Maternal administration of superoxide dismutase and catalase in phenytoin teratogenicity

Embryonic bioactivation and formation of reactive oxygen species (ROS) are implicated in the mechanism of phenytoin teratogenicity. This in vivo study in pregnant CD-1 mice evaluated whether maternal administration of the antioxidative enzymes superoxide dismutase (SOD) and/or catalase conjugated with polyethylene glycol (PEG) could reduce phenytoin teratogenicity. Initial studies showed that pretreatment with PEG-SOD alone (0.5-20 KU/kg i.p. 4 or 8 h before phenytoin) actually increased the teratogenicity of phenytoin (65 mg/kg i.p. on gestational days [GD] 11 and 12, or 12 and 13) (p < .05), and appeared to increase embryonic protein oxidation. Combined pretreatment with PEG-SOD and PEG-catalase (10 KU/kg 8 or 12 h before phenytoin) was not embryo-protective, nor was PEG-catalase alone, although PEG-catalase alone reduced phenytoin-initiated protein oxidation in maternal liver (p < .05). However, time-response studies with PEG-catalase (10 KU/kg) on GDs 11, or 11 and 12, showed maximal 50-100% increases in embryonic activity sustained for 8-24 h after maternal injection (p < .05), and dose-response studies (10-50 KU/kg) at 8 h showed maximal respective 4-fold and 2-fold increases in maternal and embryonic activities with a 50 KU/kg dose (p < .05). In controls, embryonic catalase activity was about 4% of that in maternal liver, although with catalase treatment, enhanced embryonic activity was about 2% of enhanced maternal activity (p < .05). PEG-catalase pretreatment (10-50 KU/kg 8 h before phenytoin) also produced a dose-dependent inhibition of phenytoin teratogenicity, with maximal decreases in fetal cleft palates, resorptions and postpartum lethality at a 50 KU/kg dose (p < .05). This is the first evidence that maternal administration of PEG-catalase can substantially enhance embryonic activity, and that in vivo phenytoin teratogenicity can be modulated by antioxidative enzymes. Both the SOD-mediated enhancement of phenytoin teratogenicity, and the inhibition of phenytoin teratogenicity by catalase, indicate a critical role for ROS in the teratologic mechanism, and the teratologic importance of antioxidative balance.     

Evidence supporting the role of DNA pyridyloxobutylation in rat nasal carcinogenesis by tobacco-specific nitrosamines

The tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN) both induce nasal tumors in rats and have a common metabolic activation pathway leading to pyridyloxobutylation of DNA. The role of DNA pyridyloxobutylation in rat nasal carcinogenesis has not been evaluated previously. In this study, we used gas chromatography-mass spectrometry to compare levels of 4-hydroxyl-1-(3-pyridyl)-1-butanone-releasing adducts formed by pyridyloxobutylation of rat nasal mucosa DNA after treatment with either NNK, NNN, or deuterated analogues of NNK. The latter were [4,4-D2]NNK, a stronger nasal cavity carcinogen than NNK, and [CD3]NNK, which has carcinogenic activity equivalent to NNK. We also investigated toxicity to the nasal mucosa and levels of O6-methylguanine in the DNA of this tissue in rats treated with NNK and its deuterated analogues. Rats were given three times weekly s.c. injections of the respective nitrosamines for 4 weeks and then sacrificed 24 h after the final injection. The nasal mucosa was separated into the olfactory and respiratory portions. In the rats treated with [4,4-D2]NNK, levels of O6-methylguanine in DNA from both the olfactory and respiratory portions of the nasal mucosa were significantly lower and levels of 4-hydroxy-1-(3-pyridyl)-1-butanone-releasing DNA adducts higher than in the rats treated with equivalent doses of the less carcinogenic compounds NNK or [CD3]NNK. 4-Hydroxy-1-(3-pyridyl)-1-butanone-releasing adducts were also detected in the nasal mucosa DNA of the rats treated with NNN. In the comparative study of NNK and its deuterated analogues, the histology of the nasal mucosa did not appear to be markedly different among these groups. Collectively, the results of this study provide strong evidence that DNA pyridyloxobutylation is important in rat nasal cavity carcinogenesis by NNK and NNN.     

