Alcoholic liver disease may be prevented with adequate nutrients.

Alcoholic liver disease is the result of a synergism between ethanol and nutritional factors. Therefore, it follows that the pathogenesis of alcoholic liver disease may be affected by the regulation of the intake of nutrients as well as by the regulation of the intake of ethanol.

Effect of bovine serum albumin on the extent of ortho rearrangement of N-(sulfooxy)-2-fluorenylacetamide and of enzymatically activated N-hydroxy-2-fluorenylacetamide and on the binding of reactive esters to nucleic acids.

We have investigated the effect of the bovine serum albumin (BSA)-catalyzed ortho rearrangement of synthetic and enzymatically generated N-(sulfooxy)-2-fluorenylacetamide (NSF) to the O-sulfate esters on the binding of NSF to transfer ribonucleic acid (tRNA) and to deoxyribonucleic acid (DNA). Binding of synthetic NSF to tRNA and DNA decreased approximately 90 and 70%, respectively, in the presence of BSA. Under these conditions, the ortho rearrangement, a minor reaction in the absence of BSA, was nearly quantitative. The decrease of adduct formation to nucleic acids was not attributable to the competitive binding of NSF to BSA. Binding of NSF, generated by cytosolic sulfonation of the arylhydroxamic acid, N-hydroxy-2-fluorenylacetamide, to tRNA, was diminished approximately 97% in the presence of BSA while the ortho rearrangement of the sulfonated substrate increased from less than 0.5% to approximately 50%. Adduct formation of DNA with N-hydroxy-2-fluorenylacetamide, activated by enzymatic sulfonation, was inhibited approximately 90% in the presence of BSA. In these experiments, the catalytic effect of BSA on the ortho rearrangement of enzymatically sulfonated N-hydroxy-2-fluorenylacetamide was of the same order as observed in the experiments with tRNA. The data obtained on the covalent interaction of DNA with enzymatically activated N-hydroxy-2-fluorenylacetamide indicate that, in addition to NSF, another electrophilic species accounts for binding of activated N-hydroxy-2-fluorenylacetamide to DNA. The data support the view that the reactive electrophile is N-acetoxy-2-fluorenamine, resulting from the N,O-transacetylation of N-hydroxy-2-fluorenylacetamide.(ABSTRACT TRUNCATED AT 250 WORDS)

The catalytic effect of bovine serum albumin on the ortho rearrangement of the potential ultimate carcinogen, N-(sulfooxy)-2-(acetylamino)fluorene, generated enzymatically from N-hydroxy-2-(acetylamino)fluorene and evidence for substrate specificity of the enzymatic sulfonation of arylhydroxamic acids.

This investigation examines the catalytic effect of bovine serum albumin on the ortho rearrangement of the possible ultimate carcinogen, N-(sulfooxy)-2-(acetylamino)fluorene, generated from N-hydroxy-2-(acetylamino)fluorene by the sulfotransferase(s) in the cytosol of rat liver. With various preparations of cytosol, 55-75% of the substrate, N-hydroxy-2-(acetylamino)-fluorene, was found to rearrange to the nonmutagenic and noncarcinogenic o-(sulfooxy) esters, 1- and 3-(sulfooxy)-2-(acetylamino)fluorene, in the presence of bovine serum albumin, while less than 1% of the substrate rearranged in its absence. In presence of bovine serum albumin the cytosolic reduction of N-(sulfooxy)-2-(acetylamino)fluorene to 2-(acetylamino)fluorene decreased by 60-90% and its solvolytic degradation to 4-hydroxy-2-(acetylamino)fluorene by 80-90%. The covalent interaction of enzymatically generated N-(sulfooxy)-2-(acetylamino)fluorene with the nucleophilic acceptors, N-acetyl-L-methionine and guanosine, was lowered by greater than 90% by addition of bovine serum albumin. These measurements indicated that the albumin-catalyzed ortho rearrangement controls the rates of concurrent metabolic and degradative reactions of N-(sulfooxy)-2-(acetylamino)fluorene. The results are in agreement with previous findings of a catalytic effect of serum albumin on the ortho rearrangement of synthetic N-(sulfooxy)-2-(acetylamino)fluorene. In contrast to its catalytic effect on the formation of o-(sulfooxy) esters from N-(sulfooxy)-2-(acetylamino)fluorene, bovine serum albumin had no effect on the formation of o-(acetylamino)fluorenols. To assess the substrate specificity of bovine serum albumin, its effect on the rearrangement of N-hydroxy-2-(benzoylamino)fluorene, a carcinogenic analogue of N-hydroxy-2-(acetylamino)fluorene, was analyzed under conditions of cytosolic sulfonation.(ABSTRACT TRUNCATED AT 250 WORDS)

