Alexander E. Maccubbin

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. In products containing double base lesions, guanine is converted to 8-hydroxyguanine and the pyrimidine base is degraded to a formamido remnant.

Vicinal Lesions in X-irradiated DNA?

Irradiation of the dinucleoside monophosphate d(GpT) in an oxygenated solution gives products characterized by damage on one or both guanine and thymine bases, the yields of which were proportional to radiation dose.

Tumors in fish from the Detroit River

A fish tumor survey was conducted in the Lower Detroit River during 1985 to 1987. Five species of fish were collected from sediment deposition zones and were examined for neoplasia. Neoplasms and related lesions were found in bullhead (Ictalurus nebulosus), walleye (Stizostedion vitreum), redhorse sucker (Moxostoma, sp), white sucker (Catostomus commersoni), and bowfin (Amia calva). Overall, 8.2 percent of the fish examined had oral/dermal lesions and 10.1 percent had liver lesions. Liver and skin tumors were found to be age-related in bullheads and size-related in walleye. Based on the fish surveyed, the incidence of tumors in fish from the Detroit River was similar to that observed in other chemically contaminated waterways.

Metabolites of Aromatic Hydrocarbons in the Bile of Brown Bullheads (Ictalurus nebulosus)

In vivo metabolism and biliary excretion of PAH by brown bullheads was determined using an HPLC/fluorescence method. In laboratory studies, 24 hours after feeding of PAH, bullheads had evidence for polar metabolites of the PAH in their bile as detected by HPLC’/fluorescence. These materials were detectable only at wavelengths specific for the compound that was fed. Analysis of bile from wild bullheads collected from areas with varying degrees of pollution demonstrated that bullheads from the Buffalo River had benzo[a]pyrene equivalents that were 7–148 times greater than those found in fish from less polluted areas. Similarly, levels of phenanthrene equivalents were 7–297 times greater in Buffalo River fish when compared to the other sites. These studies indicate that bullheads can rapidly metabolize dietary PAH, and wild bullheads from industrially impacted waterways such as the Buffalo River are receiving significant PAH exposure. This PAH exposure may account, in part, for the increased incidence of neoplasia observed in the bullheads.

Mutagenicity of Sediments from the Detroit River

Abstract Sediment samples from 30 sites in the lower Detroit River and one site in Lake Michigan were extracted with organic solvent and were tested for mutagenicity in the Ames test. Without metabolic activation, sediment samples were either non-mutagenic or weakly mutagenic. With metabolic activation, mutagenic responses (defined as doubling of the spontaneous mutation rate at one or more concentrations of organic extract) were observed in 16 of 31 samples. Mutation rates ranged from 20 to 370 histidine positive revertants per mg of organic extract. These results demonstrate the existence of compounds in sediments of the Detroit River that can be activated to mutagens. The sediments thus are a potential source of mutagenic and possibly carcinogenic compounds for fish and other organisms.

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. The resultant profiles are very similar, except for the appearance of 5-methyldeoxycytidine in the latter. Similar profiles are also obtained from a mixture of dinucleoside monophosphates and from DNA exposed to ionizing radiation beforehand. The 8-hydroxyguanine lesion and a formamido remnant of thymine appear in both profiles as a modified nucleoside and as modified dinucleoside monophosphate respectively. These results suggest that certain radiation induced DNA lesions can be selectively postlabelled based on their resistance to hydrolysis by nuclease P1. The nature of the nuclease P1-substrate interaction is discussed.

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 (1-2.5 mM) in vitro in a reconstituted cytochrome P-450 system at 37 degrees C for either 30 or 60 min. SupF tRNA gene insert contained in the plasmid was sequenced after replication of the drug-treated plasmid in human Ad 293 cells followed by amplification in indicator bacteria. Mutagenesis of DTIC in this system was dependent upon the presence of the cytochrome P-450 reconstituted system and NADPH. Mutations induced by DTIC included single base substitutions (35%), single base deletions (30.5%), single base insertions (19.4%) and large deletions (13.8%). Among the substitutions, transversions and transitions were in the ratio of 1:0.7. Base pairs 108 and 127 in the SupF tRNA of the pSP189 were identified as mutational hot spots.

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). The d(GpN), where N is a pyrimidine nucleoside, also yield products in which the pyrimidine base is degraded to a formamido remnant. The most interesting product is one bearing two base damages, 8-hydroxylation of the guanine base and degradation of the pyrimidine base to a formamido remnant. This double lesion was observed in both d(GpN) where N is a pyrimidine base.

Characterization of radiation-induced damage in d(TpApCpG).

The radiation chemistry of the oligomer d(TpApCpG) X-irradiated in aqueous solution containing glutathione was studied. Four products were isolated by HPLC and characterized by NMR spectroscopy. Two of the major products are isomers of a 5-hydroxy-5,6-dihydrothymine modification of d(TpApCpG). A dihydrothymine modification is also formed. The other major product is a result of strand scission. These products are different from the major products identified previously in a study of d(TpApCpG) X-irradiated in oxygenated solution. The effect of specific radiation-induced lesions on the behaviour of d(TpApCpG) as substrate to a spleen phosphodiesterase-micrococcal nuclease combination of enzymes and to nuclease P1 was studied. These enzymes are of interest because they are used in postlabelling assays of DNA damage.

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. Using this technique, we have measured monoadduct formation in the shuttle vector plasmid pSP189 and have determined mutations induced by monoadduct formation. The shuttle vector plasmid was incubated with mitomycin C under conditions favoring monofunctional activation of mitomycin C. The plasmid was then replicated in human Ad293 cells, rescued in bacteria, and analyzed for mutations in the supF tRNA gene sequence of pSP189. One major mitomycin C/DNA adduct was observed by 32P‐postla‐beling and was characterized as a monoadduct ofguanine. When pSP189 was exposed to monofunctionally activated mitomycin C, increases in adduct levels and mutation frequency were found to be related to mitomycin C concentration. The majority of the mutations involved single bases, with base substitutions making up 59.1% of the total mutations observed. Of the base substitutions, 67.2% were transversions and 32.8% were transitions, with nearly 80% of all base substitutions involving G:C base pairs. Deletions, either as single bases or large deletions, also involved G:C base pairs the majority of the time. The observed bias of mutations atG:C and the formation of a mitomycin C/DNA monoadduct involving guanine suggests that monoadduct formation may be responsible for the mutations. Environ. Mol. Mutagen. 29:143–151, 1997.