Comparison of iron-catalyzed DNA and lipid oxidation.

Lipid and DNA oxidation catalyzed by iron(II) were compared in HEPES and phosphate buffers. Lipid peroxidation was examined in a sensitive liposome system constructed with a fluorescent probe that allowed us to examine the effects of both low and high iron concentrations. With liposomes made from synthetic 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine or from rat liver microsomal lipid, lipid peroxidation increased with iron concentration up to the range of 10--20 microM iron(II), but then rates decreased with further increases in iron concentration. This may be due to the limited amount of lipid peroxides available in liposomes for oxidation of iron(II) to generate equimolar iron(III), which is thought to be important for the initation of lipid peroxidation. Addition of hydrogen peroxide to incubations with 1--10 microM iron(II) decreased rates of lipid peroxidation, whereas addition of hydrogen peroxide to incubations with higher iron concentrations increased rates of lipid peroxidation. Thus, in this liposome system, sufficient peroxide from either within the lipid or from exogenous sources must be present to generate equimolar iron(II) and iron(III). With iron-catalyzed DNA oxidation, hydrogen peroxide always stimulated product formation. Phosphate buffer, which chelates iron but still allows for generation of hydroxyl radicals, inhibited lipid peroxidation but not DNA oxidation. HEPES buffer, which scavenges hydroxyl radicals, inhibited DNA oxidation, whereas lipid peroxidation was unaffected since presumably iron(II) and iron(III) were still available for reaction with liposomes in HEPES buffer.

Apoptosis induction by a novel anti-prostate cancer compound, BMD188 (a fatty acid-containing hydroxamic acid), requires the mitochondrial respiratory chain.

We recently developed a class of novel anti-prostate cancer compounds, cyclic hydroxamates that elicit a potent apoptotic response in many tumor cells cultured in vitro (D.G. Tang et al., Biochem. Biophys. Res. Commun., 242: 380-384, 1998). The lead compound, termed BMD188, induces programmed cell death in a variety of prostate cancer cells in vitro as well as in vivo (L. Li et al., Anticancer Res., 19: 51-70, 1999). BMD188 kills androgen-independent prostate cancer cells as well as prostate cancer cells with a multidrug-resistance phenotype. The apoptotic effect of BMD188 in prostate cancer cells does not depend on cell cycle, p53 status, or its purported target, arachidonate 12-lipoxygenase, but does require caspase activation and seems to involve mitochondria. To synthesize more specific and effective anti-prostate cancer hydroxamic acid compounds, it is important to understand their mechanism(s) of action. In the present study, we studied the role of mitochondrial respiratory chain (MRC) in BMD188-induced apoptosis in androgen-independent prostate cancer PC3 cells and compared its effect with that of staurosporine (STS), a widely used apoptosis inducer. Several lines of evidence indicate that BMD188-induced cell death depends on MRC: (a) the death could be significantly inhibited by several complex-specific respiration inhibitors; (b) respiration-deficient rho0 cells were more resistant than wild-type parent cells to apoptosis induction by BMD188; and (c) BMD188 induced a rapid increase in reactive oxygen species in mitochondria, an up-regulation of cytochrome c oxidase subunits, a biphasic alteration (i.e., an early hyperpolarization, followed by later hypopolarization) in the mitochondrial membrane potential (delta psi(m)), dramatic changes in mitochondrial morphology and distribution prior to caspase activation, and an abnormal proliferation of mitochondria at the ultrastructural level. By contrast, STS-induced PC3 apoptosis seemed not to depend on MRC. Taken together, the data suggest that the MRC represents a functional target for anti-prostate cancer hydroxamates.

Gene-specific nuclear and mitochondrial repair of formamidopyrimidine DNA glycosylase-sensitive sites in Chinese hamster ovary cells.