Catalytic effect of serum albumin on the o-rearrangement of N-sulfooxy-2-acetylaminofluorene, a potential hepatocarcinogen in the rat, to nonmutagenic sulfuric acid esters of o-amidofluorenols.

Bovine serum albumin (BSA) catalyzes the o-rearrangement of the reactive electrophile, N-sulfooxy-2-acetylaminofluorene (NSF), a potential ultimate hepatocarcinogen in the rat, to the nonmutagenic sulfuric acid esters of 1- and 3-hydroxy-2-acetylaminofluorene. Conversion of NSF was proportional to BSA concentrations ranging from 0.25 to approximately 4 mg BSA/ml incubation mixture. At concentrations greater than or equal to 5 mg BSA/ml, approximately 90% of NSF was converted to the sulfuric acid esters of the o-amidofluorenols. Human serum albumin (HSA) likewise catalyzed the o-rearrangement of NSF. However, the catalytic activity of HSA was only approximately 50% of the activity of BSA. The catalytic effect of BSA was abolished by heat denaturation. However, it was not changed by dialysis or by anion exchange chromatography. These observations indicated that the catalytic effect requires intactness of the tertiary structure of BSA and is not due to a contaminant(s) of low or high molecular weight. There were no differences in the catalytic activity of three separate fractions of chromatographed BSA, suggesting that the catalytic activity is associated with the entire BSA molecule. In contrast to serum albumin, gamma-globulin (bovine or human) did not catalyze the o-rearrangement of NSF. The solvolytic degradation of NSF to 4-hydroxy-2-acetylaminofluorene, a major reaction in the absence of BSA, occurred only to a minor extent in the presence of BSA. These data indicated that the BSA-catalyzed o-rearrangement determines the rates of concurrent reactions involved in the degradation of NSF. BSA and HSA did not catalyze the o-rearrangement of N-acetoxy-2-acetylaminofluorene (N-OAC-2-AAF), the acetate ester of N-hydroxy-2-acetylaminofluorene (N-OH-2-AAF), to the acetic acid esters of the o-amidofluorenols. These findings suggest that the albumin-catalyzed o-rearrangement occurs preferentially with esters of fluorenylhydroxamic acids that readily ionize in aqueous media.

Solvolysis and metabolic degradation, by rat liver, of the ultimate carcinogen, N-sulfonoxy-2-acetylaminofluorene.

The synthetic ultimate carcinogen, N-sulfonoxy-2-acetylaminofluorene (K+ salt, N-OSO3K-2-AAF), undergoes several solvolytic and metabolic reactions that have not been reported heretofore. In aqueous media, 45-50% of N-OSO3K-2-AAF is converted to 4-hydroxy-2-acetylaminofluorene, presumably by the ionic mechanism proposed previously for the formation of the m-amidofluorenol from N-acetoxy-2-acetylaminofluorene. 4-Hydroxy-2-acetylaminofluorene was not mutagenic and did not react with the nucleophile, guanosine. In aqueous media, rearrangement of N-OSO3K-2-AAF to the biologically inactive o-amidofluorenols, 1- and 3-hydroxy-2-acetylaminofluorene, or to the respective sulfates, were only minor reactions (approximately 3% and approximately 6-8%, respectively). In the presence of bovine serum albumin, N-OSO3K-2-AAF was converted nearly quantitatively to the sulfates of 1- and 3-hydroxy-2-acetylaminofluorene. The rearrangement was abolished by heat denaturation of the bovine serum albumin. In the presence of rat liver homogenate or of hepatic cytosol of the rat, 30-40% of N-OSO3K-2-AAF was reduced to the procarcinogen, 2-acetylaminofluorene, at the expense of 4-hydroxy-2-acetylaminofluorene. The reduction appears to be catalyzed by a low molecular weight compound in the cytosol of rat liver since denaturation of cytosolic proteins by heat had no effect on the extent of the reduction. Cytosolic reduction of N-OSO3K-2-AAF to 2-acetylaminofluorene was markedly inhibited by the nucleophile, N-acetyl-L-methionine. This observation indicates that covalent interaction of N-OSO3K-2-AAF with nucleophiles and reduction of N-OSO3K-2-AAF are competing reactions.

