Peter F. Swann

Base pair mismatches and carcinogen-modified bases in DNA: an NMR study of A•C and A•O4meT pairing in dodecanucleotide duplexes

Structural features of A.C mismatches and A.O4meT pairs in the interior of oligodeoxynucleotide duplexes have been investigated by high-resolution two-dimensional proton NMR spectroscopy on the self-complementary d(C-G-C-A-A-G-C-T-C-G-C-G) duplex (designated A.C 12-mer) and and the self-complementary d(C-G-C-A-A-G-C-T-O4meT-G-C-G) duplex (designated A.O4meT 12-mer) containing A.C and A.O4meT pairs at identical positions four base pairs in from either end of and A.O4meT pairs at identical positions four base pairs in from either end of the duplex. Proton NMR shows that there are similar pH-dependent changes in the structure in the A.C 12-mer and A.O4meT 12-mer duplexes. Our studies have focused on the low-pH (pH 5.5) conformation where high-quality two-dimensional NOESY data sets were collected from the exchangeable and nonexchangeable protons in these duplexes. The spectral parameters for the A.C 12-mer and the A.O4meT 12-mer duplexes were very similar, indicating that they must have similar structures at this pH in aqueous solution. Both structures are right-handed double helices with all the bases adopting the normal anti configuration about the glycosidic bond. The same atoms are involved in hydrogen-bond pairing for the A.C mismatch and the A.O4meT pair, and these pairs have a similar spatial relationship to flanking base pairs.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of ethanol on nitrosamine metabolism and distribution: Implications for the role of nitrosamines in human cancer and for the influence of alcohol consumption on cancer incidence

For reasons that have never been explained, the consumption of alcohol is associated with an increase in the incidence of human cancer, notably that of the oesophagus. The effect of ethanol on nitrosamine metabolism and carcinogenicity is reviewed, together with new work on pharmacokinetics. This work shows that small quantities of ethanol alter the distribution and metabolism of small oral doses of N-nitrosodimethylamine and N-nitrosodiethylamine in rats, to increase by several fold the alkylation of DNA in organs that are particularly susceptible to their carcinogenic effect. It is shown that in the case of N-nitrosodimethylamine this is the result of prevention of first-pass clearance of the nitrosamine as it travels in the blood draining the gut through the liver before entering the general circulation. There is evidence that the same happens in man. These results explain the findings from various experiments in animals and they lend credence to the observation that nitrosamines occur in human blood after high-nitrate meals are taken with alcohol. The results have led to the hypothesis that the influence of alcohol consumption on human cancer may be mediated through the effect of ethanol on the pharmacokinetics of nitrosamines derived from diet, from tobacco smoke and from endogenous synthesis. The evidence for this hypothesis and its wider implications are discussed.

The accumulation of O6-methylguanine in the liver and kidney DNA of rats treated with dimethylnitrosamine for a short or a long period

A large dose of dimethylnitrosamine was administered to rats by two different dosing regimens, one being eleven intraperitoneal injections of 5 mg/kg body wt. at 12-h intervals (a dosing regimen strongly carcinogenic for the kidney but not the liver), and the other being a continuous dosing over several weeks by adding 8.5 mg of dimethylnitrosamine to each litre of drinking water giving an approximate daily dose of 0.7 mg/kg body wt. This treatment is known to be strongly carcinogenic for the liver but not the kidney. The accumulation in DNA of liver and kidney of the methylated purines 7-methylguanine and O6-methylguanine under each regimen were measured and compared. With the eleven-injection regimen there was a build up of O6-methylguanine in the DNA of the susceptible organ, the kidney, whilst in the liver virtually no accumulation was detected. Under the prolonged, low concentration regimen the liver, in spite of its susceptibility to the carcinogen did not accumulate o6-methylguanine. The results are discussed in terms of the hypothesis that production of o6-methylguanine and its persistence in the DNA of the target organ are responsible for the carcinogenic action of dimethylnitrosamine.

Metabolism of O6-alkyldeoxyguanosines and their effect on removal of O6-methylguanine from rat liver DNA.

