Oxidations of 17beta-estradiol and estrone and their interconversions catalyzed by liver, mammary gland and mammary tumor after acute and chronic treatment of rats with indole-3-carbinol or beta-naphthoflavone.

Altered cytochrome P450-catalyzed metabolism of 17beta-estradiol (E2) and estrone (E1) in the liver and (or) extrahepatic tissues may affect estrogen-sensitive tumorigenesis. We examined the effects of oral treatments of (i) indole-3-carbinol (13C) at 250 or 500 mg/kg or beta-naphthoflavone (beta-NF) at 40 mg/kg of body weight (bw)/day from 51 to 54 days of age (acute regimen), and (ii) 13C at 250 mg/kg or beta-NF at 20 mg/kg bw given 3x/week from 10 to 22 weeks of age (chronic regimen) in female Sprague-Dawley rats. We determined the effects of these treatments on the P450 content and P450 (CYP)-specific activities in the liver, P450-dependent metabolism of E2 and E1 by the liver and mammary gland, and interconversion of E1 and E2 catalyzed by 17beta-hydroxysteroid dehydrogenase (17beta-HSD) in these tissues and malignant mammary tumors. 13C at the two levels of acute regimen elicited similar responses. Acute and chronic treatments with 13C, but not beta-NF, increased P450 content approximately 2-fold. 13C, and to a lesser extent beta-NF, increased CYP1A1 and CYP1A2 probe activities in liver up to 117- and 27- fold, respectively, and after acute regimens, that of CYP3A by approximately 1.8-fold. 13C also increased activity of CYP2B up to 100-fold. Overall hepatic metabolism of E2 and E1, which was approximately 2-fold greater at 55 than 155 days of age, was increased (approximately 2.8-fold) by 13C with 2-, 4-, 16alpha-, 6alpha-, 6beta-, and 15alpha-hydroxy (OH) comprising > or = 54, 3, 2, approximately 2, approximately 5, 7, and 2%, respectively, of E1 and E2 metabolites. Acute regimens of beta-NF increased 2- and 15alpha-OH-E2 (62 and 5% of total) from E2 and 2-, 4-, and 6alpha-OH-E1 + 6beta-OH-E1 (32, 13, and 4% of total) from E1. Mammary gland metabolized E2 to E1 and small amounts of 15alpha-, 4-, 16alpha-, 6beta-, and 6alpha-OH-E2. After the acute IC3 regimen, E2 was also converted to 2-OH-E2. 17Beta-HSD-catalyzed oxidation of E2 was favored in the liver and reduction of E1 was favored in mammary gland and tumor (= 1% of hepatic activity). An increased (approximately 2-fold) ratio of reductive to oxidative activities in malignant mammary tumors by chronic 13C regimen may stimulate tumor growth. This is the first report showing that after chronic oral regimens, the 13C-, but not beta-NF-, induced changes in CYP complement led to elevated E2 and E1 metabolism. The persistent effects of increased putative carcinogenic and estrogenic 4- and 16alpha-OH as well as 6alpha- and 6beta-OH-E2 and 6beta-OH-E1 might counteract those of the less estrogenic 2-OH metabolites, thus accounting for the lack of suppression of mammary tumorigenesis by 13C in our previous study.

Post-initiation treatment of rats with indole-3-carbinol or beta-naphthoflavone does not suppress 7, 12-dimethylbenz[a]anthracene-induced mammary gland carcinogenesis.

Indole-3-carbinol (I3C) and beta-naphthoflavone (beta-NF), blocking agents of 7,12-dimethylbenz[a]anthracene (DMBA)-initiated mammary gland carcinogenesis, were examined as potential post-initiation suppressing agents. Treatment of female Sprague-Dawley rats with I3C (250 mg/kg body weight (b.w.)), beta-NF (20 mg/kg b.w.) or the vehicle ethanol:corn oil (2:3) (2.5 ml/kg b.w.), three times weekly by gavage, started 3 weeks after the initiation with one oral dose of DMBA (20 mg/rat at 7 weeks of age) and continued for up to 12 weeks. I3C- or beta-NF- or vehicle-treated groups did not differ significantly in the overall outcome of mammary tumorigenesis including cumulative mammary tumor incidences and multiplicities, latent periods and number and weight of mammary tumors per tumor-bearing rat for malignant, benign and/or malignant + benign tumors. A tendency of the I3C-treated rats to develop fewer mammary adenocarcinomas with a greater average weight per tumor per rat (2. 32+/-1.50 g) than in the beta-NF- (1.52+/-1.58 g) or vehicle- (1. 55+/-1.53 g) treated groups suggests an effect, yet to be confirmed, of I3C on tumor development and growth. A 12-week treatment with I3C or beta-NF significantly increased the P450-dependent activities of ethoxy-, methoxy-, benzyloxy- and pentoxy-(with I3C only) resorufin O-dealkylase in hepatic microsomes indicating induction of several P450s. The alterations in the P450 complement may affect endogenous estrogen metabolism and mammary gland and tumor characteristics at the molecular level, e.g. estrogen receptor status and/or proliferative activity, which require further studies.

Reductions of nitro and 9-Oxo groups of environmental nitrofluorenes by the rat mammary gland in vitro.