Interaction of the synthetic ultimate carcinogens, N-sulfonoxy- and N-acetoxy-2-acetylaminofluorene, and of enzymatically activated N-hydroxy-2-acetylaminofluorene with nucleophiles.

The interaction of four cellular nucleophiles with the putative ultimate carcinogens N-sulfonoxy-2-[ring-3H]acetylaminofluorene (N-sulfonoxy-2-AAF) and N-acetoxy-2-[ring-3H] acetylaminofluorene (N-acetoxy-2-AAF), and with N-hydroxy-2-[ring-3H]acetylaminofluorene (N-hydroxy-2-AAF) activated to the ultimate carcinogens by enzymatic sulfonation or transacetylation was determined. The adducts were isolated and adduct formation was quantified by isotope dilution. The order of nucleophilicity of the acceptors was guanosine greater than tRNA congruent to polyguanylic acid (poly G) greater than N-acetyl-L-methionine when N-sulfonoxy-2-AAF, N-acetoxy-2-AAF or N-hydroxy-2-AAF activated by transacetylation were the electrophiles. In the case of N-hydroxy-2-AAF activated by enzymatic sulfonation, the order of nucleophilicity was N-acetyl-L-methionine greater than guanosine congruent to tRNA greater than poly G. The increase in the reactivity of N-acetyl-L-methionine is hypothesized to be due to cytosolic enzyme(s) which facilitate transfer of the methionine residue from the nitrogen to carbon atoms 3 and 1 of the fluorene moiety. Of the two synthetic esters, N-sulfonoxy-2- AAF exhibited greater electrophilicity than N-acetoxy-2-AAF. The rate of adduct formation of N-sulfonoxy-2-AAF and of N-acetoxy-2-AAF with each nucleophile was a function of nucleophile concentration, indicative of a bimolecular reaction mechanism. The interaction is thought to involve attack of the nucleophile on the uncharged ultimate carcinogen, although interaction with an ion pair cannot be eliminated. The mutagenicity of N-sulfonoxy-2-AAF, N-acetoxy-2-AAF and of enzymatically activated N-hydroxy-2-AAF was evaluated by the Ames test. N-Sulfonoxy-2-AAF was virtually inactive, while N-acetoxy-2-AAF exhibited weak mutagenicity. N-Hydroxy-2-AAF activated by enzymatic sulfonation exhibited greater mutagenicity than synthetic N-sulfonoxy-2-AAF. The mutagenicity and reactivity of ultimate carcinogens derived from N-hydroxy-2-AAF by enzymatic activation do not necessarily coincide with the mutagenicity and reactivity of the synthetic ultimate carcinogens.

Interaction of nucleophiles with the enzymatically-activated carcinogen, N-hydroxy-2-acetylaminofluorene, and with the model ester, N-acetoxy-2-acetylaminofluorene.

