Chronic ethanol ingestion and the hepatocarcinogenicity of N-hydroxy-N-2-fluorenylacetamide

The effect of long-term administration of 10% and 20% ethanol solutions on the hepatocarcinogenicity of N-hydroxy-N-2-fluorenylacetamide in two strains of rats was investigated. In tests with ethanol and carcinogen, started with 4- to 6-week-old Fischer rats the size of the liver and the extent of tumor formation were slightly less with male rats, and slightly more with females, as compared to rats fed only carcinogen. Experiments beginning with 16-week-old males, or with ethanol following carcinogen, were without additional effect. In NIH black rats, less susceptible to carcinogen and with a longer experimental period of 64 weeks, the concurrent intake of 10% ethanol did not affect the liver tumor incidence, although in females there seemed to be fewer cholangiomas and more hepatomas.     

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 injectionsof proteose peptone, was examined. At 10⁶ PMNL/ml in media containinghalide (X^–), 0.14 M Cl^– ± 0.1 mM Br^–(without Ca⁺⁺ and Mg⁺⁺), addition of 10 nM phorbol myristateacetate (PMA) resulted in generation of superoxide anion andH₂O₂. Subsequent cetyltrimethylammonium Cl^– (Cetac) additionat 0.002% effected myeloperoxidase (MPO) activity release. PMNLtreated with PMA and/or Cetac did not metabolize N-OH-2-FAA(30 µM). However, 1–2 pulses of H₂O₂ (50 µM)after Cetac addition resulted in oxidation of N-OH-2-FAA toN-acetoxy-2-FAA (<0.5 µM) and 2-nitrosofluorene (2-NOF)(1–2 µM). In the presence of Br^– 2-NOF wasincreased (3–5 µM). The results are consistent withoxidation of N-OH-2-FAA by MPO/H₂O₂ and MPO/H₂O₂/X^– viatwo pathways: one electron oxidation leading to N-acetoxy-2-FAAand 2-NOF, and X^–-dependent oxidation to 2-NOF. N-Acetoxy-2-FAA(10 µM) incubated with PMNL under similar conditions wasconverted non-enzymatically to 4-OH-2-FAA (5 µM) and enzymaticallyto N-OH-2-FAA (3 µM). In the presence of H₂O₂, smalleramounts of these products were formed. Formation of N-OH-2-FAAwas prevented by paraoxon (0.1 mM) suggesting O-deacetylaseactivity. However, accountability for N-acetoxy-2-FAA decreasedwith time, presumably because of binding to cellular macromolecules.With H₂O₂ addition, 2-NOF (10 µM) was converted to 0.5or 0.25 µM 2-nitrofluorene by active PMNL or heat-inactivatedcell lysates, respectively. Low recoveries of 2-NOF were alsoattributed to binding. The results suggest that PMNL may beinvolved in activation of the carcinogenic N-arylhydroxamicacids in vivo.     

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 peritonealserosa to activate these compounds via deacylations and acyltransfers was compared to that of liver. N-Deacylations of N-OH-2-FBAand N-OH-2-FAA to N-2-fluorenylhydroxylamine (N-OH-2-FA) werefaster by liver than serosa and by microsomes than cytosols.N-Debenz-oylations of N-OH-2-FBA were 73- to 123-fold fasterthan N-deacetylations of N-OH-2-FAA. The esters, N-benzoyloxy-2-FBAand N-acetoxy-2-FAA, were O- and N-deacylated to N-OH-2-FA byliver, and the benzoate by serosa. Inhibition by paraoxon ofthe above deacylations implicated a serine carboxylesterase.Liver and serosa cytosols catalyzed acetyl CoA-, but not benzoylCoA-, dependent and iodoacetamide (IAA)-sensitive N-acylationof N-2-fluorenamine (2-FA), implicating an acetyltransferase.In hepatic microsomes this activity was LAA-insensitive andpartially inhibited by para-oxon. 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]FBAwhich was paraoxon-sensitive and increased by acetyl CoA, butnot benzoyl CoA. Binding to DNA of N-OH-2-[ring-³H]FAA catalyzedby cytosols was 22-fold greater in liver than in serosa andwas IAA-sensitive. Microsome-catalyzed binding of this compoundin both tissues was increased 2-fold by acetyl CoA. The resultssupport a two-step activation of N-OH-2-FBA in the liver consistingof esterase-catalyzed N-debenzoylation to N-OH-2-FA and an acyltrans-ferase-catalyzedO-acetylation to the putative electrophile N-acetoxy-2-FA. Inthe serosa, binding to DNA appears to be due to rapid N-debenzoylationto N-OH-2-FA, a fraction of which is O-acetylated. Whereas activationof N-OH-2-FAA by liver and serosa microsomes may also involveN-OH-2-FA and/or its O-acetate, activation by the cytosols isconsistent 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-FBAby rat liver and of both compounds by peritoneum in vitro.     

