Irbesartan, an angiotensin type 1 receptor inhibitor, regulates the vascular oxidative state in patients with coronary artery disease.

OBJECTIVES: The aim of this study was to determine the effect of angiotensin II type 1 (AT(1)) receptor antagonists on pro-oxidant species observed in the pathogenesis of atherosclerosis. Parameters such as low-density lipoprotein (LDL) susceptibility, monocyte binding capacity, superoxide generation and lipid peroxidation were examined in the presence of the AT(1) receptor antagonist irbesartan. BACKGROUND: Low-density lipoprotein oxidation is a key component in the process of atherogenesis. This modification may involve various mechanisms, including changes in nitric oxide levels and superoxide levels. Additionally, compounds that suppress these mechanisms may retard or inhibit the pathogenesis of atherosclerosis. METHODS: Forty-seven patients with documented coronary artery disease were treated with irbesartan for a 12-week period. Patients were randomized to receive irbesartan or placebo. Lipid peroxidation, superoxide levels, monocyte binding and LDL oxidation were measured at 0, 4 and 12 weeks. Findings were statistically evaluated by two-way repeated measures analysis of variance with p < 0.05 being significant. RESULTS: Treatment with irbesartan significantly decreased the pro-oxidative environment seen in our study population. Lag time for LDL oxidation increased 32% at 12 weeks, suggesting an increased resistance of LDL modification in the serum. Thiobarbituric acid reactive substances activity indicated that lipid peroxidation decreased by 36% in comparison to placebo. In addition, superoxide levels and monocyte-binding capacity were also significantly reduced in coronary artery disease patients receiving irbesartan. CONCLUSIONS: Our results indicate that irbesartan may suppress the atherosclerotic process by inhibiting the intravascular oxidative state and the production of reactive oxygen species, compounds that may cause damage to the vasculature.

Hydrocarbon carcinogens induce p53 activity in normal mouse tissue.

Previous studies led to the suggestion that a sub class of hydrocarbon carcinogens have the capacity of damage DNA and evade normal cellular defence mechanisms. To examine the ability of hydrocarbon carcinogens to induce p53 transcriptional activity in normal tissues, we have used transgenic mice in which LacZ transgene is driven by a p53 response element. We demonstrate that transcriptionally active p53 is induced in normal tissue after exposure to putative stealth carcinogens in an identical manner to non-stealth agents.

Hydrocarbon carcinogens evade cellular defense mechanism of G1 arrest in nontransformed and malignant lung cell lines.

In previous studies using human breast carcinoma cells (MCF-7) and human colon carcinoma cells (RKO) we have shown that, in response to treatment with hydrocarbon carcinogens, these cell lines failed to undergo a p53-mediated cell cycle arrest in G1 phase; rather, the cells were accumulated in the S phase with damaged DNA, a situation that may lead to replication of DNA on a damaged template, resulting in the enhanced frequency of mutations in the daughter cells. This has been termed a stealth effect. In the present work we have demonstrated that the stealth effect also pertains for lung cells. In E10 nontransformed mouse lung type II cells, two potent hydrocarbon carcinogens, benzo[a]pyrene dihydrodiol epoxide and benzo[g]chrysene dihydrodiol epoxide, damaged DNA as suggested by retardation in S phase, but did not cause G1 arrest, in contrast to the positive control, actinomycin D. Human lung adenocarcinoma A549 cells, with normal p53, likewise exhibited G1 arrest after actinomycin D, but not after treatment with the diol epoxides. Several human lung cancer cell lines with absent or mutant p53, such as H358, H1734, and H82, exhibited no G1 arrest after any of the compounds. However, lung H441 adenocarcinoma cells, with a mutation in exon 5, codon 158 of p53, exhibited partial G1 arrest after the diol epoxides as well as actinomycin D, and H2030 adenocarcinoma cells did not show G1 arrest after any of the chemicals despite a normal p53. The stealth effect of evasion of G1 arrest may contribute to initiation of lung adenocarcinomas and to progression of tumors. A role in resistance to chemotherapy by certain drugs is also likely.