Peroxidase-dependent bioactivation and oxidation of DNA and protein in benzo[a]pyrene-initiated micronucleus formation

Micronucleus formation initiated by benzo[a]pyrene (B[a]P) and related xenobiotics is widely believed to reflect potential carcinogenic initiation, yet neither a dependence upon bioactivation nor the critical enzymes have been demonstrated. Using rat skin fibroblasts, protein oxidation (carbonyl formation) and content of prostaglandin H synthase (PHS) and cytochrome P4501A1 (CYP1A1) protein were determined by Western blot/immunodetection with enhanced chemiluminescence. DNA oxidation as 8-hydroxy-2'-deoxyguanosine formation was quantified using high-performance liquid chromatography with electrochemical detection. Fibroblast CYP1A1 activity assessed as ethoxyresorufin-O-deethylase was not detectable, and even CYP1A1 protein was measurable only after induction with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). However, TCDD additionally induced prostaglandin H synthase (PHS), which also was detectable constitutively. B[a]P 10 microM initiated the oxidation of DNA and protein, and the formation of micronuclei, all of which were enhanced over 2-fold by the dual CYP1A1/PHS inducer TCDD 10 nM, as well as by other PHS inducers, 12-O-tetradecanoylphorbol-13-acetate 1 microM and interleukin-1alpha 0.625 or 1.25 ng/ml, that do not induce CYP1A1 (p < .05). Conversely, B[a]P target oxidation and micronucleus formation were abolished by 1-aminobenzotriazole 1 mM (p < .05), which was a potent inhibitor of both peroxidases and P450. These results provide the first direct evidence that B[a]P-initiated micronucleus formation, like carcinogenic initiation, requires enzymatic bioactivation, and that peroxidase-dependent, reactive oxygen species-mediated oxidation of DNA, and possibly protein, constitutes a molecular mechanism of initiation in uninduced cells. Induction of either CYP1A1 or peroxidases such as PHS substantially enhances this genotoxic initiation, which may reflect cancer risk.     

Elevated 8-hydroxy-2'-deoxyguanosine levels in lung DNA of A/J mice and F344 rats treated with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and inhibition by dietary 1,4-phenylenebis(methylene)selenocyanate

1,4-Phenylenebis(methylene)selenocyanate (p-XSC) is an effective chemopreventive agent against 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung adenoma in female A/J mice. While p-XSC can effectively inhibit NNK-induced DNA methylation in female A/J mice and in male F344 rats, its effect on NNK-induced oxidative DNA damage had not been determined. Thus, the effect of p-XSC on the levels of 8-hydroxy-2'-deoxyguanosine (8-OH-dG) in lung DNA from A/J mice and F344 rats treated with NNK was examined. Mice were given NNK by gavage (0.5 mg/mouse in 0.2 ml corn oil, three times per week for 3 weeks) or by a single i.p. injection (2 mg/mouse in 0.1 ml saline) while maintained on a control diet (AIN-76A) or control diet containing p-XSC at 10 or 15 p.p.m. (as Se) starting 1 week before NNK administration and continuing until termination. Mice were killed 2 h after the last NNK gavage in the multiple administration protocol or 2 h after the single i.p. injection. Treatment with NNK by gavage significantly elevated the levels of 8-OH-dG in lung DNA of A/J mice from 0.7 +/- 0.1 to 1.6 +/- 0.2 adducts/10(5) 2'-deoxyguanosine (dG) (P < 0.001), while dietary p-XSC (at 10 p.p.m. Se) prevented significant elevation of the levels of this lesion caused by NNK, keeping them at 0.9 +/- 0.1 adducts/10(5) dG (P < 0.003). Injection of NNK in saline also significantly increased the levels of 8-OH-dG in lung DNA of A/J mice from 1.2 +/- 0.6 to 3.6 +/- 0.8/10(5) dG adducts (P < 0.01), while dietary p-XSC (at 15 p.p.m. Se) kept these levels at 1.9 +/- 0.5 adducts/10(5) dG (P < 0.03). Rats were given a single i.p. injection of NNK (100 mg/kg body wt) in saline while being maintained on control diet (AIN-76A) or control diet containing p-XSC (15 p.p.m. as Se) starting 1 week before NNK administration and continuing until termination. The rats were killed 2 h after injection. Treatment with NNK using this protocol significantly elevated the levels of 8-OH-dG in lung DNA of F344 rats from 2.6 +/- 0.5 to 3.5 +/- 0.5 adducts/10(5) dG (P < 0.03), while dietary p-XSC (at 15 p.p.m. Se) kept the levels of this lesion at 2.2 +/- 0.6 adducts/10(5) dG (P < 0.01). Our findings suggest that the chemopreventive efficacy of p-XSC against NNK-induced lung tumorigenesis in A/J mice and F344 rats may be due in part to inhibition of oxidative DNA damage.     