Immunosuppression in primary liver and colon tumor induction with N-hydroxy-N-2-fluorenylacetamide and azoxymethane.

The question was examined as to whether immunosuppression in a rat model system would affect the carcinogenic processes leading to tumors in the liver and the large bowel. The protocols were designed to detect an increased incidence or a shorter latent period stemming from a change in immune status. Groups of rats were given injections prior to initiation of the carcinogen regimen and continuously thereafter with a purified gamma fraction of antilymphocytic serum (ALG). Appropriate controls received the gamma fraction of normal rabbit serum or 0.9% NaCl solution. Permanence of skin allografts showed that ALG was an effective immunosuppressive treatment. For liver cancer induction, rats were fed 120 ppm N-hydroxy-N-2-fluorenylacetamide in the diet for 16 weeks, then were continued on control diet. The animals given ALG developed liver tumors at a rate similar to that of controls. For cancer of the large bowel, rats received a single s.c. dose of 7.5 mg azoxymethane per kg per week for 16 weeks and were then held on control diet. With an identical ALG treatment, there were fewer intestinal tumors in the early part of the treatment, because of the important early development of liver angiosarcoma, not seen in control rats given injections of 0.9% NaCl solution. At a later time, the incidence of intestinal cancer was similar in rats on ALG or on 0.9% NaCl solution. Thus, immunosuppression had little effect on the rate of liver tumor formation with a liver carcinogen. Also, ALG led to the precocious development of liver angiosarcomas, but failed to affect intestinal cancer induction in animals given azoxymethane.     

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-hydroxybtions of a systemic mammarygland cardnogen, N-2-fluorenylaeetamide (2-FAA), by microsomalfractions of liver and mammary gland of female rats and theeffects of in vivo and/or in vitro modifiers of these oxidations.Pretreatment of lactating rats with 3-methylcholanthrene (3-MC)or ß-naphthoflavone (ß-NF) and non-lactating(50-day old virgin) rats with ß-NF showed similareffects in that the formation of 3-, 5-, 7-, 9- and N-hydroxy-2-FAAby hepatic microsomes was increased manyfold and the formationof 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 microsomesof lactating rats had capacity for N-hydroxylation which wasincreased {small tilde}3 times by pretreatment of rats with3-MC or ß-NF. All of the induced increases of metabolitesof 2-FAA in hepatic and mammary microsomes were inhibited by0.1 mM -naphthoflavone (-NF) in vitro. Pretreatment of non-lactatingrats with phenobarbital increased only the formation of 7-hydroxy-2-FAAin hepatic microsomes which was further stimulated by -NF invitro. The latter also stimulated the formation of 7- and 9-hydroxy-2-FAA by hepatic microsomes of the uninduced rats, buthad no effects in mammary microsomes, in which 9-hydroxy-2-FAAwas a major metabolite. Hence, the data showed qualitative andquantitative differences between lactating and non-lactatingrats in metabolism of 2-FAA by mammary microsomes which mayresult from differences in the levels (e.g., of cytochrome P-450)and activities of microsomal enzymes determined herein. In hepaticmicrosomes of these rats, differences in quantities of metabolitesof 2-FAA (3-, 7-, 9- and N-hydroxy-2-FAA) were found in cornoil-treated rats only. The solvent (methanol or acetone) usedfor addition of 2-FAA to the incubation mixtures altered quantitativelythe metabolite profiles in hepatic and mammary microsomes of3-MC or ß-NF treated rats. The formations of 1- and3- or 5- and 7-hydroxy-2-FAA were greater in the presence ofacetone or methanol, respectively. The results of this studysuggest that the formation of phenolic and N-hydroxy metabolitesof 2-FAA in both hepatic and mammary microsomes of lactatingrats is catalyzed by similar form(s) of cytochrome P-450 inducedby pretreatment with 3-MC or ß-NF. Lack of inductionN-hydroxylation of 2-FAA in mammary microsomes of non-lactatingrats supports our earlier conclusion that the formation of aproximate metabolite in mammary tumorigenesis by 2-FAA is accomplishedin the liver.     