This study examines the capacity of a mammalian cell to repair, at the gene level, DNA base lesions generated by photoactivation of acridine orange. Chinese hamster ovary fibroblasts were exposed to acridine orange and visible light, and gene-specific DNA repair was measured in the dihydrofolate reductase (DHFR) gene and in the mitochondrial genome. DNA lesions were recognized by Escherichia coli formamidepyrimidine-DNA glycosylase (FPG) which removes predominantly 8-oxodG and the corresponding formamidopyrimidine ring opened bases, and subsequently cleaves the DNA at the resulting apurinic site. FPG-recognized DNA lesions increased linearly with increasing photo-activation of AO, while cell survival was not affected by light alone and was negligibly affected by preincubation with AO in the dark. The frequency of induction of FPG-sensitive DNA damage by photoactivation of AO was similar in the transcribed and non-transcribed nuclear DNA as well as in the mitochondrial DNA. FPG-sensitive sites in the DHFR gene were repaired quickly, with 84% of adducts repaired within 4 h. The lesion frequency, kinetics and percent of repair of non-transcribed genomic DNA did not differ significantly from repair in the active DHFR gene up to 1 h postexposure. At late time points, transcribed DNA was repaired faster than the non-transcribed DNA. Mitochondrial DNA was efficiently repaired, at a rate similar to that in the active nuclear DNA.

p53 modulation of TFIIH-associated nucleotide excision repair activity.

p53 has pleiotropic functions including control of genomic plasticity and integrity. Here we report that p53 can bind to several transcription factor IIH-associated factors, including transcription-repair factors, XPD (Rad3) and XPB, as well as CSB involved in strand-specific DNA repair, via its C-terminal domain. We also found that wild-type, but not Arg273His mutant p53 inhibits XPD (Rad3) and XPB DNA helicase activities. Moreover, repair of UV-induced dimers is slower in Li-Fraumeni syndrome cells (heterozygote p53 mutant) than in normal human cells. Our findings indicate that p53 may play a direct role in modulating nucleotide excision repair pathways.

DNA strand bias in the repair of the p53 gene in normal human and xeroderma pigmentosum group C fibroblasts.

We have measured the gene-specific and strand-specific DNA repair of UV-induced cyclobutane pyrimidine dimers in the p53 tumor suppressor gene in a normal, repair-proficient human fibroblast strain and in fibroblasts from a patient with the repair deficient disorder xeroderma pigmentosum, complementation xeroderma pigmentosum group C (XP-C). In both cell strains, repair was measured in the p53 gene and in its individual DNA strands. For comparison, the repair also was measured in other genomic regions in these human fibroblast strains, including the housekeeping gene dihydrofolate reductase, and two inactive genomic regions, the delta globin gene, and the 754 locus of the X chromosome. In both cell strains, we find that the p53 gene is repaired faster than the dihydrofolate reductase gene and much more efficiently than the inactive genomic regions. Selective repair of the transcribed DNA strand of p53 is observed in both human cell strains; the strand bias of repair is particularly distinct in XP-C. Mutations specific to the nontranscribed strand may occur due to replication errors at the sites of unrepaired DNA damage. Therefore, our results predict that the majority of mutations in skin cancers, especially those from patients with XP-C, would occur on the nontranscribed strand of the p53 gene. Indeed, Dumasz et al. (Proc. Natl. Acad. Sci. USA, in press, 1993) report such a strand bias of p53 mutation in skin cancers from XP-C patients.

Detection and quantification of 8-hydroxydeoxyguanosine adducts in peripheral blood of people exposed to ionizing radiation.

Ionizing radiation produces a variety of damaging insults to nucleic acids, including the promutagenic lesion 8-hydroxydeoxyguanosine. In the present study, the 8-hydroxydeoxyguanosine content of peripheral blood leukocyte DNA isolated from individuals exposed to therapeutic doses of ionizing radiation was determined by a HPLC-coupled 32P-postlabeling assay. Peripheral blood leukocyte DNA from individuals irradiated with 180-200 cGy were observed to contain 2-4.5 times as much 8-hydroxydeoxyguanosine as that from unexposed individuals. These results were confirmed by the use of a HPLC-coupled electrochemical detection system. Thus, human exposure to ionizing radiation significantly increased the circulating leukocyte DNA content of 8-hydroxydeoxyguanosine.

Laboratory and epidemiologic studies of fecapentaenes.

