DNA adduct formation in precision-cut rat liver and lung slices exposed to benzo[a]pyrene.

Chemical-DNA adducts provide an integrated measure of exposure, absorption, bioactivation, detoxification, and DNA repair following exposure to a genotoxic agent. Benzo[a]pyrene (BaP), a prototypical polycyclic aromatic hydrocarbon (PAH), can be bioactivated by cytochrome P-450s (CYPs) and epoxide hydrolase to genotoxic metabolites which form covalent adducts with DNA. In this study, we utilized precision-cut rat liver and lung slices exposed to BaP to investigate tissue-specific differences in chemical absorption and formation of DNA adducts.

Double-base lesions are produced in DNA by free radicals.

Evidence has been accumulating at the oligomer level that free radical-initiated DNA damage includes lesions in which two adjacent bases are both modified. Prominent examples are lesions in which a pyrimidine base is degraded to a formamido remnant and an adjacent guanine base is oxidized. An assay has been devised to detect double-base lesions based on the fact that the phosphoester bond 3' to a nuclesoside bearing the formamido lesion is resistant to hydrolysis by nuclease P1.

Assay for reactive oxygen species-induced DNA damage: measurement of the formamido and thymine glycol lesions.

A 32P-postlabeling assay has been developed for the simultaneous detection of the thymine glycol lesion and the formamido remnant of pyrimidine bases in DNA exposed to reactive oxygen species (ROS). The formamido lesion is a principal lesion produced in X-irradiated DNA oligomers when oxygen is available to mediate the damage process. Production of the well-known thymine glycol lesion is less dependent on the concentration of oxygen.

Detection of mitomycin C-DNA adducts in vivo by 32P-postlabeling: time course for formation and removal of adducts and biochemical modulation.

Mitomycin C (MMC) is a DNA cross-linking agent that has been used in cancer chemotherapy for over 20 years, yet little is known either qualitatively or quantitatively about MMC-induced DNA adduct formation and repair in vivo. As an initial means of investigating this, we used a recently developed 32P-postlabeling assay to examine the formation and loss of MMC-DNA adducts in the tissues of a simple in vivo model test system, the chick embryo, following treatment with a chemotherapeutic dose of MMC. As early as 15 min after MMC treatment, four adducts could be detected in the liver which were tentatively identified as the (CpG) N2G-MMC-N2G interstrand cross-link, the bifunctionally activated MMC-N2G monoadduct, and two isomers (alpha and beta) of the monofunctionally activated MMC-N2G monoadduct.

Mutations induced by monofunctional and bifunctional phosphoramide mustards in supF tRNA gene.

The relative mutagenicity, nature of the mutations and the sequence specificity of mutations induced by the bifunctional alkylating agent, phosphoramide mustard (PM) and a monofunctional derivative, dechloroethyl phosphoramide mustard (dePM), were analyzed by the Ames test and by an in vitro shuttle vector mutagenesis assay. Both PM and dePM increased the mutation frequency above background in either assay. However, on an equimolar basis, dePM was less mutagenic than PM.

Altered biochemical profile and gene expression in aflatoxin B-1-transformed C3H10T1/2 cells.

A transformed cell line 7SA, obtained by transformation of C3H10T1/2 cells with irt vitro activated aflatoxin B-1 (AFB(1)), was used to investigate biochemical and molecular alterations associated with transformation by AFB(1). 7SA cells demonstrate an altered biochemical phenotype characterized by alterations in phase I and phase II enzymes in a manner that would allow these cells to survive in a hostile chemical environment. Investigations of the molecular basis of transformation revealed no mutations in codons 12/13 and 61 of ras genes (Ha-, Ki- and N-ras) and in exons 5, 6, 7 and 8 of p53 tumor suppressor gene.

Mutations induced in a shuttle vector plasmid exposed to monofunctionally activated mitomycin C.

Reductive activation of mitomycin C leads to its covalent binding to DNA, forming monoadducts and cross-links. The cytotoxicity of mitomycin C has been attributed to cross-link formation, whereas monoadducts are assumed to cause mutagenicity. We have developed a 32P-postlabeling technique to measure mitomycin C DNA adducts.

DNA damage in UVB-irradiated keratinocytes.

A DNA lesion which results from the breakdown of a pyrimidine base leaving a formamido remnant has been associated with oxidative stress. This lesion is shown to be produced in keratinocytes irradiated in culture with UVB light. The amount of formamido lesion produced is comparable to the amount of the 8-hydroxyguanine lesion.

Mutations induced by dacarbazine activated with cytochrome P-450.

The mutagenicity of the antitumor drug dacarbazine (DTIC) is due to alkylation of cellular DNA by metabolites resulting from the metabolism of this drug by the mixed function oxidase system. In the present study, we used an in vitro shuttle vector assay to study the base and sequence specificity of mutagenesis by DTIC. The shuttle vector plasmid pSP189 was treated with DTIC (1-2.

Free radical-induced double base lesions.

Evidence is presented for the formation of products in irradiated dinucleoside monophosphates in which both bases are damaged. The dinucleoside monophosphates d(GpT), d(GpC), d(TpG) and d(CpG) were X-irradiated in oxygenated aqueous solution. Product identification was by NMR spectroscopy.

Formation of 8-hydroxyguanine in DNA during mitomycin C activation.

DNA damage caused indirectly via reactive oxygen species generated during reductive activation of mitomycin C was evaluated. This oxidative DNA damage was measured by determining the formation of 8-hydroxyguanine in DNA exposed to chemically or enzymatically activated mitomycin C. The level of 8-hydroxyguanine was measured indirectly by determining formamidopyrimidine-DNA glycosylase-sensitive sites induced in plasmid DNA exposed to mitomycin C and directly by a 32P-postlabeling assay for the modified base.

