Nancy F. Fullerton

Rapid isolation, hydrolysis and chromatography of formaldehyde-modified DNA

Deoxyadenosine, deoxyguanosine, deoxycytidine and thymidine were reacted with formaldehyde. High-performance liquid chromatographic (HPLC) analysis indicated that each deoxynucleoside had formed one major product. With the exception of the thymidine product, these adducts were analyzed by nuclear magnetic resonance spectroscopy and identified as hydroxymethyl derivatives at the exocyclic amines. Calf thymus DNA was incubated with [3H]formaldehyde and, after purification, enzymatically hydrolyzed to nucleosides. HPLC analysis indicated the presence of a substantial proportion of noncovalently bound formaldehyde and the following hydroxymethyl adducts, listed in order of decreasing concentration: N6-hydroxymethyldeoxyadenosine much greater than N4-hydroxymethyldeoxycytidine greater than N2-hydroxymethyldeoxyguanosine. Incubation of Chinese hamster ovary (CHO) cells with [3H]formaldehyde resulted in metabolic incorporation of the formaldehyde into purines and pyrimidines plus an appreciable concentration of formaldehyde noncovalently associated with the DNA. However, HPLC analysis clearly indicated the presence of N6-hydroxymethyldeoxyadenosine in the CHO cell genome.

An examination of the weak mutagenic response of 1-nitropyrene in Chinese hamster ovary cells.

Incubation of suspension cultures of Chinese hamster ovary (CHO) cells with 1-nitropyrene for as long as 2.5 h failed to induce mutations at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus, while incubation with 1-nitrosopyrene, a reduced derivative of 1-nitropyrene, resulted in a strong mutagenic response. Examination of the metabolites produced during these incubations indicated that 1-nitrosopyrene was rapidly reduced to 1-aminopyrene while 1-nitropyrene was not detectably metabolized. Both compounds produced a single major DNA adduct, N-(deoxyguanosin-8-yl)-1-aminopyrene, in the CHO cells and a strong linear relationship was found between mutation induction and the extent of DNA binding. The low level of adducts produced by 1-nitropyrene was consistent with the weak mutagenic response produced by this compound. These results indicate that both 1-nitropyrene and 1-nitrosopyrene are reduced to a reactive electrophile, presumably N-hydroxy-1-aminopyrene, which produces potentially mutagenic DNA damage in CHO cells. Comparison of the relationship between N-(deoxyguanosin-8-yl)-1-aminopyrene formation and mutation induction in CHO cells with the levels of 1-nitropyrene-induced DNA damage associated with positive responses in other assays of genetic toxicity and with the number of mutations associated with the DNA adducts produced by other agents in CHO cells suggests that the CHO/HGPRT assay may be relatively insensitive to 1-nitropyrene-induced DNA damage. The poor capability of CHO cells in reducing 1-nitropyrene and the relative insensitivity of the assay to the DNA damage produced by this compound may contribute to the weak mutagenic response of 1-nitropyrene in CHO cells.

Analysis of mutations in the K-ras and p53 genes of lung tumors and in the hprt gene of 6-thioguanine-resistant T-lymphocytes from rats treated with 1,6-dinitropyrene.

