has provided the basis for speculation that agouti gene action in different regions of the body is controlled by distinct genetic loci that are closely linked. Using a combination of cDNA cloning and RNA expression studies, we find that alternative isoforms of agouti mRNA contain different noncoding first exons located 100 kb apart, whose patterns of expression indicate independent control by regulatory elements that are either ventral specific or hair cycle specific. These results demonstrate that the apparent genetic complexity of the agouti locus is explained by the existence of multiple regulatory elements exerting control over a single coding sequence and provide a conceptual basis for understanding differences in dorsal and ventral hair coloration in many mammalian species. The ventral-specific agouti isoform represents an example of a transcript whose expression is restricted to ventral skin and provide an approach to investigate the mechanisms by which dorsal-ventral differences in gene expression are established and maintained.

Age Factors↗
PubMed1994

Molecular analysis of ethyl methanesulfonate-induced mutations at the hprt gene in the ethyl methanesulfonate-sensitive Chinese hamster cell line EM-C11 and its parental line CHO9.

The Chinese hamster cell line EM-C11 has been shown to be 5 times more sensitive than its parental line CHO9, but not hypermutable, after treatment with ethyl methanesulfonate. Ethyl methanesulfonate-induced mutational spectra were determined at the hprt locus to investigate whether the same adducts are responsible for mutation induction in both cell lines. The mutational spectra for EM-C11 and CHO9 show an important difference. GC-->AT transitions were found in both cell lines at similar frequencies; however, the spectrum of CHO9 contains a class of AT-->GC transitions, which seems to be replaced by a group of deletions in EM-C11. Since the ethyl methanesulfonate-induced mutation frequency for both lines is the same at equal exposure, it is hypothesized that the lesions leading to AT-->GC transitions in CHO9 are responsible for the deletions in EM-C11. This phenomenon might be explained if the responsible adduct(s) in CHO9 is bypassed resulting in replication errors, while blocking DNA synthesis in EM-C11 causing the observed increase in cell death. In surviving EM-C11 cells, DNA strand exchanges might have occurred at the position of stalled replication forks, leading to gross molecular changes. The adduct probably responsible for the AT-->GC transitions in CHO9 and the deletions in EM-C11 is 3-ethyladenine.

Animals↗
PubMed1994

Formation and persistence of DNA adducts in pouch skin fibroblasts and liver tissue of rats exposed in vivo to the monofunctional alkylating agents N-methyl-N-nitrosourea or N-ethyl-N-nitrosourea.

Base substitutions and frameshifts induced by genotoxic agents are considered to result mainly from incomplete repair and incorrect replication of modified nucleotides in DNA. In this study, induction and persistence of O6-alkyl- and 7-alkylguanine adducts were determined by reverse phase HPLC and electrochemical detection in DNA of pouch skin fibroblasts and liver tissue of rats exposed in vivo to the monofunctional alkylating agents N-methyl-N-nitrosourea (MNU) and N-ethyl-N-nitrosourea (ENU). Although an exposure dependent increase in the level of adducts was found for both chemicals, a much lower frequency of both O6-alkylguanine and 7-alkylguanine was detected after ENU treatment than after MNU treatment, indicating that MNU is much more reactive with DNA than ENU. The persistence of O6-alkyl- and 7-alkylguanine was studied for up to 48 h at exposure levels of 60 mg/kg for MNU and 100 mg/kg for ENU. A time-dependent decline in the levels of both adducts was observed, but w6-alkylguanine was more rapidly lost than 7-alkylguanine in both pouch skin fibroblasts and liver. Furthermore, DNA adducts were faster lost from liver than from pouch skin fibroblasts. The loss of O6-alkylguanine adducts is probably mediated by the action of O6-alkylguanine-DNA alkyltransferase (AGT) in the target tissues since AGT activity was detectable in protein extracts of pouch skin fibroblasts and liver from unexposed rats and from exposed rats, 48 h but not 1 h after MNU and ENU treatment. AGT activity recovered faster in liver tissue than in pouch skin fibroblasts, and after ENU exposure an induction of AGT activity was observed in the liver but not in pouch skin fibroblasts. The difference in the level of O6-alkylguanine in DNA of pouch skin fibroblasts introduced upon exposure to MNU and ENU may explain the molecular nature of most base pair changes observed previously in spectra of hprt mutants induced in these cells in vivo. The frequency of O6-methylguanine upon MNU exposure remains relatively high with time and these adducts most likely cause GC to AT transitions. In the case of ENU, O6-ethylguanine was detected at very low frequencies resulting in a low contribution of GC to AT transitions. Rather, the ENU spectrum is dominated by base pair changes at AT base pairs.