Diverse chemical carcinogens fail to induce G(1) arrest in MCF-7 cells.

The effect of three reactive potent chemical carcinogens on the passage of MCF-7 cells through the cell cycle was investigated. While these cells, which express wild-type p53, were arrested in G(1) after treatment with actinomycin D (a positive control), treatment with anti-benzo[a]pyrene dihydrodiol epoxide, N-acetoxy-N-2-fluorenylacetamide or N-methyl-N'-nitro-N-nitrosoguanidine, at doses consistent with survival of significant numbers of cells, caused the cells to accumulate in S phase, with little increase in those in G(1). This property of these three reactive potent carcinogens, of diverse chemical types, to induce evasion of G(1) arrest (the stealth property) presumably increases the likelihood of malignant change, because DNA replication continues on a damaged template. This stealth characteristic may be a major contributor to the tumorigenicity of DNA-adducting chemical carcinogens in general.

Lack of p53-mediated G1 arrest in response to an environmental carcinogen.

The environmental carcinogen, 5-methylchrysene, is a component of cigarette smoke. Its reactive metabolite, anti-5-methylchrysene-1, 2-dihydrodiol-3,4-epoxide (5-MeCDE) mainly reacts with the N(2)-position of guanine residues in the DNA molecule. In this study, we demonstrate that the tumor suppressor protein p53 is stabilized in response to DNA damage by 5-MeCDE but fails to induce the cells' protective mechanism of G1 arrest in the human breast carcinoma cell line, MCF-7. In contrast, actinomycin D treatment of these cells did lead to G1 arrest. Western analyses revealed that, though both actinomycin D and 5-MeCDE treatment stabilized p53, only trace levels of p21(waf1/cip1) were seen in the latter case. This lack of p21(waf1/cip1) expression in 5-MeCDE-treated cells is attributed to a stealth characteristic of this environmental carcinogen that allows it to damage DNA and still escape the p53-mediated cellular defense mechanism of G1 arrest.

DNA reactions, mutagenic action and stealth properties of polycyclic aromatic hydrocarbon carcinogens (review).

A brief summary of recent research, primarily from the authors' laboratory, on polycyclic aromatic hydrocarbon carcinogens with respect to their DNA adduct formation, the mutational properties of these adducts and the effects of hydrocarbon dihydrodiol epoxide metabolites on the passage of cells through the cell cycle is presented. The concept of stealth properties of potent carcinogens, i.e. their ability to damage DNA without inducing a G1 arrest, is discussed. Also, mutation studies with dihydrodiol epoxide metabolites, the sequence-dependence of site-specific mutation, as well as the selectivity of hydrocarbon-DNA adduct formation are summarized.

DNA adduct levels associated with p53 induction and delay of MCF-7 cells in S phase after exposure to benzo[g]chrysene dihydrodiol epoxide enantiomers.

Optically active isomers of a mammary carcinogen, anti-benzo[g]chrysene 11, 12-dihydrodiol 13, 14-epoxide, react to different extents with DNA and generate DNA adducts that differ in their stereochemistry. In the study reported here, the effect of these two enantiomers on the progress of human breast carcinoma MCF-7 cells through the cell cycle was investigated. Each enantiomer caused the cells to accumulate in the S phase, but a higher dose of the benzo[g]chrysene 11S, 12R-dihydrodiol 13R, 14S-epoxide than of its enantiomer was required to induce this effect. Similarly, induction of p53 also required a higher dose of benzo[g]chrysene 11S, 12R-dihydrodiol 13R, 14S-epoxide. Postlabeling studies indicated that the latter enantiomer also caused less modification of MCF-7 cell DNA for a given level of exposure than did benzo[g]chrysene 11R, 12S-dihydrodiol 13S, 14R-epoxide. These results suggest that p53 induction and delay in the S phase are similarly related to DNA binding and that a level of binding of the order of 1 adduct per 10(5) nucleotides is associated with these effects.

Cellular response to DNA damage from a potent carcinogen involves stabilization of p53 without induction of p21(waf1/cip1).