Pyridyloxobutyl adduct O6-[4-oxo-4-(3-pyridyl)butyl]guanine is present in 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone-treated DNA and is a substrate for O6-alkylguanine-DNA alkyltransferase

The lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is activated to reactive metabolites that methylate or pyridyloxobutylate DNA. Previous studies demonstrated that pyridyloxobutylated DNA interferes with the repair of O6-methylguanine (O6-mG) by O6-alkylguanine-DNA alkyltransferase (AGT). The AGT reactivity of pyridyloxobutylated DNA was attributed to (pyridyloxobutyl)guanine adducts. One potential AGT substrate adduct, 2'-deoxy-O6-[4-oxo-4-(3-pyridyl)butyl]guanosine (O6-pobdG), was prepared. This adduct was stable at pH 7.0 for greater than 13 days and to neutral thermal hydrolysis conditions (pH 7.0, 100 degrees C, 30 min). Under mild acid hydrolysis conditions (0.1 N HCl, 80 degrees C), O6-pobdG was depurinated to yield O6-[4-oxo-4-(3-pyridyl)butyl]guanine (O6-pobG). O6-pobdG was hydrolyzed to 4-hydroxy-1-(3-pyridyl)-1-butanone and guanine under strong acid hydrolysis conditions (0.8 N HCl, 80 degrees C). O6-pobG was detected in 0.1 N HCl hydrolysates of DNA alkylated with the model pyridyloxobutylating agent 4-(acetoxymethylnitrosamino)-1-(3-[5-3H]pyridyl)-1-butanone ([5-3H]NNKOAc). When [5-3H]NNKOAc-treated DNA was incubated with either rat liver or recombinant human AGT, O6-pobG was removed, presumably a result of transfer of the pyridyloxobutyl group from the O6-position of guanine to AGT's active site.     

Coal tar residues produce both DNA adducts and oxidative DNA damage in human mammary epithelial cells

In the present study we compare the metabolic activation of coal tar, as measured by the production of both DNA adducts and oxidative DNA damage, with that of a single carcinogen that is a constituent of this complex mixture in human mammary epithelial cells (HMEC). We find that a significant level of DNA adducts, detected by 32P-postlabeling, are formed in HMEC following exposure to coal tar residues. This treatment also results in the generation of high levels of oxidative DNA damage, as measured by the production of one type of oxidative base modification, thymine glycols. The amounts of both DNA adducts and thymine varied considerably between the various coal tar residues and did not correlate with either the total amount of polycyclic aromatic hydrocarbons (PAH) or the amount of benzo[a]pyrene (B[a]P) present in the residue. Fractionating the residue from one of the sites by sequential extraction with organic solvents indicated that while the ability to produce both types of DNA damage was contained mostly in a hexane-soluble fraction, a benzene-soluble fraction produced high levels of reactive oxygens relative to the number of total DNA adducts. We find that the total amount of PAH or B[a]P present in the coal tars from the various sites was not a predictor of the level of total DNA damage formed.     

Effects of green tea and black tea on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone bioactivation, DNA methylation, and lung tumorigenesis in A/J mice

Previous studies in our laboratory showed that decaffeinated green tea and black tea extracts inhibited 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced tumorigenicity in A/J female mice. In order to understand the mechanism of the inhibitory action, we examined the effects of decaffeinated green tea, black tea, and tea components on the metabolic activation of NNK in vitro and in vivo in this animal model. When added to incubation mixtures containing mouse lung microsomes, decaffeinated green tea and black tea extracts and their fractions, at concentrations up to 0.4 mg/ml, inhibited NNK oxidation and NNK-induced DNA methylation. Among the tea components examined, (-)-epigallocatechin-3-gallate was the most potent inhibitor with 50% inhibitory concentrations of about 0.12 mM for both NNK oxidation and DNA methylation. At these concentrations, (-)-epigallocatechin-3-gallate inhibited the catalytic activities of several P450 enzymes and was more potent against P450 1A and 2B1 than 2E1. When decaffeinated green or black tea extracts were given to female A/J mice as the sole source of drinking fluid before an i.p. injection of NNK (100 mg/kg body weight), a statistically significant inhibition of lung DNA methylation, however, was not observed, although a significant reduction in lung tumor multiplicity was observed. The results suggest that, although inhibition of the metabolic activation of NNK and the subsequent DNA alkylation by tea extracts can be demonstrated in vitro, this mechanism may not be important for the inhibitory action of tea against lung tumorigenesis.     

Biodistribution of, antimutagenic efficacies in Salmonella typhimurium of, and inhibition of P450 activities by ellagic acid and one analogue