A new method was devised to study adduct formation of N-acetyl-L-methionine (N-Ac-met) with activated species of the carcinogen N-hydroxy-2-acetylaminofluorene (N-OH-2-AAF). Following degradation of the adducts, the isomeric methylthio derivatives of 2-acetylaminofluorene (2-AAF) and 2-aminofluorene (2-AF) were separated and quantified by h.p.l.c. With the use of this method the nucleophilicity of N-Ac-met toward enzymatically produced N-acetoxy and N-sulfonoxy derivatives of 2-AF and 2-AAF, respectively, and toward the synthetic model ester, N-acetoxy-2-acetylaminofluorene (N-OAc-2-AAF), was determined. For comparison, the interaction of tRNA and polyguanylic acid (poly G) with the above compounds was measured by standard procedures. tRNA was an efficient acceptor of enzymatically formed N-acetoxy and N-sulfonoxy derivatives as well as of N-OAc-2-AAF. The nucleophilicity of N-Ac-met toward enzymatically formed N-sulfonoxy-2-AAF was comparable with that of tRNA. However, its reactivity with N-OAc-2-AAF and enzymatically generated N-acetoxy-2-aminofluorene (N-OAc-2-AF) was only 20% of that of tRNA. Poly G was as reactive as tRNA toward synthetic N-OAc-2-AAF and enzymatically generated N-OAc-2-AF but was only 25% as efficient as tRNA or N-Ac-met in accepting enzymatically formed N-sulfonoxy-2-AAF. The difference in the interaction of the three nucleophiles with N-OAc-2-AAF and N-OAc-2-AF compared with N-sulfonoxy-2-AAF indicate that N-OAc-2-AAF is not a general model of the ultimate electrophilic metabolites of 2-AAF. Studies of the kinetics of the interaction of N-OAc-2-AAF with N-Ac-met, tRNA and poly G demonstrated dependence of adduct formation on nucleophile concentration, indicative of a bimolecular mechanism. Arylamidonium or nitrenium ions are therefore not necessarily the ultimate electrophilic metabolites of 2-AAF obligatory for interaction with cellular nucleophiles. There was no evidence that nitrenium ions are intermediates in the cytosolic reduction of N-OH-2-AAF to 2-AAF.

Differential effect of chronic ethanol consumption by the rat on microsomal oxidation of hepatocarcinogens and their activation to mutagens.

The effect of chronic ethanol consumption by rats on hepatic microsomal activation of the hepatocarcinogens dimethylnitrosamine (DMN) and 2-acetylaminofluorene (2-AAF) was investigated. There was a marked increase in the rate of the oxidative demethylation of DMN and its activation to a mutagen by microsomes following ethanol intake. N- and C-hydroxylation of 2-AAF were measured at substrate concentrations ranging from 2 to 70 microM. The ratio of formation of N-hydroxy-2-acetylaminofluorene to C-hydroxy-2-acetylaminofluorenes increased with decreasing substrate concentration, suggesting enhanced carcinogenic potential of 2-AAF with diminishing levels of carcinogen. Kinetic analysis indicated that N-hydroxylation as well as 7-, 5- and 3-hydroxylation of 2-AAF do not follow Michaelis-Menten kinetics. In contrast to the marked inductive effect of ethanol consumption on the metabolic activation of DMN, only a minimal random effect on the N-hydroxylation of 2-AAF was demonstrable in two separate experiments. Furthermore, N-hydroxylation of 2-AAF by microsomes from control and ethanol-treated rats followed similar kinetics. While ethanol consumption enhanced the mutagenic activation of DMN by hepatic microsomes, no such effect of ethanol consumption on the conversion of 2-AAF to a mutagen was observed. The data indicate that chronic ethanol consumption does not have a general inductive effect on the microsomal activation of hepatocarcinogens.

The kinetic properties of the ecto-ATPase of human peripheral blood lymphocytes and of chronic lymphatic leukemia cells.

This study examines whether the activity of the Mg2+-dependent ecto-ATPase of the surface membrane of the human lymphocyte is changed in chronic lymphocytic B-cell leukemia (CLL-B) and may be an indicator of malignant transformation. The ecto-ATPase activities of preparations consisting predominantly of T or B cells were compared to each other and to the ecto-ATPase of the CLL peripheral blood lymphocytes (PBL). The specific activities and kinetic constants of the ecto-ATPase of the cell preparations were determined with [gamma-32P] adenosine triphosphate (ATP) as substrate. B-enriched lymphocytes had nearly fourfold greater specific activity and apparent Vmax than T-enriched lymphocytes, while the Km values of both cell types showed no significant difference. The specific activities and kinetic constants of the ecto-ATPase of the CLL PBL were significantly higher than the corresponding values of PBL or of B-enriched lymphocytes. Judging from the kinetic constants the ecto-ATPase of the CLL-B lymphocyte appears to be an enzyme that is distinctly different from that of the normal B cell. On the basis of the kinetic properties, the ecto-ATPase of the B cell appears to be identical with that of the T cell. The differences in the maximal velocities of the hydrolysis of ATP by B and T cells are likely due to a greater number of enzymatic sites on the B cell.

Hydrolysis of uridine diphosphate N-acetyl-D-glucosamine by embryonic cells of the hamster and rat. Determination by high-pressure liquid chromatography.