Nitrofluorenes and C-9-oxidized nitrofluorenes are widespread environmental genotoxins which may be relevant for breast cancer on the basis of their carcinogenicities, particularly of 2, 7-dinitrofluorene (2,7-diNF), for the rat mammary gland. Since their metabolism to active carcinogens may involve nitroreduction, this study examined the reduction of 2-nitrofluorene (2-NF) and 2,7-diNF and their 9-oxo- and 9-hydroxy (OH) derivatives by the rat mammary gland. Cytosolic fractions catalyze NADH- and NADPH-dependent reductions of the 2-nitro and 9-oxo to the respective 2-amino and 9-OH compounds at rates 4- and >/=10-fold greater than those with microsomes. Rates of amine formation catalyzed by cytosol from 2, 7-diNF are greater than the rate from 2-NF and increase for C-9-oxidized derivatives: 9-oxo-2-NF > 9-OH-2-NF > 2-NF and 9-OH-2, 7-diNF >> 9-oxo-2,7-diNF > 2,7-diNF. Nitroreduction is inhibited by O(2) or allopurinol (20 microM), dicoumarol (100 microM), and rutin (50 microM). 9-Oxoreduction is inhibited by rutin, dicoumarol, and indomethacin (100 microM), but not by O(2) or allopurinol. Pyrazole or menadione does not inhibit nitro or 9-oxoreduction. Xanthine, hypoxanthine, 2-hydroxypyrimidine, and N'-methylnicotinamide support cytosol-catalyzed nitro, but not 9-oxo, reduction. The data suggest that the nitroreduction is catalyzed largely by a xanthine oxidase and partially by a diaphorase and 9-oxoreduction by a carbonyl reductase. The extents of the nitro and carbonyl reductions of the nitrofluorenes may determine their reactivities with DNA, and thus genotoxicities for the mammary gland.

A novel 4 S [3H]beta-naphthoflavone-binding protein in liver cytosol of female Sprague-Dawley rats treated with aryl hydrocarbon receptor agonists.

beta-Naphthoflavone (beta-NF) is a widely used inducer of phase-I and phase-II enzymes controlled by aryl hydrocarbon receptor (AhR). Studies of competitive binding with (3)H-labelled 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD), 3-methylcholanthrene (3-MC) and benzo[a]pyrene (B[a]P) have shown that beta-NF is a high-affinity ligand for AhR and also for polycyclic aromatic hydrocarbon (PAH)-binding protein, both soluble proteins of rat liver in 8 S and 4 S fractions, respectively, of sucrose gradients. This study examined binding of [(3)H]beta-NF to liver cytosolic proteins of female Sprague-Dawley rats. Treatment of rats with beta-NF, 3-MC, TCDD or alpha-naphthoflavone (alpha-NF) increased the specific [(3)H]beta-NF binding to liver cytosol up to 125-fold that of vehicle (corn oil)-treated rats (<100 fmol/mg of protein). Sucrose gradients revealed a large 4 S and a small 8 S peak of radioactivity from [(3)H]beta-NF binding to cytosols of beta-NF-, 3-MC-, TCDD- or alpha-NF-treated rats. Whereas co-incubation with the unlabelled beta-NF eliminated both peaks, co-incubation with 2,3, 7,8-tetrachlorodibenzofuran (TCDF) eliminated only the 8 S peak. The sucrose density gradient from [(3)H]TCDD binding to cytosol of beta-NF- or TCDD-treated rats yielded a small 4 S and a larger 8 S peak; only the latter was abolished by co-incubation with TCDF. Thus, the patterns of sedimentation, distribution and elimination of radioactivity from the 8 S fraction of the liver cytosols from beta-NF-, 3-MC-, TCDD- or alpha-NF-treated rats were characteristic for the AhR, whereas those from the 4 S fraction appeared specific for [(3)H]beta-NF binding. The data indicate that potent AhR agonists, TCDD, 3-MC and beta-NF, and to a lesser extent alpha-NF, a weak AhR agonist, induce a 4 S [(3)H]beta-NF-binding protein in liver cytosol of female rats. alpha-NF, beta-NF and 3-MC were effective competitors (80-85% inhibition) of the [(3)H]beta-NF-specific binding to the beta-NF-, 3 MC- or TCDD-induced 4 S protein, whereas several PAHs including B[a]P and benzo[e]pyrene were only weak competitors. The increased [(3)H]beta-NF binding was not associated with glycine N-methyltransferase activity. Hence, the 4 S [(3)H]beta-NF-binding protein described herein differs from the constitutive 4 S PAH-binding protein of rat liver cytosols in the inducibility by beta-NF and 3-MC, ligand-binding characteristics, and lack of glycine N-methyltransferase activity. Gel filtration on Sephacryl of liver cytosols from beta-NF-treated rats indicated a molecular mass of approximately 42 kDa for [(3)H]beta-NF-bound protein and suggested that it was derived from a large mass component that before the radioligand binding was eluted with the void volume of the gel and sedimented in a 7 S fraction of the sucrose gradient. The [(3)H]beta-NF binding activity was not eluted with glutathione S-transferase Ya, aldehyde-3-dehydrogenase or DT-diaphorase [NAD(P)H: quinone oxidoreductase] activities, which are AhR-controlled and beta-NF-inducible. Further studies are needed to determine the identity and function of this novel protein which may be involved either directly or indirectly (as a carrier protein) in xenobiotic metabolism in vivo.

Potent carcinogenicity of 2,7-dinitrofluorene, an environmental pollutant, for the mammary gland of female Sprague-Dawley rats.

