n(7)-hydroxyethylguanine and 1-(2-hydroxyethyl)-1-nitrosourea

We have investigated the genotoxic effects of 1-(2-hydroxyethyl)-1-nitrosourea (HENU). We have chosen this agent because of its demonstrated ability to produce N7-(2-hydroxyethyl) guanine (N7-HOEtG) and O(6)-(2-hydroxyethyl) 2'-deoxyguanosine (O(6)-HOEtdG); two of the DNA alkylation products produced by 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU). For these studies, we have used the Big Blue Rat-2 cell line that contains a lambda/lacI shuttle vector. Treatment of these cells with HENU produced a dose dependent increase in the levels of N7-HOEtG and O(6)-HOEtdG as quantified by HPLC with electrochemical detection. Treatment of Big Blue Rat-2 cells with either 0, 1 or 5mM HENU resulted in mutation frequencies of 7.2+/-2.2x10(-5), 45.2+/-2.9x10(-5) and 120.3+/-24.4x10(-5), respectively. Comparison of the mutation frequencies demonstrates that 1 and 5mM HENU treatments have increased the mutation frequency by 6- and 16-fold, respectively. This increase in mutation frequency was statistically significant (P<0.001). Sequence analysis of HENU-induced mutations have revealed primarily G:C-->A:T transitions (52%) and a significant number of A:T-->T:A transversions (16%). We propose that the observed G:C-->A:T transitions are produced by the DNA alkylation product O(6)-HOEtdG. These results suggest that the formation of O(6)-HOEtdG by BCNU treatment contributes to its observed mutagenic properties.

Topics: Animals; Bacterial Proteins; Base Sequence; Carmustine; Cell Line; Deoxyguanosine; DNA Adducts; DNA Mutational Analysis; Escherichia coli Proteins; Ethylnitrosourea; Guanine; Lac Repressors; Mutagenicity Tests; Mutagens; Mutation; Rats; Repressor Proteins

Young adult male Lewis rats were exposed to ethylene oxide (EO) via single intraperitoneal (i.p.) injections (10-80 mg kg-1) or drinking water (4 weeks at concentrations of 2, 5, and 10 mM) or inhalation (50, 100 or 200 ppm for 4 weeks, 5 days week-1, 6 h day-1) to measure induction of HPRT mutations in lymphocytes from spleen by means of a cloning assay. N-ethyl-N-nitrosourea (ENU) and N-(2-hydroxyethyl)-N-nitrosourea (HOENU) were used as positive controls. Levels of N-(2-hydroxyethyl)valine (HOEtVal) adducts in haemoglobin (expressed in nmol g-1 globin) were measured to determine blood doses of EO (mmol kg-1 h, mM h). Blood doses were used as a common denominator for comparison of mutagenic effects of EO administered via the three routes. The mean HPRT mutant frequency (MF) of the historical control was 4.3 x 10(-6). Maximal mean MFs for ENU (100 mg kg-1) and HOENU (75 mg kg-1) were 243 x 10(-6) and 93 x 10(-6), respectively. In two independent experiments, EO injections led to a statistically significant dose-dependent induction of mutations, with a maximal increase in MF by 2.3-fold over the background. Administration of EO via drinking water gave statistically significant increases of MFs in two independent experiments. Effects were, at most, 2.5-fold above the concurrent control. Finally, inhalation exposure also caused a statistically significant maximal increase in MF by 1.4-fold over the background. Plotting of mutagenicity data (i.e., selected data pertaining to expression times where maximal mutagenic effects were found) for the three exposure routes against blood dose as common denominator indicated that, at equal blood doses, acute i.p. exposure led to higher observed MFs than drinking water treatment, which was more mutagenic than exposure via inhalation. In the injection experiments, there was evidence for a saturation of detoxification processes at the highest doses. This was not seen after subchronic administration of EO. The resulting HPRT mutagenicity data suggest that EO is a relatively weak mutagen in T-lymphocytes of rats following exposure(s) by i.p. injection, in drinking water or by inhalation.

Topics: Administration, Inhalation; Administration, Oral; Animals; Carcinogens; Chromosome Aberrations; DNA Adducts; Erythrocytes; Ethylene Oxide; Ethylnitrosourea; Guanine; Hemoglobins; Hypoxanthine Phosphoribosyltransferase; Injections, Intraperitoneal; Lymphocytes; Male; Micronucleus Tests; Mutation; Rats; Rats, Inbred Lew; Sister Chromatid Exchange; Spleen

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.

Topics: Adenine; Animals; Base Composition; Base Sequence; Carcinogens; DNA Adducts; DNA, Complementary; Drug Resistance; Ethyl Methanesulfonate; Ethylnitrosourea; Guanine; Hypoxanthine Phosphoribosyltransferase; In Vitro Techniques; Male; Molecular Sequence Data; Mutagenesis; Point Mutation; Polymerase Chain Reaction; Rats; Rats, Inbred Lew; T-Lymphocytes; Thioguanine; Thymidine