Fecapentaenes are a class of conjugated ether lipids which have been identified as the major component of human fecal mutagenicity in the Ames Salmonella mutagenesis assay. Human epidemiologic data have indicated that most healthy North American populations eating a western diet do excrete detectable levels of fecapentaenes. Excreted fecapentaene levels seem to reflect levels throughout the colonic lumen, and levels vary characteristically between individuals. Those individuals found to excrete high levels of fecapentaene appear, based on limited data, to be at decreased risk of colorectal neoplasia. Carcinogenicity studies in rats and mice have been predominantly negative, however, increased tumor incidence in mice exposed to fecapentaenes as neonates has recently been reported. Fecapentaenes are direct-acting genotoxins, which may react with DNA through free radical mechanisms, and/or aldehyde formation. Mechanisms by which fecapentaene-induced DNA damage may mediate carcinogenesis are discussed.

Generation of reactive intermediates from the tumor promoter butylated hydroxytoluene hydroperoxide in isolated murine keratinocytes or by hematin.

BHTOOH (2,6-di-tert-butyl-4-hydroperoxyl-4-methyl-2,5-cyclohexadienone), a metabolite of the food antioxidant BHT (2,6-di-tert-butyl-4-methylphenol), has previously been shown to function as a tumor promoter in mouse skin. The metabolism of BHTOOH was examined to assess the role of reactive intermediates in mediating tumor promotion in this tissue. Free radical metabolites of BHTOOH were characterized in either isolated neonatal mouse keratinocytes or a cell-free hematin system using electron paramagnetic resonance (EPR) spectroscopy while non-radical, EPR silent products were characterized using HPLC separation coupled with UV or mass spectral detection. Incubation of BHTOOH with keratinocytes or hematin resulted in the generation of the BHT-phenoxyl radical detectable by EPR spectroscopy. Formation of the BHT-phenoxyl radical was prevented by heat inactivation of the cells prior to exposure to BHTOOH. Only one non-radical metabolite of BHTOOH was detected in keratinocytes: BHT-quinol (2,6-di-tert-butyl-4-methyl-4-hydroxyl-2,5-cyclohexadienone), while incubation of BHTOOH with hematin produced several metabolites: oxacyclopentenone (2,5-di-tert-butyl-5-(2'-oxopropyl)-4-oxa-2-cyclopentenone), BHT-quinone (2,6-di-tert-butyl-p-benzoquinone), BHT, BHT-stilbenequinone (3,5,3',5'-tetra-tert-butylstilbene-4,4'-quinone), and BHT-quinone methide (2,6-di-tert-butyl-4-methylene-2,5-cyclohexadienone). Thus, radical as well as non-radical reactive intermediates can be formed during metabolism of BHTOOH. Several of the stable metabolites of BHTOOH were evaluated for possible promoter activity in a short-term bioassay, induction of ornithine decarboxylase (ODC) activity in mouse skin. In contrast to the action of BHTOOH, topical application of equimolar doses of BHT-quinol, BHT-quinone, BHT-stilbenequinone, as well as BHT itself, did not induce epidermal ODC activity. Thus, reactive metabolites of BHTOOH such as the BHT-phenoxyl radical or BHT-quinone methide may be involved in the molecular mechanisms of action of this hydroperoxide tumor promoter.

Tumor promotion by a hydroperoxide metabolite of butylated hydroxytoluene, 2,6-di-tert-butyl-4-hydroperoxy-4-methyl-2,5-cyclohexadienone, in mouse skin.

The nature of the skin tumor promotion response to a hydroperoxide metabolite of butylated hydroxytoluene, 2,6-di-tert-butyl-4-hydroperoxyl-2,5-cyclohexadienone (BHTOOH), was examined in SENCAR mice. BHTOOH was an effective inducer of epidermal ornithine decarboxylase (ODC) activity. Maximal induction of ODC activity was observed 12 hours after a single application of BHTOOH. Dose-dependent increases were seen between 2 and 20 mumol while higher amounts were less effective. A similar dose-response relationship for papilloma and carcinoma formation was observed when BHTOOH was applied twice weekly for 50 weeks to mice previously initiated with 7,12-dimethylbenz[a]anthracene. Doses of 2, 8, and 20 mumol BHTOOH gave maximal papilloma responses of 0.1, 0.6, and 3.6 papillomas/mouse, respectively. The progression of papillomas to carcinomas was examined and at 60 weeks the incidence of carcinomas was 0, 17, and 28% for the three treatment groups. The carcinoma:papilloma ratios were 0.08 and 0.40 for the high and intermediate BHTOOH dose groups. The data suggest that BHTOOH, unlike butylated hydroxytoluene, is an effective tumor promoter in mouse skin. Additionally, BHTOOH may enhance the conversion of papillomas to carcinomas. However, BHTOOH is not a complete carcinogen in that no papillomas or carcinomas were observed in uninitiated mice treated with BHTOOH only.

