The cytoprotective aminothiol WR1065 activates p21waf-1 and down regulates cell cycle progression through a p53-dependent pathway.

The phosphoaminothiol WR1065, the active metabolite of the pro-drug amifostine (WR2721), protects cultured cells and tissues against cytotoxic exposure to radiation or chemotherapeutic agents. We show here that WR1065 and the pro-drug WR2721 activate the p53 tumor suppressor protein and induce the expression of the cyclin-dependent kinase inhibitor p21waf-1 in the breast cancer cell line MCF-7, and in the mouse fibroblast cell line balb/c 3T3. Using two MCF-7 derived cell lines, MN1 and MDD2, we show that induction of p21waf-1 is detectable in MN1 (expressing a functional p53) but not in MDD2 (p53 disabled). These effects are observed at concentrations of WR1065 (0.5 to 1 mM) identical to those required to protect against cytotoxicity by hydrogen peroxide. Induction of p53 is not prevented by addition of aminoguanidine, an inhibitor of Cu-dependent amine-oxidases which blocks the extra-cellular degradation of WR1065 into toxic metabolites. Moreover, spermidine, a natural polyamine structurally related to amifostine, does not activate p53. Induction of p53 by WR1065 results in a delay in the G1/S transition in MCF-7 and MN-1 cells, but not in the p53 disabled cells MDD2. These data indicate that WR1065, a polyamine analog with thiol anti-oxidant properties, activates a cell cycle check-point involving p53.

Metabolic activation of vinyl chloride by rat liver microsomes: low-dose kinetics and involvement of cytochrome P450 2E1.

The metabolism and pharmacokinetics of vinyl chloride (VC) have been extensively studied in rodents and humans, but the maximum velocity (Vmax) and Michaelis constant (K(m)) for the activation of VC by microsomal monooxygenases in vitro have not yet been determined. Using a new sensitive assay, the epoxidation of VC by rat liver microsomes (adult Sprague-Dawley) at concentrations from 1 ppm to 10(6) ppm in the gas phase was measured. In the assay, the reactive VC metabolites chloroethylene oxide and 2-chloroacetaldehyde were trapped with excess cAMP, yielding, 1,N6-etheno-cAMP (epsilon cAMP) which was quantitated by HPLC fluorimetry. The trapping efficiency of electrophilic VC metabolites by cAMP was close to 10%. The specificity of the method was confirmed by purification of epsilon cAMP on an immunogel. The VC concentration in the gas phase was measured by GC/flame ionization detection, while in the aqueous phase it was calculated from the partition coefficient between air and the microsomal suspension. Activation of VC by rat liver microsomes followed Michaelis-Menten kinetics with K(m) = 7.42 +/- 0.37 (+/- SD) microM and Vmax = 4674 +/- 46 pmol.mg protein-1.min-1. Inhibitor studies and immunoinhibition assays showed that VC was activated by cytochrome P450 (CYP) 2E1 down to 1 ppm in the air phase. Based on the metabolic parameters determined, the uptake of VC by rats in vivo can be accurately predicted.

Formation and accumulation of DNA ethenobases in adult Sprague-Dawley rats exposed to vinyl chloride.

DNA ethenobases are promutagenic lesions formed by carcinogens such as vinyl chloride (VC). Their formation was investigated in 6-week old, male Sprague-Dawley rats exposed to 500 p.p.m. VC by inhalation (4 h/day, 5 days/ week) for 1, 2, 4 or 8 weeks and in 7- and 14-week old, matched control animals. 1,N6-Ethenoadenine (epsilon A) and 3, N4-ethenocytosine (epsilon C) deoxyribonucleotides were analysed by immunoaffinity purification and 32P-postlabelling. This postlabelling method was compared with a radio-immunoassay method, which yielded similar results. Background levels of ethenobases were found in DNA from the liver, lungs, kidneys and circulating lymphocytes of unexposed, control rats. In the liver, the following background molar ratios of ethenobase to parent base in DNA were detected (mean values x 10(-8)): epsilon A/A, 0.04-0.05; epsilon C/C, 0.06-0.07. In the lungs, kidneys and circulating lymphocytes, background levels of epsilon A and epsilon C ranged from 1.7 to 4.2 x 10(-8) and from 4.8 to 11.2 x 10(-8), respectively. Following a 5-day exposure to VC, a significant increase of epsilon A and epsilon C was measured in hepatic DNA from rats sacrificed immediately after treatment. Further, a dose-dependent increase of both etheno adducts was observed in liver DNA of VC-treated rats. Compared to the 5-day exposure, approximately 4-fold higher levels of epsilon A and epsilon C were observed in the liver of animals after 8 weeks of exposure. In contrast, there was an accumulation of epsilon C but not of epsilon A in lungs and kidneys. In circulating lymphocytes, no significant increase of ethenobase levels above control values was observed after 2 months of exposure to VC. Both etheno adducts were found to be persistent in liver DNA, after 2 months following the termination of VC exposure. These results further support the notion that DNA etheno-bases are critical lesions in VC-induced carcinogenesis. The possible contribution of lipid peroxidation products that also yield ethenobases, on the formation and persistence of these DNA adducts, remains to be clarified.