The hydrolysis of UDP-N-acetylglucosamine by extracts of rat embryo cells has been compared to the hydrolysis of this sugar nucleotide by extracts of hamster embryo cells. A method utilizing high-pressure liquid chromatography was developed to separate and quantify the products of the hydrolysis. Rat embryo cells as well as hamster embryo cells hydrolyzed UDP-N-acetylglucosamine virtually completely under the experimental conditions. The principal product of the hydrolysis of UDP-N-acetylglucosamine by hamster embryo cells was N-acetylglucosamine. The sugar moiety of N-acetyglucosamine, produced by hamster embryo cells, was identified by thin-layer chromatography to be glucose. By contrast, the major metabolite (60--70%) of the hydrolysis of UDP-N-acetylglucosamine by rat embryo cells was identified as a phosphorylated acetylamino sugar. Only about 30--40% of the substrate was degraded to the free acetylamino sugar. Examination of the sugar moiety of the phosphorylated and of the free acetylamino sugar by thin-layer chromatography indicated that it was a mixture of glucose and of the epimers, mannose or galactose. The identity of the epimers has as yet not been established. Thus, unlike hamster embryo cells, rat embryo cells contain an active epimerase. However, the phosphohydrolase of the rat embryo cell which degrades the phosphorylated acetylamino sugars to the free acetylamino sugars seems to be less active than the enzyme in the hamster embryo cell. On the basis of the data of this study a comparison of the pattern of hydrolysis of UDP-N-acetylglucosamine by normal and transformed embryonic cells of the rat and of the hamster appears feasible.

Studies on the transformation of rat embryo cells of low passage by carcinogenic fluorenylhydroxamic acids and their acetate esters.

Rat embryo cells of low passage subjected to a single treatment with certain carcinogenic fluorenylhydroxamic acids and their respective acetate esters showed signs of transformation in vitro, such as changes in phenotype, growth in soft agar and agglutination with concanavalin A. In addition, certain changes in karyotype and loss of diploidy were observed. There was no evidence, either by electron microscopy or by assay of RNA-dependent DNA polymerase, for the presence of virus. None of these cell lines produced tumors after inoculation into the isologous host. The results of these study lead us to suggest that malignant transformation is a multistep process and that certain criteria of transformation of rat embryo cells are associated with the initial stage(s) in which the cells are transformed without being tumorigenic. The ultimate test for malignant transformation of rat embryo cells remains the production of tumors in a susceptible host after inoculation of treated cells.

Mammary carcinogenesis in the rat by topical application of fluorenylhydroxamic acids and their acetates.

This work confirms the previous observation that a single application of N-hydroxy-2-fluorenylacetamide or N-hydroxy-3-fluorenylacetamide to the mammary gland of the rat induced a high incidence of tumors, whereas the corresponding arylamides, N-2-fluorenylacetamide (2-FAA) and N-3-fluorenylacetamide, were only weakly active. The results suggested N-hydroxylation of the arylamides as a prerequisite for mammary carcinogenesis. Since N-hydroxylation of 2-FAA by hepatic microsomes is catalyzed by the mixed-function oxidase containing cytochrome P-450 or the 2-methylcholanthrene-inducible cytochrome P1-450, we examined whether these cytochromes are present in mammary microsomes. In contrast to liver, neither cytochrome nor N-hydroxylation of 2-FAA was detected in the mammary gland of normal and 3-methylcholanthrene-treated rats. These experiments indicated that the N-hydroxylation of 2-FAA, although obligatory for induction of mammary neoplasia, is not performed in the mammary gland but may take place in the liver. We also examined the carcinogenicity of N-acetoxy-2-fluorenylacetamide and N-acetoxy-3-fluorenylacetamide for the mammary gland upon topical application. Since both acetates were carcinogenic and since the acetyl group of acetyl coenzyme A is transferred to fluorenylhydroxamic acids at pH 7.4, these esters may be ultimate carciogens in mammary carcinogenesis. Ovariectomized rats did not develop mammary tumors after a single application of the fluorenylhydroxamic acids, and administration of estradiol and fluorenylhydroxamic acids to the ovariectomized rats did not improve the tumor yield. These results indicate that induction of mammary tumors by fluorenylhydroxamic acids is under hormonal control.