Ellagic acid (EA) is generated by hydrolysis of ellagitannins present in fruit berries and edible nuts and grapes. Large doses of EA prevent lung tumorigenesis induced by the tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in A/J mice. In this study, we document the efficacies of the EA structural analogue (3,4,7,8-tetrahydroxy-6H-benzo[b,d]pyran-6-one) (analogue 1) to inhibit specific P450 activities, pulmonary metabolism of NNK in A/J mice, and NNK-induced mutations in Salmonella typhimurium. Mouse lung microsomes metabolized benzyloxyresorufin, a marker of cytochrome P450 2B1 activity, more extensively than methoxyresorufin or ethoxyresorufin. The EA analogue was more effective than EA in inhibiting dealkylation of the three alkoxyresorufins, suggesting that it is a nonspecific inhibitor of P450s. Mouse lung microsomes hydroxylate testosterone in the 7alpha and 6beta positions, suggesting contributions of P450 2A1 and P450 3A2 isozymes, respectively. Inhibition of both pathways was more effective with the EA analogue than with EA. Mouse lung explants metabolized NNK by alpha-carbon hydroxylation (activation) and pyridine N-oxidation (deactivation). Both pathways were inhibited when 100 microM EA was added to the culture medium. The EA analogue was a better inhibitor of the activation of NNK to electrophilic species than EA. Mouse lung microsomes activate NNK to intermediates mutagenic to S. typhimurium. Inhibition of NNK mutagenicity by EA or the EA analogue was 20 or 65%, respectively. The distribution of the EA analogue in lung and liver was determined following gavage with 1.7 mmol of the EA analogue. In the lung, a maximal level of EA analogue corresponding to 105 nmol was observed 30 min after administration of the analogue. The level in liver tissues was 4-fold lower than in the lung. Results of this study demonstrate that the EA analogue is more effective than EA in inhibiting the pulmonary activation of NNK and suggest that the EA analogue could be effective in preventing lung tumorigenesis.     

Induction of microsomal epoxide hydrolase activity in inbred mice by chronic phenytoin exposure

A lower microsomal epoxide hydrolase (mEH) activity has been associated with increased likelihood of fetal hydantoin syndrome. While phenytoin anticonvulsive regimens are long-term, there are no data regarding induction of mEH by chronic phenytoin exposure. Two inbred mouse strains which differ in their susceptibility (A/J > C57BL/6J) to phenytoin-induced oral clefting were treated with an oral gavage of phenytoin for 14 consecutive days. The mice were sacrificed on the 15th day, and hepatic microsomes were prepared. mEH activity was determined using benzo[a]pyrene-4,5-oxide. The dihydrodiol product was separated by HPLC and quantified. There was no significant difference (P = 0.15) in the phenytoin plasma level between the two strains on Day 15. There was no significant difference (P = 0.07) between control and sham control groups within each strain, so they were combined for further analysis. There was a significant strain difference (P = 0.0001) between the control and phenytoin-exposed group means, with the C57BL/6J strain having the greater activity before and after phenytoin exposure. The A/J phenytoin-exposed group activity was 51% higher (P = 0.01) than the A/J control, while the C57BL/6J phenytoin-exposed group activity was 78% higher (P = 0.001) than the C57BL/6J control. The greater mEH activity in the phenytoin-induced clefting resistant strain (C57BL/6J) before and after phenytoin exposure is consistent with a putative oxidative metabolism mechanism of phenytoin teratogenecity. Chronic phenytoin exposure induced mEH activity in both strains, although the strain with the greater enzyme activity prior to the exposure continued to have the greater activity following induction.     

Modulation of embryonic glutathione peroxidase activity and phenytoin teratogenicity by dietary deprivation of selenium in CD-1 mice

Selenium (Se)-dependent and -independent glutathione (GSH) peroxidases detoxify H2O2 and lipid hydroperoxides, which may mediate the teratogenicity of phenytoin and related xenobiotics. To test this hypothesis, CD-1 mice were placed on Se-deficient diets for 15, 25 or 40 days and bred so that the day of analysis corresponded to gestational day 11. In Se-replete control animals, embryonic peroxidase activities were only 5% of activities in maternal liver (P < .05). After 15 days of Se deprivation, maternal activities for H2O2 (reflecting Se-dependent peroxidase) and cumene hydroperoxide (CmOOH) (reflecting both Se-dependent and -independent peroxidases) were reduced to 20% (P < .05) and 35% of controls, respectively. At this time, the incidence of fetal cleft palates initiated by phenytoin (55 mg/kg intraperitoneally on gestational days 11, 12 and 13) was doubled, from 12% to 25% (P < .05). Selenite rescue (Na2SeO3, 350 micrograms/kg intraperitoneally on day 9) restored maternal and embryonic peroxidase activities and completely inhibited phenytoin-initiated postpartum lethality and fetal resorptions in animals that had been Se depleted for 15 days. After 40 days of Se deprivation, maternal and embryonic peroxidase/H2O2 activities were reduced to < 1% and 27% of Se-replete controls, respectively. In contrast, maternal peroxidase/CmOOH activity was increased to 70% of controls, reflecting induction of Se-independent peroxidase, compared with that with 15 days' depletion. Phenytoin-initiated cleft palates with 40 days' depletion appeared to be reduced (16%) compared with Se-replete controls (24%) (P < .07). In 40-day Se-depleted animals given selenite rescue, the 10% incidence of cleft palates was significantly lower than that in the 40-day Se-replete group (24%) but not the Se-depleted group (16%). This is the first demonstration of reduced Se-dependent GSH peroxidase activities in embryonic tissues with dietary Se-deprivation. The results implicate reactive oxygen species and lipid hydroperoxides in the mechanism of phenytoin teratogenicity and suggest that GSH peroxidases are important embryoprotective enzymes.     