O6-Methyldeoxyguanosine and O6-ethyldeoxyguanosine are weak inhibitors (of approximately equal potency) of the removal of O6-methylguanine from methylated DNA by a rat liver enzyme in vitro. When administered to rats, O6-ethyldeoxyguanosine retarded the removal from liver DNA of the O6-methylguanine which had been produced by pretreatment with dimethylnitrosamine, but the effect was short lived. O6-Methyldeoxyguanosine was much less effective. When cells in culture were grown in a medium containing radioactive O6-methylguanine or O6-methyldeoxyguanosine there was negligible incorporation of the methylated base into DNA, but substantial conversion to guanine which was incorporated. When these substances were injected into rats after partial hepatectomy, a very small incorporation of O6-methylguanine into DNA apparently occurred. Both O6-ethyldeoxyguanosine and O6-methyldeoxyguanosine were dealkylated by rat liver extracts, but the methylated derivative was metabolized much more rapidly. O6-Methylguanosine and O6-ethylguanosine were also dealkylated by rat liver extracts, but the corresponding bases were not attacked. This reaction was probably carried out by the adenosine deaminase in the extracts because it could be prevented by addition of erythro-9-(2-hydroxy-3-nonyl)adenine, a potent adenosine deaminase inhibitor, and could also be effected by purified calf intestinal adenosine deaminase. The Km for the demethylation of O6-methyldeoxyguanosine by calf intestinal adenosine deaminase was comparable to that for adenosine, whereas the Km for O6-ethyldeoxyguanosine was ten times greater. The V for O6-methyldeoxyguanosine was about 11% that for adenosine, but that for O6-ethyldeoxyguanosine was only 0.3%. The higher Km and the slower V for O6-ethyldeoxyguanosine may contribute to the slower dealkylation of this nucleoside by liver extracts and could account for its greater effect on slowing O6-methylguanine excision from DNA in vivo.

The persistence in Xenopus laevis DNA of O6-methylguanine produced by exposure to N-methyl-N-nitrosourea

Abstract Xenopus laevis (the South African clawed toad) appears to be insensitive to the carcinogenic action of the alkylation agent N -methyl- N -nitrosourea (NMU), which induces tumours in many tissues of the rat, mouse, and other mammals. The amounts of N 7 -methylguanine and O 6 -methylguanine produced in Xenopus liver and kidney DNA by NMU injection were measured; since NMU is most effective as a carcinogen in rat tissues which cannot remove the O 6 -methylguanine from their DNA, the persistence of these methylated purines in the Xenopus DNA was also studied. In Xenopus liver and kidney DNA the amounts of N 7 -methylguanine and O 6 -methylguanine found 5 hr after injection were comparable with the amounts found in rats after a carcinogenic dose of NMU. There was no evidence of loss of N 7 -methylguanine or excision of O 6 -methylguanine from either Xenopus liver or kidney DNA: there were no marked differences between the amounts of these purines present in liver and kidney DNA at 5 hr and at 72 hr after NMU injection.

The Possible Role of Nitrosamines in the Link between Alcohol Consumption and Esophageal Cancer in Man

Ethanol in amounts equivalent to a man drinking a pint of beer has a dramatic effect on the metabolism and distribution of nitrosamines in rats. It prevents the first pass clearance of dimethylnitrosamine and thus exposes the extrahepatic organs to oral doses of this carcinogen. By selectively inhibiting metabolism in liver and kidney, ethanol increases the amount of diethylnitrosamine activated in the esophagus between 1.8- and 4.6-fold. It is suggested that there may be a link between these observations and the increase in human esophageal cancer which is associated with alcohol consumption.

Clearance of N-nitrosodimethylamine and N-nitrosodiethylamine by the perfused rat liver: Relationship to the Km, and Vmax for nitrosamine metabolism

The first-pass clearance of dietary N-nitrosodimethylamine (NDMA) by the liver is the most important factor in the pharmacokinetics of this carcinogen in the rat, but is less important in the pharmacokinetics of N-nitrosodiethylamine (NDEA). The reason for the difference in clearance of these two nitrosamines is not known. These experiments were carried out to see whether the general characteristics of the clearance of these two carcinogens in vivo could be reproduced in the perfused liver, and whether the clearance could be correlated with the Michaelis-Menten parameters Km and Vmax for their metabolism. If this could be done one would be able to predict the possible extent of first-pass clearance of nitrosamines in man from measurement of Km and Vmax for nitrosamine metabolism by the human liver. The Km (22 microM) and Vmax (10.2 and 13.4 nmol/g liver/min) for the metabolism of NDMA by slices from two human livers, the inhibition of that metabolism by ethanol (Ki 0.5 microM), and the rate of N-7 methylation of DNA when slices are incubated with NDMA, were measured. These results are similar to those reported previously with rat liver. The Km (27 microM) for the metabolism of NDEA by rat liver slices and the inhibition of that metabolism by ethanol (Ki 1 microM) were estimated from the rate of ethylation of the DNA of the slices. The clearance of both these nitrosamines by the perfused rat liver was measured, and the results appeared to parallel those in vivo with a striking difference between the clearance of NDMA and NDEA. The maximal rate of clearance of NDMA was 11.2 nmol/g liver/min and of NDEA 8.9 nmol/g liver/min, similar to the Vmax for metabolism of NDMA by liver slices and to the estimated maximal rate of liver metabolism of both nitrosamines in the living rat. However, although the Km for metabolism of these two nitrosamines by liver slices is similar (about 25 microM), the logarithmic mean sinusoidal concentration [see Bass and Keiding, Biochem Pharmacol 37: 1425-1431, 1988] giving half maximal clearance during perfusion (the equivalent to Km) was 2.3 microM for NDMA and 10.6 microM for NDEA. The almost 5-fold difference between these two values is the basis for the difference between the clearance of the two nitrosamines.(ABSTRACT TRUNCATED AT 400 WORDS)