Biomedical subjects

J P Plastaras

Publications and source records attributed to J P Plastaras.

7 recordsLinked to original sources

PubMed2001

Endogenous DNA damage and mutation.

In humans, approximately 10(7) cells divide per second. Estimates suggest that spontaneous mutations arise in about a third of those cells. These mutations arise as mistakes in DNA replication and when DNA polymerases copy damaged templates. The latter result from chemical hydrolysis of nucleoside bases or by reaction of DNA with electrophiles or reactive free radicals generated during metabolism (endogenous DNA damaging agents). This article highlights recent discoveries and emerging opportunities in the study of endogenous DNA damage and mutation.

Animals↗

PubMed2001

Cyclooxygenase-2, malondialdehyde and pyrimidopurinone adducts of deoxyguanosine in human colon cells.

Cyclooxygenases (COX) catalyse the oxygenation of arachidonic acid to prostaglandin (PG) endoperoxides. Activity of one of the COX isoforms, COX-2, results in production of prostaglandin E(2) (PGE(2)) via the endoperoxide PGH(2). COX-2 has been implicated in the pathogenesis of colorectal cancer. Malondialdehyde (MDA) is a mutagen produced by spontaneous and enzymatic breakdown of PGH(2). MDA reacts with DNA to form adducts, predominantly the pyrimidopurinone adduct of deoxyguanosine (M(1)G). Here the hypothesis was tested that COX-2 activity in human colon cells results in formation of MDA and generation of M(1)G adducts. M(1)G was detected in basal cultures of human non-malignant colon epithelial (HCEC) and malignant SW48, SW480, HT29 and HCA-7 colon cells, at levels from 77 to 148 adducts/10(8) nucleotides. Only HCA-7 and HT29 cells expressed COX-2 protein. Levels of M(1)G correlated significantly (r = 0.98, P < 0.001) with those of intracellular MDA determined colorimetrically in the four malignant cell types, but neither parameter correlated with expression of COX-2 or PG biosynthesis. Induction of COX-2 expression by phorbol 12-myristate 13-acetate in HCEC cells increased PGE(2) production 20-fold and MDA concentration 3-fold. Selective inhibition of COX-2 activity in HCA-7 cells by NS-398 significantly inhibited PGE(2) production, but altered neither MDA nor M(1)G levels. Malondialdehyde treatment of HCEC cells resulted in a doubling of M(1)G levels. These results show for the first time in human colon cells that COX-2 activity is associated with formation of the endogenous mutagen, MDA. Moreover, they demonstrate the correlation between MDA concentration and M(1)G adduct levels in malignant cells.

Colon↗

PubMed2000

Xenobiotic-metabolizing cytochromes P450 convert prostaglandin endoperoxide to hydroxyheptadecatrienoic acid and the mutagen, malondialdehyde.

Cyclooxygenases catalyze the oxygenation of arachidonic acid to prostaglandin endoperoxides. Cyclooxygenase-2- and the xenobiotic-metabolizing cytochrome P450s 1A and 3A are all aberrantly expressed during colorectal carcinogenesis. To probe for a role of P450s in prostaglandin endoperoxide metabolism, we studied the 12-hydroxyheptadecatrienoate (HHT)/malondialdehyde (MDA) synthase activity of human liver microsomes and purified P450s. We found that human liver microsomes have HHT/MDA synthase activity that is concentration-dependent and inhibited by the P450 inhibitors, ketoconazole and clotrimazole with IC(50) values of 1 and 0.4 microM, respectively. This activity does not require P450 reductase. HHT/MDA synthase activity was present in purified P450s but not in heme alone or other heme proteins. The catalytic activities of various purified P450s were determined by measuring rates of MDA production from prostaglandin endoperoxide. At 50 microM substrate, the catalytic activities of purified human P450s varied from 10 +/- 1 to 0.62 +/- 0.02 min(-1), 3A4 >> 2E1 > 1A2. Oxabicycloheptane analogs of prostaglandin endoperoxide, U-44069 and U-46619, induced spectral changes in human P450 3A4 with K(s) values of 240 +/- 20 and 130 +/- 10 microM, respectively. These results suggest that co-expression of cyclooxygenase-2 and P450s in developing cancers may contribute to genomic instability due to production of the endogenous mutagen, MDA.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗

PubMed2000

Reactivity and mutagenicity of endogenous DNA oxopropenylating agents: base propenals, malondialdehyde, and N(epsilon)-oxopropenyllysine.