Ca2+-independent activation of the endothelial nitric oxide synthase in response to tyrosine phosphatase inhibitors and fluid shear stress

Chinese hamster V79 cell lines were constructed for stable expression of human cytochrome P450 1B1 (P450 1B1) in order to study its role in the metabolic activation of chemicals and toxicological consequences. The new V79 cell lines were applied to studies on DNA adduct formation of the polycyclic aromatic hydrocarbon (PAH) dibenzo[a,l]pyrene (DB[a,l]P). This compound has been found to be an environmental pollutant, and in rodent bioassays it is the most carcinogenic PAH yet discovered. Activation of DB[a,l]P in various metabolizing systems occurs via fjord region DB[a,l]P-11, 12-dihydrodiol 13,14-epoxides (DB[a,l]PDE): we found that DB[a,l]P is stereoselectively metabolized in human mammary carcinoma MCF-7 cells to the (-)-anti- and (+)-syn-DB[a,l]PDE which both bind extensively to cellular DNA. To follow up this study and to relate specific DNA adducts to activation by individual P450 isoforms, the newly established V79 cells stably expressing human P450 1B1 were compared with those expressing human P450 1A1. DNA adduct formation in both V79 cell lines differed distinctively after incubation with DB[a,l]P or its enantiomeric 11,12-dihydrodiols. Human P450 1A1 catalyzed the formation of DB[a,l]PDE-DNA adducts as well as several highly polar DNA adducts as yet unidentified. The proportion of these highly polar adducts to DB[a,l]PDE adducts was dependent upon both the concentration of DB[a,l]P and the time of exposure. In contrast, V79 cells stably expressing human P450 1B1 generated exclusively DB[a,l]PDE-DNA adducts. Differences in the total level of DNA binding were also observed. Exposure to 0.1 microM DB[a,l]P for 6 h caused a significantly higher level of DNA adducts in V79 cells stably expressing human P450 1B1 (370 pmol/mg of DNA) compared to those with human P450 1A1 (35 pmol/mg of DNA). A 4-fold higher extent of DNA binding was catalyzed by human P450 1B1 (506 pmol/mg of DNA) compared to human P450 1A1 (130 pmol/mg of DNA) 6 h after treatment with 0.05 microM (-)-(11R,12R)-dihydrodiol. In cells stably expressing human P450 1B1 the DNA adducts were derived exclusively from the (-)-anti-DB[a,l]PDE. These results indicate that human P450 1B1 and P450 1A1 differ in their regio- and stereochemical selectivity of activation of DB[a,l]P with P450 1B1 forming a higher proportion of the highly carcinogenic (-)-anti-(11R, 12S,13S,14R)-DB[a,l]PDE metabolite.     

Stress, hormonal changes, alcohol, food constituents and drugs: factors that advance the incidence of tobacco smoke-related cancer?

The genotoxicity of the most potent carcinogen in cigarette smoke [4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)] is dependent on the relationship between its activation by cytochrome P450 enzymes and its detoxification by carbonyl reduction to NNK alcohol (NNAL) followed by glucuronidation. Recently, '11 beta-hydroxysteroid dehydrogenase' (11 beta-HSD 1) was identified to be responsible for NNK carbonyl reduction. It is now speculated that differences in tissue expression of 11 beta-HSD 1, as well as genetic polymorphisms, may have profound influences on the organospecificity and potency of NNK-induced cancerogenesis. Moreover, endogenous and exogenous substrates or inhibitors of 11 beta-HSD 1 may shift the NNK/NNAL equilibrium and favour NNK toxification in a variety of physiological and therapeutic situations. These issues are discussed here by Edmund Maser, who also describes how recent observations could provide the experimental base for epidemiological or clinical studies, which focus on polymorphisms in 11 beta-HSD 1 enzyme expression, as well as on implications of exposure to 11 beta-HSD 1 modulators and concurrent smoking.     

Lung tumor induction in A/J mice by the tobacco smoke carcinogens 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and benzo[a]pyrene: a potentially useful model for evaluation of chemopreventive agents

