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.

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)

Laparoscopic-guided biopsy for diagnosis of hepatic candidiasis.

Histopathological evaluation of infected tissue is critical in the diagnosis of hepatic candidiasis since cultures are unreliable. Percutaneous techniques are inaccurate because lesions often are small and multifocal, and open biopsy is not always well-tolerated in acutely ill patients. The authors investigated the feasibility of laparoscopically guided biopsy in patients suspected of having hepatic candidiasis. Preliminary results suggest that laparoscopically guided biopsy is highly accurate and less invasive than open biopsy.

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.

Metabolite profile in milk of lactating rats after treatment with a carcinogen, N-2-fluorenylacetamide.

Metabolites were determined in milk and urine of lactating rats 4 to 5 hr after each of 3 to 6 ip injections of N-2-fluorenylacetamide (2-FAA) at 0.2 mmol/kg of body weight. Milk contained 2-FAA as the major free compound, variable amounts of N-2-fluorenamine (2-FA) and phenols (7- greater than 5- greater than 3-hydroxy-2-FAA) chiefly as glucuronides, and very small amounts of the glucuronide of N-hydroxy-2-FAA. Urine contained large amounts of the phenols and N-hydroxy-2-FAA as free and conjugated compounds, but in contrast to milk, only small amounts of 2-FAA and no 2-FA. Pretreatment of rats with beta-napthoflavone, an inducer of microsomal C- and N-hydroxylations of 2-FAA, increased the amounts of 3- and 5-hydroxy-2-FAA in milk and of 3-hydroxy-2-FAA in urine. However, the total amounts of the compounds excreted in 1 ml of milk or urine, i.e. 0.05 to 0.13% or 0.6% of the dose of 2-FAA, respectively, were similar in the uninduced and induced groups. Protein hydrolysates of milk of 2-FAA-treated rats and of milk interacted with 2-nitrosofluorene (2-NOF) in vitro both contained 2-FA and 9-oxo-2-FA. This suggested formation of 2-NOF in vivo possibly by peroxidative metabolism of N-OH-2-FAA. Since 2-NOF has been reported to form adducts with unsaturated lipids, the effect of treatment of lactating rats with 2-FAA on the fatty acid composition of milk lipids was examined.(ABSTRACT TRUNCATED AT 250 WORDS)

Peroxidative metabolism of a carcinogen, N-hydroxy-N-2-fluorenylacetamide, by rat uterus and mammary gland in vitro.

Lactoperoxidase-catalyzed metabolism of N-hydroxy-N-2-fluorenylacetamide (N-OH-2-FAA) may be via one-electron oxidation to nitroxyl free radical which dismutates to equimolar N-acetoxy-N-2-fluorenylacetamide and 2-nitrosofluorene (2-NOF) and/or a Br(-)-dependent oxidative cleavage to 2-NOF. Hence, the 2-NOF:N-acetoxy-N-2-fluorenylacetamide ratios reflect the relative contributions of the two peroxidative pathways to the metabolism of N-OH-2-FAA. Peroxidative activities of rat uterus (UT) and mammary gland (MG) were extracted with a cationic detergent, cetyltrimethylammonium bromide (Cetab). MG extracts had 1 to 5% the specific activity of UT extracts when assayed with guaiacol as hydrogen donor. At 0.004% Cetab, which in the incubation media corresponds to the approximate physiological levels of 0.1 mM Br(-), oxidation of N-OH-2-FAA by UT extracts yielded a product ratio indicative of both peroxidative pathways with Br(-)-dependent oxidation prevailing. At 0.4% Cetab, one-electron oxidation was negligible and Br(-)-dependent conversion of N-OH-2-FAA to 2-NOF was markedly enhanced. At both 0.004 and 0.4% Cetab, MG extracts yielded only 2-NOF, suggesting solely Br(-)-dependent oxidation. With equivalent guaiacol units of peroxidative activities, MG extracts produced much lower amounts of 2-NOF than did UT extracts. The low specific activities of MG extracts necessitated the use of larger amounts of protein, which might have interfered with peroxidative metabolism. At 0.004% Cetab, formation of 2-NOF by the Br(-)-dependent pathway was greater at pH 5.5 than at 7.4. At acid pH, small amounts of 2-nitrofluorene were also formed by UT and MG extracts and could be attributed to further oxidation of 2-NOF. Peroxidative activities of the UT and MG extracts may be of granular leukocyte origin and their potential role in carcinogen activation and tumorigenesis is discussed.

Microsomal metabolism of the carcinogen, N-2-fluorenylacetamide, by the mammary gland and liver of female rats. I. Ring- and N-hydroxylations of N-2-fluorenylacetamide.