Nitrofluorene compounds are environmental pollutants chiefly from incomplete combustion. This study examined carcinogenicities after one intramammary injection of 2-nitrofluorene (2-NF), 2, 7-dinitrofluorene (2,7-diNF) or dimethyl sulfoxide (DMSO) (solvent control) to 30-day-old and of 2-NF, 9-OH-2-NF, 9-oxo-2-NF, 2,7-diNF, 9-oxo-2,7-diNF, 2,5-dinitrofluorene, 9-oxo-2,4,7-trinitrofluorene, N-OH-2-acetylaminofluorene (N-OH-2-AAF) (carcinogen control) or DMSO to 50-day-old female Sprague-Dawley rats. In 30- and 50-day-old rats 6 and 8 glands/rat, respectively, were injected with 2.04 micromol of compound in 50 microliter/gland of DMSO. Whereas all compounds including DMSO yielded combined malignant and benign mammary tumor incidences of 33-87% by week 82 after injection, 2,7-diNF produced 100 and 93% incidences significantly (P < 0.001) sooner than did DMSO, i.e. by weeks 23-49 and 18-48 after treatment of 30- and 50-day-old rats, respectively. Rats treated with 2,7-diNF and 9-oxo-2,7-diNF had significantly (P < 0.0001) and marginally (P = 0. 0536) more mammary tumors, respectively, than DMSO-treated rats. In 2,7-diNF-treated rats, the ratio of malignant to benign mammary tumors was 5.4, whereas in all other groups it was <0.5. N-OH-2-AAF, a potent tumorigen when applied to the mammary gland as a solid or in suspension, did not yield the expected tumorigenicity here. The contrasting tumorigenic potencies of 2,7-diNF and N-OH-2-AAF may have been prompted by differences in their solubilities in DMSO. Thus, the poorly soluble 2,7-diNF was slowly absorbed from the injection sites since residues (up to 0.9% of the dose injected) were recovered even after 45 weeks. The data indicate prolonged exposure of the mammary gland to 2,7-diNF and suggest that contamination of the environment with 2,7-diNF, even at low levels, poses substantial carcinogenic risk.

Nitroreduction of nitrated and C-9 oxidized fluorenes in vitro.

Widespread environmental pollution with mutagenic and carcinogenic nitrofluorenes contributes to human health risks. Since nitroreduction leads to activation of many nitro compounds, nitroreduction of the nitrofluorene (NF) derivatives by one- and two-electron reductants was examined. Rates of nitroreduction catalyzed by xanthine oxidase (XO)/hypoxanthine and measured via stimulation of acetylated cytochrome c reduction increased with the number of nitro groups and oxidation at C-9: 9-oxo-2,4,7-triNF > 9-oxo-2,7-diNF > 2,7-diNF > 9-oxo-2-NF = 2,5-diNF > 9-hydroxy-2-NF > 2-NF. Ascorbate catalyzed one-electron reduction to nitro anion radicals which reacted with molecular O2 to yield superoxide. Rates of O2 uptake with 9-oxo-2,4,7-triNF and 9-oxo-2,7-diNF were 63 and 0.17 times those, respectively, with equivalent concentrations of nitrofurazone, a classical substrate. Superoxide formation was indicated by the approximately 75% regeneration of O2 upon addition of superoxide dismutase and catalase. 9-Oxo-2,4,7-triNF stimulated O2 uptake in the presence of XO/NADH with typical Michaelis-Menten kinetics with an apparent Km of 0.476 +/- 0.054 microM versus a Km of 6.18 +/- 0.719 microM for nitrofurazone. HPLC analyses of products from reduction catalyzed by XO or diaphorase of Clostridium with NADH showed the following trends for the rates of amine formation from 9-oxo-2,7-diNF > 2,7-diNF; 9-oxo-2-NF > 9-hydroxy-2-NF > 2-NF; 2,7-diNF > 2-NF; and 9-oxo-2,7-diNF > 9-oxo-2-NF. Little or no amine was formed in 95% O2, suggesting O2-labile intermediates. The data herein suggest that oxidation at C-9 and multiple nitro groups increase the potential for nitroreduction of the nitrofluorenes in vivo which may lead to genotoxic effects.

Modulation by beta-naphthoflavone of ovarian hormone dependent responses in rat uterus and liver in vivo.

The potential of an aryl hydrocarbon receptor (AhR) agonist, beta-naphthoflavone (beta-NF), to modulate ovarian hormone responses in the uterus and liver of 50-day-old Sprague-Dawley rats was examined. Treatment with beta-NF at 40 mg/kg of body weight consisted of 3 or 9 intraperitoneal injections in corn oil administered to ovariectomized (OVX) and sham-treated (SH) rats on day 5 through 7 or 1 through 9 after surgery performed on day 42 or 40 of age, respectively. Treatment of SH rats with either dose regimen of beta-NF effected a decrease (approximately 80%) in the uterine peroxidase activity, which was similar to that effected by ovariectomy (> 93%). By contrast, treatment of rats with alpha-naphthoflavone, an AhR antagonist, did not decrease the peroxidase activity. After the 9-dose treatment with beta-NF, decreases (approximately 70%) in hepatic estrogen receptor (ER) levels in both SH and OVX rats exceeded those effected by ovariectomy (30%). However, treatment with beta-NF partially prevented the ovariectomy-effected increase (approximately 1.5-fold) in body weight gain, decrease (approximately 67%) in uterine weight, and increase (3-fold) in uterine ER level. In both SH and OVX rats, treatment with beta-NF increased (1.7-fold) uterine progesterone receptor (PR) levels, which were unaffected by ovariectomy. Thus, the results suggest that the effect to of treatment with beta-NF is both mimicking and counteracting the effects of estrogen. Since beta-NF itself or upon conversion to metabolites by liver microsomes was shown herein not to be a ligand for uterine ER and PR, the aforementioned effects of beta-NF resembled those of certain halogenated polycyclic hydrocarbons, and thus may be mediated via AhR.

Aryl sulfotransferase IV deficiency in rat liver carcinogenesis initiated with diethylnitrosamine and promoted with N-2-fluorenylacetamide or its C-9-oxidized metabolites.