The purpose of this study was to establish a lung tumor model for the evaluation of chemopreventive agents against lung cancer in smokers. Lung tumor induction in A/J mice by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and benzo[a]pyrene (BaP) was studied using protocols in which these two tobacco smoke carcinogens were given individually or in combination. Groups of female A/J mice were treated by either intragastric gavage (i.g.) or by intraperitoneal injection (i.p.) with various doses of NNK and/or BaP for 8 consecutive weeks. The mice were killed either 9 or 19 weeks later and tumors of the lung and forestomach were counted. The i.g. route of administration proved to be more satisfactory than i.p. administration, because it avoided complications due to tumor formation at the injection site and associated mortality. A dose-response relationship for lung tumor induction by i.g. administration of NNK and BaP in combination was established in the mice killed 9 or 19 weeks after completion of carcinogen treatment. The highest total doses of NNK and BaP (a total of 24 mumol of each) induced more lung tumors than would have been expected by extrapolation from the lower doses. Comparisons of NNK and BaP given individually showed that BaP was more tumorigenic to the lung than NNK when given by the i.g. route; i.p. administrations of BaP were complicated by local tumor formation and mortality. The most favorable dosing regimen of NNK and BaP for evaluation of chemopreventive agents appears to be a total dose of 24 mumol of each, administered in eight weekly subdoses i.g., with sacrifice 9 weeks after completion of dosing. This regimen induced 10.5 +/- 4.4 lung adenomas/mouse. A combination of benzyl isothiocyanate and phenethyl isothiocyanate, given 2 h prior to each gavage of NNK and BaP, was found to be an effective inhibitor of lung tumor formation, reducing the tumor multiplicity to 5.9 +/- 5.7 lung adenomas/mouse (P < 0.001) and completely inhibiting forestomach tumor development. The results of this study provide a convenient model for assessing the efficacy of chemopreventive agents against lung cancer induction by tobacco smoke carcinogens.     

Modulation of DNA repair by various inhibitors of DNA synthesis following 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) induced DNA damage

The tobacco specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is present in tobacco smoke and is hepatocarcinogenic in rats. Its bioactivation in rat hepatocytes leads to methylation and pyridyloxobutylation of DNA. Rat hepatocytes were cultured in serum-free William medium E on collagen-coated dishes. We demonstrated that some enzymes of the base and/or excision-repair pathways were involved in repair of NNK-induced DNA damage, measured by [methyl-3H] thymidine incorporation. Unscheduled DNA synthesis (UDS) induced by N-methyl-N-nitrosourea (MNU), NNK, N'-nitrosonornicotine (NNN) and 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNKOAc) increased 2.9-, 2.8-, 1.5- and 3.5-fold, respectively, suggesting that methylated and/or pyridyloxobutylated-DNA by these four nitroso compounds is repaired by the excision pathway. Moreover, levels of NNK-induced UDS were dose (1-3 mM) and time (1-18 h) dependent. Enzymes involved in the excision repair pathways were selectively inhibited. Inhibitors of DNA topoisomerase I (camptothecin) and topoisomerase II (etoposide, nalidixic acid) did not decrease the induction of UDS, suggesting that topoisomerases are not involved in the repair of NNK-induced damage. While aphidicolin and arabinocytidine (DNA polymerase alpha, delta, epsilon inhibitors) totally inhibited NNK- and NNKOAc-induced UDS, dideoxythymidine (DNA polymerase beta inhibitor) inhibited NNK- and NNKOAc-induced UDS by 40 and 33%, respectively. We conclude that DNA polymerase alpha, delta or epsilon and to a lesser degree polymerase beta are involved in the repair of pyridyloxobutylated DNA. Previous studies showed that inhibition of poly(ADP-ribosyl) polymerase (PARP) by 3-aminobenzamide (3-ab) facilitated DNA ligation. Our results demonstrate that 3-ab increased NNK-induced UDS, but does not affect NNKOAc-induced UDS. These observations suggest that the ligation step is rate limiting in the repair of methylated DNA but not of pyridyloxobutylated DNA.     

Absence of metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) by flavin-containing monooxygenase (FMO)

The N-nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent lung carcinogen present in tobacco and tobacco smoke. Carbonyl reduction, alpha-carbon hydroxylation (activation) and N-oxidation of the pyridyl ring (detoxification) are the three main pathways of metabolism of NNK. In this study, metabolism of NNK was studied with lung and liver microsomes from F344 rats, Syrian golden hamsters and pigs and cloned flavin-containing monooxygenases (FMOs) from human and rabbit liver. Thermal inactivation at 45 degrees C for 2 min reduced FMO S-oxygenating activity but did not affect N-oxidation of NNK, leading to the conclusion that FMOs are not implicated in the detoxification of NNK. Detoxification of NNK was not increased by n-octylamine or by incubation at pH 8.4, supporting the conclusion that FMOs are not involved in the metabolism of NNK. SKF-525A (1 mM) significantly reduced N-oxidation and alpha-carbon hydroxylation, suggesting that these two pathways were catalyzed by cytochromes P450. Metabolism of NNK was lower with lung microsomes than with liver microsomes. Inhibition of metabolism of NNK by SKF-525A was also observed with rat lung microsomes, leading to the conclusion that cytochromes P450 are involved in pulmonary metabolism of NNK. Cloned FMOs did not metabolize NNK. In conclusion, cytochromes P450 rather than FMOs are involved in N-oxidation of NNK. The high capacity of hamster liver microsomes to activate NNK does not correlate with the resistance of this tissue to NNK-induced hepatocarcinogenesis.     