Direct pulmonary instillation of 1,6-dinitropyrene (DNP) into male Fischer 344 rats results in a dose-dependent induction of lung tumors and 6-thioguanine-resistant (TGr) T-lymphocytes. The treatment also results in DNP binding to dG in the lung and in T-lymphocytes. In the present study, we have examined the types of mutations associated with these responses to DNP. Sequencing of DNA amplification products from 20 DNP-induced lung tumors identified 5 mutations in K-ras codon 12, 4 GGT-->TGT transversions and one GGT-->GAT transition. No mutations were found in K-ras codons 13 or 61. Single-strand conformation polymorphism analysis of p53 exons 5-8 revealed mobility shifts indicative of mutation in 9 of the 20 tumor samples. Eight of the mutations were substitutions at G:C base pairs, and one was a deletion of a single G:C base pair. DNA from 161 TGr lymphocyte colonies cultured from DNP-treated rats was examined for point mutations by amplification of hprt exons 2, 3, and 8, and screening the products for mutant: wild-type heteroduplex formation by denaturing gradient-gel electrophoresis. Only three mutations were found, a G-->T transversion in exon 3, a G-->A transition in exon 8, and a complex mutation consisting of a tandem G-->T transversion and a one base deletion in exon 3. The mutations identified in the DNP-induced lung tumors and TGr T-lymphocytes are consistent with the formation of dG-DNA adducts by DNP. The extremely low recovery of point mutations from TGr lymphocytes suggests that DNP induces a substantial number of mutations by other mechanisms.

Hprt lymphocyte mutant frequency in relation to DNA adduct formation in rats fed the hepatocarcinogen 2-acetylaminofluorene

The lymphocyte hypoxanthine-guanine phosphoribosyltransferase (Hprt) assay is frequently used as a biomarker for the exposure of both humans and laboratory animals to potentially carcinogenic agents. To obtain information concerning the sensitivity of the rat Hprt lymphocyte assay toward aromatic amine carcinogens, male F344 rats were fed 0.02% 2-acetylaminofluorene (2-AAF) for 1 month and then returned to control diet for 2 months. At 4, 27, 48, 62, and 90 days after the initiation of 2-AAF-feeding, the frequency of mutants in the Hprt gene was determined. In addition, DNA was isolated from liver nuclei, spleen lymphocytes, bone marrow, and thymus, and DNA adducts were analyzed by 32P-postlabeling. 2-AAF feeding resulted in a significant induction of 6-thioguanine-resistant T-lymphocytes and the mutant frequency continued to increase after the 2-AAF feeding was stopped. The same major DNA adduct, N-(deoxyguanosin-8-yl)-2-aminofluorene, was detected in liver, spleen lymphocytes, bone marrow, and thymus. DNA adduct levels were greatest in the tumor target tissue (liver) but occurred in all T-lymphocyte compartments, being highest in spleen lymphocytes. The DNA adduct levels were highest at the end of the 1-month 2-AAF feeding period and decreased rapidly in all tissues. The data indicate that the Hprt lymphocyte mutagenesis assay detects arylamine carcinogens, but with relatively low sensitivity.

DNA Adduct Formation During Chronic Administration of an Aromatic Amine

DNA adduct formation is considered to play an integral part in the initiation of tumors. Since most human exposure to chemical carcinogens is chronic, it is important to investigate DNA adduct formation in relation to preneoplasia and tumorigenesis in animal models using a similar type of exposure. In this communication, two different experimental systems are described in which rodents were continuously fed 2-acetylaminofluorene (AAF). In both experiments, DNA adducts were detected with antisera specific for the acetylated [N-(deoxyguanosin-8-уl)-2-acetylaminofluorene; dG-C8-AAF] and deacetylated [N-(deoxyguanosin-8-y1)-2-aminofluorene; dG-C8-AF] C8-substituted deoxyguanosine adducts of AAF. The first study compared the tumor incidence in the livers and bladders of mice chronically fed seven different doses of AAF for up to 33 months with the DNA adducts in these tissues after one month of feeding AAF at similar doses. In the second investigation, four different hepatocarcinogenesis protocols were used to induce enzyme-altered, preneoplastic foci in the livers of rats. The animals were then fed AAF for five to six days and the concentration of AAF-DNA adducts was determined in the foci and adjacent tissues. Taken together, these studies suggest that initiating events involving DNA adducts occur relatively early in the carcinogenic process, and that cells progressing to form tumors may not necessarily contain or be able to form DNA adducts. Furthermore, the number of adduct-related events required for tumor initiation appears to differ among tissues of the same animal.