We determined ring- and N-hydroxylations of a systemic mammary gland carcinogen, N-2-fluorenylacetamide (2-FAA), by microsomal fractions of liver and mammary gland of female rats and the effects of in vivo and/or in vitro modifiers of these oxidations. Pretreatment of lactating rats with 3-methylcholanthrene (3-MC) or beta-naphthoflavone (beta-NF) and non-lactating (50-day old virgin) rats with beta-NF showed similar effects in that the formation of 3-, 5-, 7-, 9- and N-hydroxy-2-FAA by hepatic microsomes was increased manyfold and the formation of 1-hydroxy-2-FAA was induced. In mammary gland microsomes, the formation of 3-, 5- and 7-hydroxy-2-FAA was likewise increased, but of 9-hydroxy-2-FAA was unaffected. Only mammary microsomes of lactating rats had capacity for N-hydroxylation which was increased approximately 3 times by pretreatment of rats with 3-MC or beta-NF. All of the induced increases of metabolites of 2-FAA in hepatic and mammary microsomes were inhibited by 0.1 mM alpha-naphthoflavone (alpha-NF) in vitro. Pretreatment of non-lactating rats with phenobarbital increased only the formation of 7-hydroxy-2-FAA in hepatic microsomes which was further stimulated by alpha-NF in vitro. The latter also stimulated the formation of 7- and 9- hydroxy-2-FAA by hepatic microsomes of the uninduced rats, but had no effects in mammary microsomes, in which 9-hydroxy-2-FAA was a major metabolite. Hence, the data showed qualitative and quantitative differences between lactating and non-lactating rats in metabolism of 2-FAA by mammary microsomes which may result from differences in the levels (e.g., of cytochrome P-450) and activities of microsomal enzymes determined herein. In hepatic microsomes of these rats, differences in quantities of metabolites of 2-FAA (3-, 7-, 9- and N-hydroxy-2-FAA) were found in corn oil-treated rats only. The solvent (methanol or acetone) used for addition of 2-FAA to the incubation mixtures altered quantitatively the metabolite profiles in hepatic and mammary microsomes of 3-MC or beta-NF treated rats. The formations of 1- and 3- or 5- and 7-hydroxy-2-FAA were greater in the presence of acetone or methanol, respectively. The results of this study suggest that the formation of phenolic and N-hydroxy metabolites of 2-FAA in both hepatic and mammary microsomes of lactating rats is catalyzed by similar form(s) of cytochrome P-450 induced by pretreatment with 3-MC or beta-NF.(ABSTRACT TRUNCATED AT 400 WORDS)

Modifications of carcinogen metabolism in hepatic microsomes of suckling young by 3-methylcholanthrene or beta-naphthoflavone administered to lactating rats.

The effects of treating lactating rats with 3-methylcholanthrene (3-MC) or beta-naphthoflavone (beta-NF) (three i.p. injections of 20 or 40 mg compound/kg of body weight) on hepatic microsomal enzymes of their suckling young were examined. This treatment had no apparent effect on the contents of cytochromes P-450 and b5 or on the activities of NADH- and NADPH-cytochrome c reductases in hepatic microsomes of the pups. However, these microsomes had 8- and 6-fold increased capacities for hydroxylations of benzo[a]pyrene (B[a]P) and N-2-fluorenylacetamide (2-FAA) respectively. These increases were about 5-fold greater in the hepatic microsomes of the dams, in which they were inhibited by 0.1 mM alpha-naphthoflavone (alpha-NF) in vitro 72-81 and 89-95% and by 0.1 mM beta-NF in vitro 12-41 and 60-76% respectively. In the pups, the induced activities were also inhibited, whereas the basal hydroxylations of B[a]P and 2-FAA were stimulated by alpha-NF 2.7- and 5.0-fold and by beta-NF 1.4- and 2.4-fold respectively. The inhibition of the induced hydroxylations by alpha-NF and beta-NF may be explained by their higher affinities (Ks, 0.14 and 0.28 microM, respectively) than those of B[a]P and 2-FAA (Ks, 4.4 to 8.8 and 2.4 to 3.1 microM, respectively) for cytochrome P-450. Whereas beta-NF gave a type I binding spectrum, alpha-NF gave a spectrum composed of type I and reverse-type I elements. Analysis of metabolites of 2-FAA showed differences in their type and amounts formed by hepatic microsomes of beta-NF-treated lactating rats and their pups. Thus, in the dams the formation of 1-, 3-, 5-, 7-, 9- and N-hydroxy-2-FAA was increased by 9-, 30-, 40-, 5-, 20- and 5-fold respectively. In the pups, the formation of 1-, 3-, 5-, 7- and N-hydroxy-2-FAA was increased by 2-, 30-, 18-, 4- and 27-fold respectively. All these increased hydroxylations were inhibited by 0.1 mM alpha-NF in vitro. In the hepatic microsomes of pups from the corn oil-treated dams, alpha-NF stimulated all ring-hydroxylations, but not N-hydroxylation of 2-FAA. The results support earlier findings that microsomal enzymes differ in immature and mature rat liver and suggest that N-hydroxylation of 2-FAA, the activation required for carcinogenesis, and specific ring-hydroxylations are catalyzed by different cytochrome P-450 isozymes.(ABSTRACT TRUNCATED AT 400 WORDS)

Binding of daunomycin to DNA and the inhibition of RNA and DNA synthesis.

With synchronized tissue culture cells (L929), daunomycin had the greatest inhibitory effect on cell growth when the drug was administered during the later stages of cell division (late S, G2, and M). The level of binding of daunomycin to DNA was not found to be influenced by the phase of the cell cycle. The highest level of radioactivity from [eH]-daunomycin was bound to DNA of the heterochromatin fraction. Both RNA and DNA syntheses were inhibited in isolated enzyme systems when daunomycin-treated DNA, from which the unbound drug was removed by passage through Sephadex column, was used. DNA polymerase was reduced to one-fifth of the control activity, while that of RNA polymerase was reduced to one-half. Similar experiments with daunomycin-treated RNA and DNA polymerase preparations showed that the drug had no effect on the activities of the enzymes per se. Hence, the reduction of RNA and DNA polymerase activities could be accounted for by the loss of template activity of the drug-treated DNA. Daunomycin caused by a marked drop in the formation of a complex between RNA polymerase and DNA, indicating that the binding of daunomycin to DNA may give rise to steric hindrance effects that interfere with the association of the template to RNA polymerase enzyme. Sedimentation profile in alkaline sucrose density gradient of DNA that had been treated with daunomycin showed that no change in the molecular weight could be demonstrated.

Rifting in iceland: new geodetic data.

Small but measurable lengthening of several survey lines within the eastern rift zone of Iceland occurred between 1967 and 1970. The changes can be interpreted as a widening of the rift by 6 to 7 centimeters, possibly during the 1970 eruption of Hekla volcano.