Down regulation of aryl sulfotransferase IV (AST IV) in promotion/progression of liver carcinogenesis by N-2-fluorenylacetamide (2-FAA) has been established. This study examined whether the C-9 oxidized metabolites of 2-FAA, which have recently been shown to promote diethylnitrosamine (DEN)-initiated liver carcinogenesis in male Sprague-Dawley rats, effect the above change. Hence, in DEN-initiated rats, the effects of promoting regimens of 9-OH-2-FAA or 9-oxo-2-FAA, 15 oral doses at 50 and 100 mumol/kg of body weight, were compared to those of 2-FAA at 50 mumol/kg of body weight and of the vehicle on the activity of N-hydroxy(OH)-2-FAA sulfotransferase (ST), an isozyme of AST IV and AST IV expression and distribution. Relative to the vehicle, treatment with the fluorenyl compounds led to decreased levels in hepatic N-OH-2-FAA ST activity and development of hepatic nodules and tumors which had still lower levels of the ST activity than the respective remnant livers. At approximately 8 months after treatment with the C-9-oxidized compounds at doses twice that of 2-FAA, the extents of decreases in the hepatic N-OH-2-FAA ST activity and cytosolic AST IV protein in tumors were comparable to those with 2-FAA. Immunocytochemical analysis showed close association of AST IV deficiency with neoplastic liver lesions. In comparison to N-OH-2-FAA, 9-OH-2-FAA had only low and 9-oxo-2-FAA lacked sulfate acceptor activity in the presence of male rat liver cytosol or AST IV. At 3.3-fold greater concentration than N-OH-2-FAA, 9-oxo-2-FAA inhibited (27%) the sulfate acceptor activity of N-OH-2-FAA in the presence of AST IV, which suggested interference by 9-oxo-2-FAA at the active site. Although the C-9-oxidized compounds do not appear to be substrates for N-OH-2-FAA ST, their ability to cause a decrease in N-OH-2-FAA ST activity and protein similar to that of 2-FAA supports their role in hepatocarcinogenesis. Whereas 9-OH-2-FAA had a 3.9-fold greater sulfate acceptor activity in the presence of female than male rat liver cytosol and inhibited dehydroepiandrosterone ST activity of female rat liver, N-OH-2-FAA and 9-oxo-2-FAA inhibited estrone ST activity of male rat liver, suggesting that the C-9-oxidized compounds as well as N-OH-2-FAA are substrates for STs other than AST IV.

Effect of ovariectomy on the in vitro and in vivo activation of carcinogenic N-2-fluorenylhydroxamic acids by rat mammary gland and liver.

N-Hydroxy-N-2-fluorenylacetamide (N-OH-2-FAA) and its benzamide analogue N-OH-2-FBA are mammary gland carcinogens in the female Sprague-Dawley rat. Ovariectomy inhibits tumorigenicity of topically applied N-OH-2-FAA suggesting modulation of carcinogen-activating enzymes in the gland. This study concerned the activation of N-OH-2-FAA and N-OH-2-FBA by the mammary gland and liver, a chief site of metabolism, from 50-day-old female rats and effects on the activation of ovariectomy performed at 22 days of age. The levels of N-debenzolyation of N-OH-2-FBA to N-hydroxy-N-2-fluorenamine (N-OH-2-FA), catalyzed by microsomal carboxylesterases in mammary gland and liver were similar and increased 1.5- and 1.7-fold, respectively, by ovariectomy. N-Debenzoylating activity in cytosols of both tissues appeared to be partially of microsomal origin. Mammary gland cytosol contained N-, O- and N,O-acyltransferase activities at levels 40-50% those of liver. N-Acyltransferase activity was determined via acetyl coenzyme A (AcCoA)-dependent acetylation of 2-FA and a new assay, N-OH-2-FAA-dependent acetylation of 9-oxo-2-FA. The latter activity was decreased in mammary gland by ovariectomy. Microsomal N-acyltransferase activities were <36% those of cytosols. AcCoA-dependent binding of N-OH-2-[ring-[3H]FBA to DNA, catalyzed by cytosol, was consistent with a two-step activation of N-OH-2-FBA involving esterase-catalyzed N-debenzoylation to N-OH-2-FA and its O-acyltransferase-catalyzed acetylation to the electrophilic N-acetoxy-2-FA. O-Acetyltransfer by mammary gland appeared to be rate-limiting since ovariectomy-dependent increases in N-debenzoylation did not increase binding with S9 fraction. Little or no sulfotransferase-catalyzed binding of N-OH-2-[ring-3H]FBA-derived N-OH-2-[ring-3H]FA was detected in the liver or mammary gland cytosol, respectively. The level of binding of N-OH-2-[ring-3H]FAA to DNA catalyzed by cytosolic N,O-acyltransferase was decreased approximately 23% in mammary gland and increased 1.2-fold in liver by ovariectomy. 32P-Postlabeling analyses indicated a single adduct N-(deoxyguanosin-8-yl)-2-fluorenamine in DNA of both tissues 24 h after one intraperitoneal injection of N-OH-2-FBA or N-OH-2-FAA. Respective levels were 3.6- and 5.5-fold greater in liver than mammary gland. After ovariectomy, the adduct levels from N-OH-2-FBA increased 1.8-fold in mammary gland and from N-OH-2-FAA decreased approximately 50% in both tissues. Thus, the ovariectomy-dependent changes in levels of enzymes activating N-OH-2-FBA and N-OH-2-FAA were consistent with in vivo DNA adduct levels in the target mammary gland, but not in the liver.

Debenzoylating and deacetylating activities of rat liver and mammary gland microsomes. Effect of ovariectomy.