Alkylating Agents↗
PubMed1994

Molecular analysis of hprt gene mutations in skin fibroblasts of rats exposed in vivo to N-methyl-N-nitrosourea or N-ethyl-N-nitrosourea.

The granuloma pouch assay in the rat is a model system in which relative frequencies of genetic and (pre-) neoplastic changes induced in vivo by carcinogenic agents can be determined within the same target tissue. The target is granuloma pouch tissue and consists of a population of (transient) proliferating fibroblasts which can be cultured in vitro. hprt gene mutations were studied in granuloma pouch tissue of rats treated with single doses of direct acting alkylating agents N-methyl-N-nitrosourea (MNU) or N-ethyl-N-nitrosourea (ENU). Both agents showed an exposure-dependent increase in the hprt mutant frequency. Thirty-seven MNU (60 mg/kg)- and 43 ENU (100 mg/kg)-induced hprt mutant cell clones were analyzed at the molecular level. Twenty-two MNU-induced and 36 ENU-induced mutants carried a single base pair change in exon sequences of the hprt gene. The predominant base pair alterations induced by MNU were GC to AT transitions (18 of 22), which are probably caused by O6-methylguanine lesions. For most of the GC to AT transitions (16 of 18), the G was located in the nontranscribed strand, suggesting a strand bias in the repair of O6-methylguanine lesions. ENU-induced mutations occurred predominantly at AT base pairs (28 of 36), being mostly AT to TA and AT to CG transversions, and are probably caused by O2-ethylthymidine. Also here, DNA repair processes seem to act with different rates/efficiencies on DNA adducts in the 2 strands of the hprt gene, since all the 24 transversions observed at AT base pairs had the thymidine residue in the nontranscribed strand. GC to AT transitions were only present at a low frequency among ENU-induced mutations, suggesting that O6-ethylguanine lesions were repaired efficiently before mutations were fixed during replication. The mutational spectra of MNU- and ENU-induced hprt mutant clones were different from spontaneously occurring hprt mutant clones. These results indicate that MNU and ENU induce different mutational spectra in vivo and that DNA repair systems remove O6-methylguanine, O2, and/or O4-ethylthymidine much faster from the transcribed strand than the nontranscribed strand of the hprt gene in these rat fibroblasts.

Amino Acid Sequence↗
PubMed1994

Blocking of in vitro DNA replication by deoxycytidine adducts of the mutagen and clastogen 2-bromoacrolein.

Calf thymus single-stranded DNA was modified with 2-bromoacrolein (2BA), a genotoxic metabolite of tris(2,3-dibromopropyl)phosphate. This DNA was used as a template for in vitro DNA replication by T7-polymerase and Klenow fragment of Escherichia coli DNA polymerase I. Increasing levels of 2BA modification led to decreased DNA synthesis as measured by [methyl-3H]dTTP incorporation. M13 mp19 single-stranded DNA template modified with 2BA was used to determine the sites of termination of DNA replication by T7 polymerase and Klenow fragment of Escherichia coli DNA polymerase I. It was found that DNA replication stopped frequently before and occasionally opposite deoxycytidine nucleotides. These results indicated that an as yet unidentified deoxycytidine:2BA adduct may have been formed in the reaction of 2BA with M13 DNA. To investigate if such adducts were formed, we reacted 2BA with deoxycytidine in vitro at pH 4.4, and putative deoxycytidine:2BA adducts were isolated by high-performance liquid chromatography. They were characterized by 1H and 13C nuclear magnetic resonance and with fast atom bombardment mass spectrometry as two diastereomeric 3-bromo-7-(beta-D-deoxyribofuranosyl)- 3,4-dihydro-2-hydroxy-(2H,7H)[1,6-a]pyrimidin-6-one adducts and a 3-bromo-7-(beta-deoxyribofuranosyl)-(4H,7H)-pyrimido[1,6-a]pyrimidin-6 -one adduct. Only the latter adduct, however, was formed in the reaction of 2BA with calf thymus single-stranded DNA in vitro. Tris(2,3-dibromopropyl)phosphate is clastogenic. Because clastogenicity may result from DNA adducts that block replication, the role of the presently identified deoxycytidine adducts of the reaction metabolite 2BA in the clastogenicity of tris(2,3-dibromopropyl)phosphate is discussed.

Acrolein↗