Mutations in a shuttle vector exposed to activated mitomycin C.

The cytotoxicity of the potent antibiotic and antitumor agent mitomycin C (MMC) is due to its irreversible binding to DNA. Alkylating species generated by bioreductive activation of MMC are known to cause monoadducts and cross-links in DNA by specifically binding to guanine residues. To gain insight into how these lesions lead to base- and sequence-specific mutations, shuttle vector pSP189 was treated with MMC chemically reduced by treatment with sodium borohydride, replicated in human Ad293 cells, rescued in bacteria, and analyzed for mutations in the supF tRNA gene sequence.

Altered expression of cytochrome P450 mRNA during chemical-induced hepatocarcinogenesis and following partial hepatectomy.

Levels of various cytochrome P450 proteins have been reported to be decreased to varying degrees in chemically induced hepatocyte nodules and following partial hepatectomy (PH). By screening a rat liver lambda ZAP cDNA expression library with antibodies raised against a partially purified preparation of cytochrome P450 isolated from untreated male Fischer 344 rats, we have isolated a 1.1-kb cDNA.

Characterization of the products of dinucleoside monophosphates d(GpN) irradiated in aqueous solutions.

The dinucleoside monophosphates d(GpT), d(GpC), and d(GpA) were X-irradiated in oxygenated aqueous solution. The principal products were identified in the intact modified dinucleoside monophosphates using NMR spectroscopy and FAB mass spectrometry. The 8-hydroxyguanine modification is a major product in each of the d(GpN).

Interaction of the cyclophosphamide metabolite, acrolein, with lactate dehydrogenase-m homolog.

The cyclophosphamide metabolite, acrolein, binds to both nucleic acids and proteins. In this report, acrolein is shown to interact preferentially with a rat liver 35KD DNA-binding protein. The 35KD protein was purified to homogeneity from a hepatic microsomal fraction using (NH4)2SO4 precipitation, DEAE-Sepharose chromatography, cation exchange HPLC, and DNA-Sepharose affinity chromatography.

The differential lysis of phosphoester bonds by nuclease P1.

The hydrolysis by nuclease P1 of the 16 common deoxydinucleoside monophosphates was examined. The rates of hydrolysis of phosphodiester bond differ by more than two orders of magnitude; dinucleotide monophosphates of the type d(TpN) being most resistant and d(GpN) being next most resistant. The profiles of a mixture of the 16 common dinucleoside monophosphates and of DNA after partial hydrolysis by nuclease P1 and simultaneous treatment with acid phosphatase were compared.

32P-postlabeling assay for free radical-induced DNA damage: the formamido remnant of thymine.

A prominent lesion in DNA exposed to oxidative free radicals results from the degradation of thymine leaving a formamido remnant. A 32P-postlabeling assay has been developed for the detection of the formamido lesion. The assay is based on the circumstance that the lesion prevents hydrolysis by nuclease P1 of the phosphoester bond 3' to the damaged nucleoside.

Analysis of DNA damage at the dinucleoside monophosphate level: application to the formamido lesion.

The dinucleoside monophosphates d(TpG), d(TpC), and d(TpT) were X-irradiated in oxygenated solution. In each case the modification of the dinucleoside in which the thymine base is degraded to a formamido remnant was observed as a principal product. The hydrolysis of the phosphoester bond of formamido-modified dinucleosides is much slower than that of the corresponding unmodified dinucleosides.

32P-postlabeling of acrolein-deoxyguanosine adducts in DNA after nuclease P1 digestion.

In order to study the relationship between the level of acrolein-DNA adducts and their biological effects, sensitive methods are needed to quantitate DNA adducts. 32P-postlabeling is one such method that has been widely used and we have adapted the technique to detect acrolein-deoxyguanosine adducts. Adducts formed by the reaction of acrolein and deoxyguanosine-3'-monophosphate were isolated by HPLC.

Characterization of two radiation-induced lesions from DNA: studies using nuclease P1.

Exposure of DNA oligomers to ionizing radiation in oxygenated solution reveals that the two lesions formed in highest yield are the 8-hydroxy modification of guanine and the formamido remnant of thymine. The effect of these lesions on the hydrolysis of the phosphodiester bond by nuclease P1 was studied. Whereas the 8-hydroxyguanine lesion does not affect hydrolysis of the adjacent 3' phosphoester bond by nuclease P1, the formamido lesion markedly retards hydrolysis.

Enzymatic excision of radiation-induced lesions from DNA model compounds.

Dinucleoside monophosphates in which the 5' nucleoside contained a radiation-modified base were tested as substrates to bovine spleen phosphodiesterase (SPD) and snake venom phosphodiesterase. The radiation-modified bases included thymine glycols, 5-hydroxymethyluracil, 8-hydroxyguanine, and a formamido remnant of thymine. The lesions had widely different effects on diesterase action, varying from little inhibition, as in the case of digestion of dT*pA by SPD, where T* is the hydroxymethyluracil modification, to severe inhibition, as in the case of digestion of dG*pC by SPD, where G* is the 8-hydroxyguanine modification.

A cyclophosphamide/DNA phosphoester adduct formed in vitro and in vivo.

The antitumor activity of cyclophosphamide is thought to be due to the alkylating activity of phosphoramide mustard, a metabolite of cyclophosphamide. Reaction of 2'-deoxyguanosine 3'-monophosphate and phosphoramide mustard resulted in the formation of several adducts that could be detected by high performance liquid chromatography (HPLC). One of these adducts, isolated and purified by HPLC, could be detected by 32P postlabeling.