This study compared the rates of N-deacylations of N-hydroxy-N-2-fluorenylbenzamide (N-OH-2-FBA) with those of its analogue, N-hydroxy-N-2-fluorenylacetamide (N-OH-2-FAA), by the mammary gland (tumor target for both compounds) and the liver of female Sprague-Dawley rats and examined the effect of ovariectomy on these activities. N-Debenzoylation of N-OH-2-FBA was catalyzed by the mammary and liver microsomes of 50-day-old female rats at similar rates (approximately 24 nmol/min/mg). The activity of both tissues increased (up to 1.8 times) after ovariectomy at 42, 32 and, especially, 22 days of age. The rapid hydrolysis appeared to be unique for the benzoyl group since N-OH-2-FAA was deacylated only approximately 0.05 and 0.004 times as fast by the liver and mammary microsomes, respectively, and these low rates were unaffected by ovariectomy. Since such substrate specificity would be of significance in the metabolism of xenobiotics and drug design, esterase activity and its sensitivity to ovariectomy at 22 days of age were examined with several acetylated and benzoylated substrates in the liver and mammary microsomes and compared with those of male liver. Tissues of rats of both sexes had a greater capacity to hydrolyze carboxyl esters than amides. Expect for N-2-fluorenylacetamide (2-FAA) and o-nitrophenylacetate (o-NPA), all substrates were hydrolyzed by liver microsomes of the male up to 3.9 times faster than by those of the female. Microsomes of female liver hydrolyzed acetylated substrates 1.2 to 25 times faster than benzoylated analogues except for N-OH-2-FBA and benzamide. By contrast, mammary gland microsomes hydrolyzed benzoylated compounds 1.4 to 333 times faster except for 2-naphthyl benzoate. Respective rates of hydrolysis of o-NPA by microsomes of liver and mammary gland were 1.7 and 0.6 times those of p-NPA. After ovariectomy, deacylating activities increased (up to 1.6 times) except for those of 2-FAA and acetanilide. All deacylations were > 98% inhibited by 0.1 mM paraoxon, indicating catalysis by serine hydrolases. The results suggest involvement of multiple carboxylesterases and indicate that certain benzoylated xenobiotics may have a greater effect on the mammary gland than acetylated xenobiotics because of their greater vulnerability to hydrolysis by esterases of mammary gland.

Detection of N-(deoxyguanosin-8-yl)-2-fluorenamine in DNA of peritoneal serosa and liver after intraperitoneal exposure of rats to N-hydroxy-N-2-fluorenylbenzamide or N-hydroxy-N-2-fluorenylacetamide.

DNA adduct formation was examined in rat peritoneal serosa, a tumor target for i.p. administered aqueous suspensions of N-hydroxy-N-2-fluorenylbenzamide (N-OH-2-FBA) and N-hydroxy-N-2-fluorenylacetamide (N-OH-2-FAA), and compared to that in the liver, which is a tumor target for N-OH-2-FAA in the male rat. 32P-Postlabeling analyses showed the presence of a single adduct, N-(deoxyguanosin-8-yl)-2-fluorenamine (dG-C8-FA), from activation of both hydroxamic acids by the serosa and liver in vitro and in vivo. The relatively low levels of dG-C8-FA (60-80 fmol/micrograms DNA) from N-OH-2-FBA in vitro were increased 2.7- and 35-fold upon the addition of acetyl coenzyme A (AcCoA) to the serosal cytosol and hepatic cytosol or microsomes respectively. By contrast, addition of AcCoA led to a decrease (approximately 34%) in the high level of dG-C8-FA (4330 fmol/micrograms DNA) from activation of N-OH-2-FAA by hepatic cytosol and did not alter the levels from activation by hepatic microsomes and serosal cytosols (530 and 78.3 fmol/micrograms DNA respectively). These data and the previously reported hydroxamic acid activation enzyme activities in the serosa and liver indicated that the precursor of dG-C8-FA, N-acetoxy-N-2-fluorenamine, was formed from N-OH-2-FAA chiefly via an intramolecular N,O-acetyltransfer and from N-OH-2-FBA via a two-step sequence of N-debenzoylation and AcCoA-dependent O-acetylation. The levels of dG-C8-FA were approximately 2- to 3-fold higher in the serosal DNA (up to 515 and 1012 fmol/micrograms DNA) after one (30 mumol/rat) and ten or eleven (cumulative dose of approximately 275 mumol/rat) injections of N-OH-2-FBA or N-OH-2-FAA than in the hepatic DNA. This correlated with the carcinogenicities of the hydroxamic acids, but was inversely proportional to the rates and extents of their activation in vitro. Multiple injections affected hepatic enzyme activities related to the activation of the hydroxamic acids in that the cytosolic N-debenzoylation of N-OH-2-FBA increased (approximately 1.7-fold) whereas N-OH-2-FAA acetyltransferase and sulfotransferase activities decreased. The effect of treatment with N-OH-2-FBA was greater than that with N-OH-2-FAA and was greater on the sulfotransferase activity (approximately 88% decrease). The latter suggested that N-OH-2-FBA, although a poor acceptor for an enzymatic sulfate transfer, may be carcinogenic for the rat liver.(ABSTRACT TRUNCATED AT 400 WORDS)

Modulations of hepatic gamma-glutamyltranspeptidase and N-hydroxy-N-2-fluorenylacetamide sulfotransferase activities following treatment of rats with a hepatocarcinogenic regimen: effect of partial hepatectomy.

The feasibility of using biochemical assays of gamma-glutamyltranspeptidase (gamma-GTP) and N-hydroxy-N-2-fluorenylacetamide sulfotransferase (N-OH-2-FAA ST) activities to monitor the effects of treatment of male Sprague-Dawley rats with a two-stage hepatocarcinogenic regimen was investigated. One week after initiation with diethylnitrosamine (200 mg/kg of bw), the rats were treated with 10 oral doses within 2 weeks of N-2-fluorenylacetamide (2-FAA) at 0.05 mmole/kg or vehicle (corn oil) at 5 ml/kg of body weight. After five doses of 2-FAA or corn oil, half of the rats in each group underwent partial (70%) hepatectomy (PH). Three days after completion of 2-FAA treatment, gamma-GTP activity increased approximately 8-fold in the livers of both the nonhepatectomized (-PH) and hepatectomized (+PH) groups. After 17 days, the enzyme activity decreased to the control level in the -PH group but increased 3.1-fold above the control level in the +PH group. After 31, 66, and 87 days, gamma-GTP activity increased only 1.4- to 2.6-fold in the -PH group, whereas that of +PH group increased 15- to 32-fold. N-OH-2-FAA ST activity, determined 3 days after completion of 2-FAA treatment, decreased by approximately 60% in the -PH and +PH groups. After 17 days, the effect of PH became evident in that the losses of N-OH-2-FAA ST activity were smaller (20%) in the -PH than in the +PH group (45.5%). After 31, 66, and 87 days, the respective decreases of 27, 29, and 41% in the +PH group were significant.(ABSTRACT TRUNCATED AT 250 WORDS)

Peroxidative metabolism of carcinogenic N-arylhydroxamic acids: implications for tumorigenesis.