Malondialdehyde (MDA), a mutagenic product of lipid peroxidation, reacts with DNA to form the premutagenic lesion, pyrimido[1, 2-a]purin-10(3H)-one (M(1)G). M(1)G is present in normal human tissues, but the contribution of other endogenously produced MDA analogues is poorly understood. Oxidation of the DNA backbone can cause strand breaks and release base propenals, and MDA condensation with proteins yields N(epsilon)-oxopropenyllysine. Here we compare the M(1)G-forming ability and Salmonella typhimurium mutagenicity of MDA with adenine, thymine, and cytosine propenals and N(alpha)-acetyl-N(epsilon)-oxopropenyllysine methyl ester. Base propenals are 30-150 times more potent than MDA in M(1)G formation and are 30-60 times more mutagenic than MDA. In addition, the Fe-bleomycin complex, which generates base propenals, induced M(1)G, but gamma-radiation, which generates mostly MDA, did not. M(1)G formation by MDA and base propenals was concentration-dependent, time-dependent, and enhanced by acidic conditions. N(alpha)-Acetyl-N(epsilon)-oxopropenyllysine methyl ester was less reactive and less mutagenic than MDA. These differences in potency are consistent with differences in leaving group ability. This work supports a role for other MDA analogues, especially base propenals, in the formation of endogenous M(1)G adducts.

Animals↗

PubMed1999

A sensitive immunoslot-blot assay for detection of malondialdehyde-deoxyguanosine in human DNA.

As part of a large programme on food risk assessment, we have become Interested in the endogenous production of genotoxic agents from dietary precursors. Malondialdehyde (MDA), a product of lipid peroxidation and prostaglandin biosynthesis, is mutagenic in bacterial and mammalian systems. MDA reacts with DNA, and the major adduct (M1-dG) has been detected in healthy human liver and leukocyte DNA. Analytical methods used so far for the detection of M1-dG have not been applied to large numbers of individuals or a large variety of samples. Often, only a few micrograms of DNA from human tissues are available for analysis, and a very sensitive assay is needed to detect background levels of M1-dG in very small amounts of DNA. In this paper, we describe the development of an immunoslot-blot (ISB) assay for the measurement of M1-dG in 1 microgram of DNA. The limit of detection of the assay is about 5 adducts per 10(8) bases. The advantages of ISB over other assays for DNA adduct detection, such as the possibility of analysing 1 microgram DNA per sample and the fact that it is less time-consuming and laborious, mean that it can be more easily used for routine analysis of large numbers of samples in biomonitoring. A series of human samples was analysed, and levels of 0.3-6.43 M1-dG per 10(7) normal bases were detected in 42 gastric biopsy samples and 0.7-16.65 M1-dG per 10(7) normal bases in 28 samples of leukocyte DNA. In an initial study in five human volunteers on standardized diets, the levels of M1-dG in leukocyte DNA changed in relation to meat, vegetable and tea intake.

Biomarkers↗

PubMed1998

Indirect mutagenesis by oxidative DNA damage: formation of the pyrimidopurinone adduct of deoxyguanosine by base propenal.

Oxidation of endogenous macromolecules can generate electrophiles capable of forming mutagenic adducts in DNA. The lipid peroxidation product malondialdehyde, for example, reacts with DNA to form M1G, the mutagenic pyrimidopurinone adduct of deoxyguanosine. In addition to free radical attack of lipids, DNA is also continuously subjected to oxidative damage. Among the products of oxidative DNA damage are base propenals. We hypothesized that these structural analogs of malondialdehyde would react with DNA to form M1G. Consistent with this hypothesis, we detected a dose-dependent increase in M1G in DNA treated with calicheamicin and bleomycin, oxidizing agents known to produce base propenal. The hypothesis was proven when we determined that 9-(3-oxoprop-1-enyl)adenine gives rise to the M1G adduct with greater efficiency than malondialdehyde itself. The reactivity of base propenals to form M1G and their presence in the target DNA suggest that base propenals derived from oxidative DNA damage may contribute to the mutagenic burden of a cell.

Adenine↗

PubMed1998

Determination of malondialdehyde-induced DNA damage in human tissues using an immunoslot blot assay.

Malondialdehyde (MDA) is a product of lipid peroxidation and prostaglandin biosynthesis. It is mutagenic and carcinogenic and the major adduct formed by reaction with DNA, a highly fluorescent pyrimidopurinone (M1-dG), has been detected in healthy human liver and leukocyte DNA. Analytical methods used so far for the detection of M1-dG have not been applied to a large number of individuals or variety of samples. Often, only a few microg of DNA from human tissues are available for analysis and a very sensitive assay is needed in order to detect background levels of M1-dG in very small amounts of DNA. In this paper, the development of an immunoslot blot (ISB) assay for the measurement of MI-dG in 1 microg of DNA is described. The limit of detection of the assay is 2.5 adducts per 10(8) bases. A series of human samples were analysed and levels of 5.6-9.5 (n = 8) and 3.1-64.3 (n = 42) of M1-dG per 10(8) normal bases were detected in white blood cell and gastric biopsy DNA, respectively. Results on four human samples were compared with those obtained using an HPLC/32P-post-labelling (HPLC/PPL) method previously developed and indicated a high correlation between M1-dG levels measured by the two assays. The advantages of ISB over other assays including HPLC/PPL, such as the possibility of analysing 1 microg DNA/sample and the fact that it is less time-consuming and laborious, means that it can be more easily used for routine analysis of a large number of samples in biomonitoring studies.

Animals↗