Role of inflammatory cells in the metabolic activation of polycyclic aromatic hydrocarbons in mouse skin.

Oxidants, such as those generated by activated polymorphonuclear leukocytes (PMNs) during inflammation, have been implicated in the metabolic activation of procarcinogens to their ultimate carcinogenic form. In this study we examined the effect of inflammation on the metabolic activation of (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (BP 7,8-dihydrodiol) to a covalent binding species in mouse epidermis. Interaction of BP 7,8-dihydrodiol with 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated murine leukocytes resulted in the generation of both a chemiluminescent intermediate and one that covalently bound to the DNA of cocultured epidermal keratinocytes. Topical treatment of mouse skin with TPA led to an influx of PMNs into the skin beginning several hours after application. Myeloperoxidase activity, a marker for neutrophils, increased 15-fold in the skin by 16 hr after TPA treatment. Dual applications of TPA at both 16 hr before and concurrently with administration of [3H]BP 7,8-dihydrodiol led to a 50% enhancement of the level of carcinogen that was covalently bound to epidermal DNA. However, a single application of TPA, either 16 hr before or concurrently with BP 7,8-dihydrodiol administration, had no enhancing effect, suggesting that both initial recruitment of PMNs into the skin and subsequent stimulation of oxidant production by the PMNs were required to enhance carcinogen binding. By contrast, no enhancement of benzo[a]pyrene binding was observed by TPA treatments in vivo. However, TPA-stimulated neutrophils did not activate this procarcinogen to a chemiluminescent metabolite in vitro. These results suggest that oxidants generated by metabolically stimulated PMNs can activate penultimate polycyclic aromatic hydrocarbons, such as BP 7,8-dihydrodiol, to potentially genotoxic metabolites in vivo and further define a role for inflammation in carcinogenesis.

Generation of free radicals from organic hydroperoxide tumor promoters in isolated mouse keratinocytes. Formation of alkyl and alkoxyl radicals from tert-butyl hydroperoxide and cumene hydroperoxide.

The organic hydroperoxides tert-butyl hydroperoxide and cumene hydroperoxide are tumor promoters in the skin of SENCAR mice, and this activity is presumed to be mediated through the activation of the hydroperoxides to free radical species. In this study we have assessed the generation of free radicals from organic hydroperoxides in the target cell (the murine basal keratinocyte) using electron spin resonance. Incubation of primary isolates of keratinocytes from SENCAR mice in the presence of spin traps (5,5-dimethyl-1-pyrroline N-oxide or 2-methyl-2-nitrosopropane) and either tert-butyl hydroperoxide or cumene hydroperoxide resulted in the generation and detection of radical adducts of these spin traps. tert-Butyl alkoxyl and alkyl radical adducts of 5,5-dimethyl-1-pyrroline N-oxide were detected shortly after addition of tert-butyl hydroperoxide, whereas only alkyl radical adducts were observed with cumene hydroperoxide. Spin trapping of the alkyl radicals with 2-methyl-2-nitrosopropane led to the identification of methyl and ethyl radical adducts following both tert-butyl hydroperoxide and cumene hydroperoxide exposures. Prior heating of the cells to 100 degrees C for 30 min prevented radical formation. The radical generating capacity of subcellular fractions of these epidermal cells was examined using 5,5-dimethyl-1-pyrroline N-oxide and cumene hydroperoxide, and this activity was confined to the 105,000 X g supernatant fraction.