Peroxidative oxidations of chemical carcinogens including N-substituted aryl compounds could result in their metabolic activation because the products react with cellular molecules and lead to cytotoxicity, mutagenicity, and carcinogenicity. In vivo, peroxidative activities are chiefly of neutrophilic leukocyte origin. Neutrophils may be attracted to the site(s) of exposure to carcinogen and, via phagocytosis and respiratory burst, release oxidants that catalyze carcinogen activation and/or cause DNA damage. Our studies, presented herein, concern oxidations of carcinogenic N-arylhydroxamic acids, N-hydroxy-N-2-fluorenylacetamide (N-OH-2-FAA), and N-hydroxy-N-2-fluorenylbenzamide (N-OH-2-FBA), by enzymatic and chemical systems simulating those of neutrophils, myeloperoxidase and hydrogen peroxide (H2O2) +/- halide, and hypohalous acid and halide at the physiologic concentrations (0.1 M Cl- and/or 0.1 mM Br-) and the pH (4-6.5) of phagocytosis. Studies also concern oxidations of the hydroxamic acids by rat peritoneal neutrophils stimulated to undergo respiratory burst and release myeloperoxidase in medium-containing 0.14 M Cl- +/- 0.1 mM Br-. The metabolites formed in the presence of exogenous H2O2 are consistent with two peroxidative mechanisms: one electron-oxidation to a radical that dismutates to equimolar 2-nitrosofluorene (2-NOF) and the ester of the respective hydroxamic acid and halide-dependent oxidative cleavage, especially efficient in the presence of Br-, to equimolar 2-NOF and the respective acyl moiety. 2-NOF and the esters undergo further enzymatic and nonenzymatic conversions to unreactive products and/or may bind to cellular macromolecules. The results suggest that peroxidative metabolism of N-arylhydroxamic acids by neutrophils, yielding the potent direct mutagen 2-NOF and the electrophilic esters, occurs in vivo and is involved in the activation and thus local tumorigenicities of the hydroxamic acids at the site(s) of application.

Activation of the carcinogens N-hydroxy-N-2-fluorenylbenzamide and N-hydroxy-N-2-fluorenylacetamide via deacylations and acetyl transfers by rat peritoneal serosa and liver.

Intraperitoneally administered N-hydroxy-N-2-fluorenylbenzamide (N-OH-2-FBA) and N-hydroxy-N-2-fluorenylacetamide (N-OH-2-FAA) are carcinogenic for rat peritoneum. The potential of peritoneal serosa to activate these compounds via deacylations and acyl transfers was compared to that of liver. N-Deacylations of N-OH-2-FBA and N-OH-2-FAA to N-2-fluorenylhydroxylamine (N-OH-2-FA) were faster by liver than serosa and by microsomes than cytosols. N-Debenzoylations of N-OH-2-FBA were 73- to 123-fold faster than N-deacetylations of N-OH-2-FAA. The esters, N-benzoyloxy-2-FBA and N-acetoxy-2-FAA, were O- and N-deacylated to N-OH-2-FA by liver, and the benzoate by serosa. Inhibition by paraoxon of the above deacylations implicated a serine carboxylesterase. Liver and serosa cytosols catalyzed acetyl CoA-, but not benzoyl CoA-, dependent and iodoacetamide (IAA)-sensitive N-acylation of N-2-fluorenamine (2-FA), implicating an acetyltransferase. In hepatic microsomes this activity was IAA-insensitive and partially inhibited by paraoxon. Liver cytosol, but not microsomes, used N-OH-2-FAA as an acyl donor and neither used N-OH-2-FBA. Liver and serosa catalyzed binding to DNA of N-OH-2-[ring-3H]FBA which was paraoxon-sensitive and increased by acetyl CoA, but not benzoyl CoA. Binding to DNA of N-OH-2-[ring-3H]FAA catalyzed by cytosols was approximately 22-fold greater in liver than in serosa and was IAA-sensitive. Microsome-catalyzed binding of this compound in both tissues was increased approximately 2-fold by acetyl CoA. The results support a two-step activation of N-OH-2-FBA in the liver consisting of esterase-catalyzed N-debenzoylation to N-OH-2-FA and an acyltransferase-catalyzed O-acetylation to the putative electrophile N-acetoxy-2-FA. In the serosa, binding to DNA appears to be due to rapid N-debenzoylation to N-OH-2-FA, a fraction of which is O-acetylated. Whereas activation of N-OH-2-FAA by liver and serosa microsomes may also involve N-OH-2-FA and/or its O-acetate, activation by the cytosols is consistent with N,O-acetyltransfer of N-OH-2-FAA to yield N-acetoxy-2-FA. The study provides first evidence for activation of N-OH-2-FBA by rat liver and of both compounds by peritoneum in vitro.

Metabolism of the carcinogen N-hydroxy-N-2-fluorenylacetamide by rat peritoneal neutrophils.