Biliary excretion of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in the rat

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent pancreas carcinogen in rats. The biliary excretion of NNK was therefore studied in anesthetized female Sprague-Dawley rats following i.p. administration of 0.7 mumol/kg [carbonyl-14C]NNK. The concentration of radioactivity peaked within 30 min and decreased thereafter exponentially. Cumulative excretion of radioactivity reached a plateau at 6-9% of the total dose. HPLC analysis revealed the presence of 4-hydroxy-4-(3-pyridyl)butyric acid (hydroxy acid), 4-oxo-4-(3-pyridyl)-butyric acid (keto acid), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butyl beta-D-glucopyranosiduronic acid (NNAL Glu), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and NNK. NNAL Glu was the major metabolite contributing 34 +/- 4% of total radioactivity in bile at 30 min and 58 +/- 4% at 5 h. The percentage of acidic metabolites remained constant at approximately 20%. In contrast, the percentage of NNK and NNAL decreased within the first 2 h to < 5% and < 10% respectively. The elimination kinetics of NNK and its metabolites fitted into a one-compartment model with a half-life of 37 min for NNK, 52 min for NNAL and 110 min for NNAL Glu and acidic metabolites. In three rats dosed with 240 mumol/kg NNK i.p., the concentration of radioactivity peaked after 1-2 h and decreased very slowly thereafter. After 5-8 h a total of 12-17% of the dose has been excreted in the bile with no indication of a plateau. At all time points NNAL Glu was the major metabolite contributing up to 95% of total radioactivity in bile. The percentage of acidic metabolites was < 5% throughout the experiment. Whereas NNK contributed one-third of the radioactivity at 30 min and decreased rapidly, the percentage of NNAL in bile remained rather constant at approximately 5-10%. In conclusion, the detection of NNK, NNAL and NNAL Glu gives support to the hypothesis that tobacco-specific carcinogens could reach the pancreas retrograde from the bile, especially at high NNK concentrations.     

Structure, conformations, and repair of DNA adducts from dibenzo[a, l]pyrene: 32P-postlabeling and fluorescence studies

The nature of stable DNA adducts derived from the very potent carcinogen dibenzo[a,l]pyrene (DB[a,l]P) in the presence of rat liver microsomes in vitro and in mouse skin in vivo has been studied using 32P-postlabeling and laser-based fluorescence techniques. Analysis of DB[a,l]P-DNA adducts via 32P-postlabeling has been obtained by comparison of the adduct patterns to those obtained from reactions of synthetic (+/-)-anti-, (+)-anti-, (-)-anti-, and (+/-)-syn-DB[a,l]P-11,12-diol 13,14-epoxide (DB[a,l]PDE) with single nucleotides and calf thymus DNA. anti-DB[a,l]PDE-dA adducts derived from the (-)-enantiomer are the major adducts formed in calf thymus DNA and in mouse skin DNA. The ratio of deoxyadenosine to deoxyguanosine modification is approximately 2:1 in mouse skin exposed to DB[a,l]P; activation by rat liver microsomes leads to a similar profile of adducts but with two additional spots. The conformations of DB[a,l]P adducts in native DNA, as well as the possibility of conformation-dependent repair, have been explored by low-temperature fluorescence spectroscopy. These studies have been performed using polynucleotides and calf thymus DNA reacted in vitro with DB[a,l]PDE and native DNA from mouse epidermis exposed to DB[a, l]P. The results show that adducts are heterogeneous, possess different structures, and adopt different conformations. External, external but base-stacked and intercalated adduct conformations are observed in calf thymus DNA and in mouse skin DNA samples. Differences in adduct repair rates are also revealed; namely, the analysis of mouse skin DNA samples obtained at 24 and 48 h after exposure to DB[a,l]P clearly shows that external adducts are repaired more efficiently than intercalated adducts. These results, taken together with those for B[a]P-DNA adducts [Suh et al. (1995) Carcinogenesis 16, 2561-2569], indicate that the repair of DNA damage resulting from PAH diol epoxides is conformation-dependent.     