The effect of a potent mammary carcinogen, anti benzo[g]chrysene 11,12-dihydrodiol 13,14-epoxide, on the progress of human mammary carcinoma MCF-7 cells through the cell cycle was investigated. While these cells, which express wild-type p53, were arrested in G1 after treatment with actinomycin D (a positive control), treatment with the mammary carcinogen did not cause G1 arrest but instead delayed the cells in the DNA synthesis phase. In concert with the absence of a G1 arrest, it was found that though both chemical treatments led to increased levels of p53, only the p53 induced by actinomycin D was transcriptionally active and increased the levels of the cyclin dependent kinase inhibitor, p21(waf1/cip1). Since treatment of the cells with the mammary carcinogen did not abrogate the G1 arrest induced by actinomycin D, the lack of p21(waf1/cip1) and of G1 arrest, resulting from treatment with the mammary carcinogen alone, was not due to some general inhibition of transcription or translation. An analogous difference between these two chemicals was demonstrated also in other human cell systems. The stealth-like property of the mammary carcinogen that allows it to damage DNA without turning on the cells' 'guardian of the genome' defense mechanism presumably increases the likelihood of malignant change because DNA replication continues on a damaged template. It is suggested that this stealth characteristic may be a major contributor to the high carcinogenic potency of this mammary carcinogen and possibly to that of other highly potent carcinogens.

Mutagenicity of furfural in plasmid DNA.

Furfural is recognized as a dietary mutagen and is present in various foods and beverages. We have examined the mutagenic effect of furfural induced lesions in plasmid pBluescript SK(+/-). There was a progressive decrease in the transformation capacity of the plasmid as a function of furfural concentration with a concomitant increase in the number of mutant plasmids. Several mutant plasmids with reduced transformation capacity and a molecular size similar to the parental plasmid were isolated. A stretch of DNA of 108 basepairs within the multiple cloning region was sequenced. It was observed that the number of mutagenic events in the case of furfural damaged plasmid was not significantly greater than in spontaneously arisen mutants. These results were interpreted to indicate that furfural mediated DNA damage is efficiently repaired.

Inactivation and repair of bacteriophage lambda by furfural.

Furfural is a dietary mutagen and is present in various frequently consumed food products. In earlier work we have shown that it induces single strand breaks in duplex DNA which occur preferentially in AT base pairs. Experiments on the conservation of EcoRI cleavage site in mutant plasmids further established that repair of furfural damaged plasmid DNA occurs on propagation in the host cells. In this paper it is shown that the strand scissions induced by furfural in DNA account for its biological activity as assayed by inactivation of bacteriophage lambda. Using various repair defective mutants of E. coli we have shown that DNA repair predominantly involves the polA pathway.

Effect of furfural on plasmid DNA.

Furfural is a dietary mutagen and is present in various food products and beverages. We have previously shown that it induces single strand breaks in double stranded DNA that occur mainly in AT sequences. In this paper, we have examined the mutagenic effect of furfural induced lesions in the plasmid pBR322. Plasmid DNA was treated with furfural before transformation in E. coli. There was a progressive decrease in the transformation capacity of the plasmid both as a function of furfural concentration and time of reaction. Several mutants were isolated from plasmid preparations whose transforming capacity had been decreased by treatment with furfural. Depending on the concentration of furfural used, these were found to have undergone DNA alterations including deletions. Experiments on the conservation of EcoRI cleavage site in mutant plasmids indicated that repair of furfural damaged plasmid DNA takes place on propagation in host cells.

Toxoplasmosis among pregnant women in northern parts of Pakistan.

Three hundred and twenty four serum samples, collected from different areas of Punjab, NWFP and Azad Kashmir, were screened for Toxo IgG and IgM antibody titres utilizing the Enzyme linked immunosorbent assay (ELISA). Of the total 149 (46%) samples were positive for IgG antibody. Seven (2.1%) showed titre greater than 130. Ninety sera (27.7%) were positive for IgM antibody, twenty six (8%) had a titre of more than 40 and the uncertain positive percentage was 19.7%. Infection rate was highest in Punjab (63%) followed by Azad Kashmir (48%) and NWFP (38%).