The in vitro metabolism of a locally carcinogenic N-hydroxy-N-2-fluorenylacetamide (N-OH-2-FAA) by rat peritoneal polymorphonuclear leukocytes (PMNL), chiefly neutrophils, elicited with intraperitoneal injections of proteose peptone, was examined. At 10(6) PMNL/ml in media containing halide (X-), 0.14 M Cl- +/- 0.1 mM Br- (without Ca++ and Mg++), addition of 10 nM phorbol myristate acetate (PMA) resulted in generation of superoxide anion and H2O2. Subsequent cetyltrimethylammonium Cl- (Cetac) addition at 0.002% effected myeloperoxidase (MPO) activity release. PMNL treated with PMA and/or Cetac did not metabolize N-OH-2-FAA (30 microM). However, 1-2 pulses of H2O2 (50 microM) after Cetac addition resulted in oxidation of N-OH-2-FAA to N-acetoxy-2-FAA (< 0.5 microM) and 2-nitrosofluorene (2-NOF) (1-2 microM). In the presence of Br- 2-NOF was increased (3-5 microM). The results are consistent with oxidation of N-OH-2-FAA by MPO/H2O2 and MPO/H2O2/X- via two pathways: one electron oxidation leading to N-acetoxy-2-FAA and 2-NOF, and X(-)-dependent oxidation to 2-NOF. N-Acetoxy-2-FAA (10 microM) incubated with PMNL under similar conditions was converted non-enzymatically to 4-OH-2-FAA (< or = 5 microM) and enzymatically to N-OH-2-FAA (< or = 3 microM). In the presence of H2O2, smaller amounts of these products were formed. Formation of N-OH-2-FAA was prevented by paraoxon (0.1 mM) suggesting O-deacetylase activity. However, accountability for N-acetoxy-2-FAA decreased with time, presumably because of binding to cellular macromolecules. With H2O2 addition, 2-NOF (10 microM) was converted to 0.5 or 0.25 microM 2-nitrofluorene by active PMNL or heat-inactivated cell lysates, respectively. Low recoveries of 2-NOF were also attributed to binding. The results suggest that PMNL may be involved in activation of the carcinogenic N-arylhydroxamic acids in vivo.

Activation of the carcinogen N-hydroxy-N-(2-fluorenyl)benzamide via chemical and enzymatic oxidations. Comparison to oxidations of the structural analogue N-hydroxy-N-(2-fluorenyl)acetamide.

Chemical or enzymatic oxidations of the carcinogen N-hydroxy-N-(2- fluorenyl)benzamide (N-OH-2-FBA) were investigated under the conditions facilitating one-electron oxidation or oxidative cleavage of N-hydroxy-N-(2-fluorenyl)acetamide (N-OH-2-FAA). HPLC methods were developed for separation and quantitation of the above hydroxamic acids and their respective oxidation products. To identify the products of oxidation of N-OH-2-FBA, N-(benzoyloxy)-2-FBA (N-BzO-2-FBA) was synthesized and shown to undergo ortho rearrangement to 1- and 3-BzO-2-FBA. Oxidation of N-OH-2-FBA (4.88 mM) with alkaline K3Fe(CN)6 in benzene was complete and yielded equimolar amounts of 2-nitrosofluorene (2-NOF) and the ester (chiefly N-BzO-2-FBA), indicative of one-electron oxidation to nitroxyl free radical which undergoes bimolecular dismutation. However, one-electron oxidation of N-OH-2-FBA (30 or 10 microM) by horseradish peroxidase/H2O2 at pH 7 or myeloperoxidase/H2O2 at pH 6.5 yielded only approximately 10% as much product as N-OH-2-FAA (30 microM). The addition of 0.1 mM Br- +/- 0.1 M Cl- at pH 4 to 6.5 increased 2-NOF formation in MPO/H2O2-catalyzed oxidations. Simulations of these oxidations with HOCl/Cl- or HOBr/Br- showed that the latter was more efficient, converting N-OH-2-FAA almost completely and less than or equal to 62% of N-OH-2-FBA to 2-NOF. The amounts of the ester (N- and o-BzO-2-FBA), which by itself did not contribute to 2-NOF formation or significant substrate regeneration, indicated that approximately 10% of 2-NOF originated from one-electron oxidation.(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction of C-nitroso aromatics with polyunsaturated fatty acids: route to lipid peroxidation.

The possibility that the interaction of C-nitroso aromatics with polyunsaturated fatty acids (PUFA) causes lipid peroxidation was investigated through determination of conjugated diene and malodialdehyde (MDA) formation after anaerobic/aerobic vs. aerobic incubations of nitrosobenzene (NOB) or 2-nitrosofluorene (2-NOF) with linoleic, linolenic or arachidonic acid or methyl linolenate. Anaerobic incubation of NOB or 2-NOF with linolenic acid at the molar ratio of 1:1 for 24 h yielded approximately 5.5-13% of the PUFA as conjugated diene which appeared stable upon exposure to air. Interaction of PUFA and 2-NOF or NOB yielded MDA, the amounts of which were significantly greater when 24-h anaerobic preceded 1-6-h aerobic incubation. Furthermore, the differences in the amounts of MDA resulting from 24- and 0-h anaerobic incubations were significantly greater when the molar ratio of 2-NOF (or NOB) to PUFA was increased (2.0 greater than 1.0 greater than 0.5). Superoxide dismutase or catalase had no effect on the yields of MDA following either anaerobic/aerobic or aerobic incubations of PUFA and 2-NOF. EDTA (1 or 10 microM) had no effect on the yields of MDA from aerobic incubations, but it decreased the amounts of MDA (by approximately 30 or 60%, respectively) from anaerobic/aerobic incubations. The data suggested that inhibition by EDTA was due to chelation of trace iron, which following anaerobic interaction of PUFA and 2-NOF might have been reduced to Fe2+ and contributed to the enhanced lipid peroxidation. Thus, adduction of C-nitroso aromatics to PUFA yields radical species which directly and/or via reaction with trace iron lead to lipid peroxidation. The lipophilicity of C-nitroso aromatics suggests that this process may be of consequence in their mutagenesis/carcinogenesis.