The effect of oxygen free radicals on calcium current and dihydropyridine binding sites in guinea-pig ventricular myocytes

1. We used electrophysiological and binding techniques to determine the effects of oxygen free radicals (OFRs) generated by dihydroxyfumaric acid (DHF, 5 mM) on calcium current and dihydropyridine binding sites in guinea-pig isolated ventricular myocytes. 2. Binding of [3H]-PN200-110 to isolated ventricular myocytes revealed one population of binding sites with a KD of 0.11 +/- 0.01 nM and Bmax of 139.1 +/- 6.9 fmol mg-1 protein (n = 24). After 15 min of exposure to DHF, the density, but not the affinity of [3H]-PN200-110 binding sites was significantly (P < 0.01) reduced to 35% of the control value (Bmax = 49.4 +/- 3.7 fmol mg-1 protein, KD = 0.11 +/- 0.01 nM, n = 15). In the presence of superoxide dismutase (SOD) and catalase (CAT) the reduction in [3H]-PN200-110 binding sites was almost completely prevented (Bmax = 120.5 +/- 7.4 in control, n = 4 and 98.8 +/- 7.4 fmol mg-1 protein in DHF plus SOD and CAT, n = 4). KD values were not modified (0.08 +/- 0.01 in control and 0.09 +/- 0.01 nM in DHF plus SOD and CAT). 3. The time-course of the reduction of [3H]-PN200-110 binding sites by OFRs was paralleled by the decrease in L-type calcium current (Ica,L) measured in patch-clamped guinea-pig ventricular myocytes either in the absence or in the presence of EGTA in the patch pipette. In the former conditions OFRs induced the appearance of calcium-dependent alterations, i.e. the transient inward current, within 10 min. After 30 min of incubation with DHF, [3H]-PN200-110 binding sites were reduced to 25% of the control value. 4. In myocytes incubated with the antilipoperoxidant agent, butylated hydroxytoluene (BHT, 50 microM), the decrease in [3H]-PN200-110 binding sites caused by DHF was partially prevented (Bmax values after 30 min exposure to DHF were 55.5 +/- 1.9 and 23.7 +/- 5.9 fmol mg-1 protein in the presence and in the absence of BHT respectively, P < 0.05). BHT did not affect the decrease in [3H]-PN200-110 binding sites during the first 15 min of exposure to DHF, but was able to prevent completely the further decrease occurring during the following 15 min of incubation with OFRs. 5. Our results demonstrate that the OFR-induced decrease in calcium current is associated with a reduction in DHP binding sites. The decrease in calcium current and in calcium channels may be implicated in the mechanical dysfunction associated with oxidative stress.     

Power imbalance--evolving theory in need of research

CuZn superoxide dismutase (SOD) secretion was detected in media of [35S]cysteine-labeled human neuroblastoma SK-N-BE cells precipitated with antihuman CuZn SOD antibodies. The ability of Fe2+/ascorbate oxidative stress to induce CuZn SOD in SK-N-BE cells was evaluated by Western blot analysis. The results showed that, like human hepatocarcinoma cells and human fibroblasts, SK-N-BE cells secrete CuZn SOD. In addition, the CuZn SOD concentration was higher in cells subjected to oxidative stress than in unstressed cells. The secretion of CuZn SOD and the ability of Fe2+/ascorbate to increase its protein content in SK-N-BE cells indicates that this enzyme protects the brain from damage induced by oxidative stress.     

Comparative analysis of 8-oxo-2' -deoxyguanosine in DNA by 32P- and 33P-postlabeling and electrochemical detection

8-Oxo-2'-deoxyguanosine (8-oxo-dG) is emerging as a useful marker for oxidative DNA damage. Reported basal levels determined by 32P-postlabeling (PPL) method were 10-fold or more higher than those obtained with HPLC/electrochemical detection (ECD). This discrepancy was investigated. In commercial calf thymus DNA, levels of 4 +/- 1 and 64 +/- 14 8-oxo-dG per 10(6) 2'-deoxynucleosides (dN) were measured by the standard HPLC/ECD and PPL methods, respectively. DNA digestion by micrococcal nuclease/spleen phosphodiesterase and nuclease P1 (as used in the standard PPL method), followed by ECD analysis resulted in a level of 8 +/- 3. In calf thymus DNA spiked with chemically synthesized 8-oxo-dGp to give an increment of 9 8-oxo-dG/10(6) dN, the added standard produced a significant increase with HPLC/ECD but not PPL. After spiking the DNA with 90 8-oxo-dG/10(6) dN, the added 8-oxo-dGp was detectable also with PPL, with a labeling efficiency of 65%. In order to investigate the role of ionizing radiation from 32P for the higher 8-oxo-dG levels in PPL, incubation times and amounts of radioactivity in the phosphorylation reaction with commercial dGp were increased, and external irradiation of commercial dG with 32P was investigated. All modifications resulted in higher values of 8-oxo-dG measured, but the effect was not large enough to fully explain the discrepancy between PPL and HPLC/ECD. Using [gamma-33P]ATP instead of [gamma-32P]ATP or adding [33P]phosphate to a 32P-PPL assay resulted in even higher levels of 8-oxo-dG measured. The increase in 8-oxo-dG levels during the PPL workup is attributed to the presence and oxidation of unmodified dGp in the reaction mixture. For a determination of true basal levels, the PPL method will have to be modified, including the removal of dGp prior to the phosphorylation reaction.