Microsomal metabolism of the carcinogen, N-2-fluorenylacetamide, by the mammary gland and liver of female rats. II. Glucuronidation of ring- and N-hydroxylated metabolites of N-2-fluorenylacetamide

We determined UDP-glucuronyltransferse (UDP-GT) activities ofhepatic and mammary gland microsomes of female rats with p-nitrophenoland the ring- and N-hydroxylated metabolites of N-2-fluorenylacetamlde(2-FAA) and the effects of hepatic inducers of UDP-GT's on theseglucuronidations. Pre-treatment of non-lactating (NL)and lactating(L) rats with ß-naphthoflavone (ß-NF) significantlyincreased glucuronidations, of p-nitrophenil, aphenolic metabolitesof 2-FAA, especially of 5-hydroxy 2-FAA and also of N-hydrosy-2-FAAby hepatic microsomes. Pre-treatment of L rats with ß-NFor 3-methyl cholanthrene (3-MC) significantly incresed glucuronidationsof these compounds by mammary gland microsomes suggesting thatboth liver and mammary gland of L rats possess similar UDP-GTactivities. In NL rats, UDP-GT activities of mammary microsomestoward phenols were greater than in L rats, and except for thatof 5- and 7- hydroxy-2-FAA, were not inducible with ß-NF.The data obtained with L rats, the greater magnitude of stimulationof the hepatic UDP-GT of NL rats by ß-NF than by phenobarbital, and the lack of effect of the latter on UDP-GT ofmammary microsomes suggested that the phenolic metab olitesof 2-FAA and N-hydroxy-2-FAA share chiefly the characteristicsof substrates for group 1 UDP-GT activities (i.e., those induciblewith ß-NF or 3-MC). Neither inducer increased glucuronidationof 9-hydroxy-2-FAA, a relatively poor substrate for UDP-GT ofmammary or hepatic micro somes. In contrast to hepatic microsomeswhich formed considerable amounts of the glucuronide of N-hydroxy-2-FAA,mammary gland microsomes glucuronidated this substrate to aminor extent only. This suggested that glu curonide of N-hydroxy-2-FAAmay play a role in systemic, but not in local mammary tumorigenesisby N-hydroxy-2-FAA.     

The effect of neonatal administration of sex hormones on ribonucleic acid metabolism in the liver of male and female rats

Fifteen minutes after the intraperitoneal injection of ³²P labelled phosphate, normal adult male rats show a higher incorporation of isotope into their liver nuclear RNA than do females. A single injection of testosterone into neonatal female rats causes a higher uptake of ³²P in adult life, while a single injection of oestradiol into male neonates lowers the incorporation in adult life. Gonadectomy at 4 weeks of age has only a small effect on the subsequent incorporation of ³²P into nuclear RNA either in control rats or in rats injected with sex hormones immediately after birth, showing that this effect of liver metabolism is mainly determined by the hormanal pattern at about the time of birth. The possible relevance of this sex difference in RNA metabolism to the different sex incidence of spontaneous or induced liver cancer is discussed.