Oxidations of the carcinogen N-hydroxy-N-(2-fluorenyl)acetamide by enzymatically or chemically generated oxidants of chloride and bromide.

The oxidations of the carcinogen N-hydroxy-N-(2-fluorenyl)acetamide (N-OH-2-FAA) via one-electron (1e-) oxidation to equimolar 2-nitrosofluorene (2-NOF) and N-acetoxy-2-FAA and via oxidative cleavage to 2-NOF by chemically and myeloperoxidase (MPO)/H2O2 generated oxidants of Cl- and/or Br- were investigated. 2-NOF was determined spectrophotometrically in the reaction mixtures and by HPLC of their extracts; N-acetoxy-2-FAA was determined by HPLC. In the presence of individual or mixed halides at their physiologic concentrations (0.1 M Cl- and/or 0.1 mM Br-) and pH 4-6, MPO/H2O2-catalyzed oxidation of N-OH-2-FAA to 2-NOF via oxidative cleavage was much greater than 1e- oxidation. At the respective pH optima, oxidation was much more rapid with Br- and Br- + Cl- than with Cl-. HOBr or HOCl + Br- oxidized N-OH-2-FAA more rapidly than HOCl, also chiefly via oxidative cleavage. This suggested that, in the presence of MPO/H2O2 + Cl- + Br-, oxidation was due to HOBr from HOCl oxidation of Br- and/or oxidation of Br- by MPO/H2O2. In the presence of taurine (1 or 10 mM), a scavenger of hypohalous acids, MPO/H2O2 catalysis of oxidative cleavage was unaffected with Br-, prevented with Cl-, and partially prevented with Cl- + Br-. These results were linked to N-halotaurine formation since it was found that N-bromotaurine, but not N-chlorotaurine, oxidized N-OH-2-FAA chiefly to 2-NOF. With time N-chlorotaurine and N-bromotaurine appeared to undergo a pH-dependent halide exchange with Br- and Cl-, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Modifications of hepatic microsomal 9-oxidation of N-2-fluorenylacetamide in response to gonadectomy and treatment of rats with phenobarbital or diethylnitrosamine.

1. The hepatic microsomal 9-hydroxylation of N-2-fluorenylacetamide (2-FAA) is greater in the presence of male, or absence of female, hormones. Thus, 9-hydroxy-2-FAA was the major microsomal metabolite of male rats, which formed 6-fold greater amounts than did female rats. One week after gonadectomy, the amount of 9-hydroxy-2-FAA formed by male rats was decreased by 61%, whereas that formed by female rats was increased 1.4-fold. 2. Treatment of rats with phenobarbital (PB) increased 2- to 3-fold the capacities of hepatic microsomes of both sexes (normal and gonadectomized) for 9-hydroxylation of 2-FAA. 3. Hepatic microsomes of male rats also had greater capacities to form 9-oxo-2-FAA, the metabolite of 9-hydroxy-2-FAA, and 6-hydroxy-2-FAA, a newly identified microsomal metabolite of 2-FAA. These metabolites were also decreased by orchidectomy and induced by PB. 4. 9-Hydroxy-2-FAA was a poor substrate for hepatic microsomal UDP-glucuronyltransferase, and conjugation was not induced by treatment of rats with PB. This indicated retention of 9-hydroxy-2-FAA in the liver and/or further metabolism (e.g. to 9-oxo-2-FAA). 5. The formation of 9-oxo-2-FAA from 2-FAA or 9-hydroxy-2-FAA was increased (1.5-fold) two weeks after treatment of male rats with a single i.p. dose of diethylnitrosamine (200 mg/kg), an initiator of hepatocarcinogenesis. 6. Based on the data we suggest that 9-oxidized metabolites of 2-FAA, the preferential formation of which coincides with the susceptibility of the rat to hepatocarcinogenesis, are promoters.

Sex hormone-mediated effects on the phase I and phase II metabolism of N-2-fluorenylacetamide. Modulation of 9-hydroxylation.

Sex differences in the phase I (cytochrome P-450-catalyzed hydroxylations) and phase II (conjugations) metabolism of N-2-fluorenylacetamide (2-FAA) by the livers of 50-day-old Sprague-Dawley rats and effects of gonadectomy were determined. The higher level (1.4 times) of cytochrome P-450 in the microsomes of male rats correlated with their 8 and 1.3 times greater capacities to form 9-hydroxy(OH)-2-FAA and 7-OH-2-FAA respectively. One week after gonadectomy, the formation of 9-OH-2-FAA, the major metabolite in the male, was decreased by 70%, whereas in the female it was increased 1.3 times. Treatment of male rats with beta-naphthoflavone (beta-NF) increased the formation of phenolic metabolites and N-OH-2-FAA, but decreased that of 9-OH-2-FAA. The amounts of 9-OH-2-FAA were increased, however, in beta-NF-treated female and gonadectomized male rats. These sex hormone- and beta-NF-mediated differences in the extent of 9-hydroxylation of 2-FAA are discussed in relation to the fluctuations in the levels of specific cytochrome P-450 isozymes. In contrast to the phenolic metabolites and N-OH-2-FAA, 9-OH-2-FAA was a poor substrate for UDP-glucuronyltransferase; this conjugation was not induced by treatment of male rats with beta-NF. Hence, in the presence of male hormones, relatively large amounts of 9-OH-2-FAA were formed and possibly retained in the liver. A role of this alcohol as a potential promoter in hepatocarcinogenesis by 2-FAA is suggested.