Formation of 1,N6-ethenoadenine and 3,N4-ethenocytosine by lipid peroxidation products and nucleic acid bases.

Lipid peroxidation (LPO) products are known to interact with DNA, yielding several types of adduct with nucleobases. In this study, we demonstrate the formation of two ethenobase adducts, 1,N6-ethenoadenine and 3,N4-ethenocytosine, by reaction of LPO products with nucleic acid bases. Rat liver microsomes were incubated at 37 degrees C for 30 min in the presence of inducers of LPO [Fe(II) or cumene hydroperoxide] and adenine or cytosine nucleotides or nucleosides, followed by further heating at 80 degrees C for 30 min to complete the reactions. The etheno adducts detected after immunoaffinity chromatography were 1,N6-etheno-cAMP and 1,N6-etheno-2'-deoxyadenosine (HPLC/fluorimetry), 3,N4-etheno-2'-deoxycytidine (competitive radioimmunoassay), and 1,N6-etheno-2'-deoxyadenosine 3'-monophosphate and 3,N4-etheno-2'-deoxycytidine 3'-monophosphate (32P-postlabeling). Incubation of arachidonic acid supplemented with Fe(II) also led to the formation of the 1,N6-etheno adduct from cAMP. LPO intermediates that may be involved are discussed. These data suggest that etheno adducts may be markers of DNA damage associated with LPO.

Dietary fibre, fat and beef modulation of colonic nuclear aberrations and microcapsule-trapped gastrointestinal metabolites of benzo[a]pyrene-treated C57/B6 mice consuming human diets.

Male C57/B6 mice were adapted to human diets of British origin that had 3-fold differences in either dietary fibre, fat or beef protein within the normal human range, and were then treated p.o. with 200 mg/kg benzo[a]pyrene (B[a]P) to induce colonic nuclear aberrations. [14C]B[a]P was included in the dose that followed 2 h after a gavage of magnetic PEI microcapsules. Untreated control groups were fed mouse chow or the baseline human diet which was low in all three dietary components (LLL). After the animals were killed at 24 h, large reductions (P less than 0.05) in colonic nuclear aberrations, and alterations to faecally excreted, microcapsule-trapped B[a]P metabolites were found for elevations of all three human diet components. Compared to untreated LLL control, B[a]P treatment gave an 8-fold increase in total nuclear aberrations, which was decreased 2- to 3-fold by increased fibre or fat. HPLC assay of B[a]P metabolites desorbed from microcapsules showed dietary fibre and beef protein to increase B[a]P diols and phenols but almost abolish B[a]P diones, consistent with a shift to enzymatic metabolism from non-specific oxidation. Increased fat considerably altered B[a]P metabolite disposition and microcapsule trapping, and comparison with microcapsules removed from colon contents indicated an altered enterohepatic circulation. Although it was not possible to attribute nuclear aberrations to individual B[a]P metabolites, a possible role of B[a]P diones seemed indicated, this being in line with previous microcapsule studies. These results show that microcapsules and human diets can be used in monitoring modulations of xenobiotic agents linked to mucosal chromosomal damage, with the eventual aim of human microcapsule biomonitoring.

Exposure to N-nitrosamines and other risk factors for gastric cancer in Costa Rican children.

The hypothesis that endogenous chemical nitrosation in the normal stomach in early life could play a crucial role in inducing chronic atrophic gastritis/intestinal metaplasia in later life was tested by applying the N-nitrosoproline (NPRO) test to 12-h urine samples from about 50 children (aged 8-14 years) living in high- and low-risk areas for stomach cancer. The median values of NPRO and the sum of four nitrosamino acids analysed were 0.28-0.84 micrograms/12 h and 0.75-1.75 micrograms/12 h, respectively. The NPRO level after proline intake was significantly higher in children from a high-risk area than in those from a low-risk area (p less than 0.04), and markedly reduced after ingestion of ascorbic acid and proline (p less than 0.05). Urinary nitrate level was lower than that of adults. NPRO levels on the day of proline intake, however, correlated well with nitrate levels (p less than 0.001), indicating that children in a high-risk area in Costa Rica have high endogenous nitrosation potential. Blood samples were also collected from about 300 children (aged 7-20 years) and analysed for antibodies against Campylobacter pylori, a suspected gastritis-causing bacteria. About 71% of children in both high- and low-risk areas for stomach cancer had antibodies. In addition, raw and cooked beans, which are consumed very frequently in Costa Rica, were collected from families in both areas and analysed for levels of nitrite/nitrate, total N-nitroso compounds and genotoxicity in the SOS chromotest.(ABSTRACT TRUNCATED AT 250 WORDS)