Metabolism and activation of chemical carcinogens

Summary A great deal of effort has gone into research on the mechanisms of action of chemical carcinogens. The action of the host on the carcinogen represents one approach — to determine what metabolic products may be involved. Study of the action of the carcinogen on the host requires a thorough comparison of changes wrought in cellular constituents by the carcinogen. Meshing of these two approaches increases the potential to discover the detailed mechanisms involved. Despite the increase in knowledge, many crucial molecular events and their consequences in the process of carcinogenesis still remain a mystery.     

An overview of bioactivation of chemical carcinogens

Most environmental carcinogens require metabolic activation to reactive intermediates and are mutagenic in appropriate test systems. During the last decade, the cDNAs of numerous xenobiotic-metabolizing enzymes have been cloned. The individually expressed enzymes were used to study their substrate specificities and their inhibition by other compounds. Various enzymes were expressed directly in target cells of in vitro mutagenicity tests. This is illustrated in the present study for rat and human sulphotransferases (SULTs) expressed in Salmonella typhimurium TA1538. Numerous compounds were mutagenic in the new test system. Some of these promutagens were activated by several different SULT forms, whereas many other promutagens were activated with high selectivity by a specific enzyme form, but not by genetically closely related forms from the same species (e.g. allelic variants) or orthologous enzymes from other species. Similar findings have been made using recombinant test systems for specific forms of other classes of enzymes (e.g. cytochromes P450). This high selectivity in activation (and inactivation) may explain some organotropisms as well as species and inter-individual differences in the action of carcinogens. Many carcinogen-metabolizing enzymes are induced or inhibited by other xenobiotics. Such interactions can be exploited for chemo-prevention, which however may be carcinogen- and tissue-dependent.     

Hyperketonaemia markedly modulates the metabolic activation of chemical carcinogens

Male Wistar albino rats were rendered hyperketonaemic by oral administration of medium chain triacylglycerols or by a single intraperitoneal injection of the diabetogenic agent streptozotocin. Hepatic post-mitochondrial preparations from these animals were employed as activation systems in the Ames mutagenicity test. Activation systems from both groups of hyperketonaemic rats were more efficient than those of control rats in metabolically converting the precarcinogens Glu-P-1, Trp-P-1, Trp-P-2, N-nitrosopiperidine and N-nitrosopyrrolidine to mutagens. In contrast, when 2-aminofluorene was used as the precarcinogen, the preparations from the hyperketonaemic animals were less efficient than controls in activating this carcinogen. In all cases, the preparations from streptozotocin-treated rats displayed a more pronounced effect than those from triacylglycerol-treated rats, possibly reflecting the greater extent of hyperketonaemia in the former group. It is concluded that hyperketonaemia modulates the bioactivation of chemical carcinogens.     

Histofluorescence technique for the intracellular localization of chemical carcinogens

Summary Many carcinogens exhibit a fluorescent emission when excited with ultraviolet light. Advantage has been taken of this property to develop a technique that can detect microquantities of these carcinogens on conventional microscopic tissue preparations. This work describes the localization of aflatoxin B₁, N-2-fluorenylacetamide and benzo(a)pyrene both in the cell cytoplasm and nucleus afterin vivo administration of these compounds.     

Streptozotocin-induced diabetes modulates the metabolic activation of chemical carcinogens

The effect of chemically-induced diabetes on the hepatic microsomal mixed-function oxidase system and the activation of chemical carcinogens was investigated in animals treated with streptozotocin (STZ). In order to distinguish between the effects of the diabetogenic chemical per se and that of the diabetic state, groups of STZ-treated animals received either nicotinamide simultaneously with STZ to prevent the onset of diabetes, or daily treatment with insulin in order to reverse the effects of diabetes. STZ-treated animals exhibited higher pentoxyresorufin O-dealkylase, ethoxy-resorufin O-deethylase, ethoxycoumarin O-deethylase, aniline p-hydroxylase and NADPH-cytochrome c reductase activities; similarly, increases were seen in cytochrome P-450 and b5 levels. All of these effects were prevented by nicotinamide and, at least partly, antagonised by insulin therapy. Treatment of animals with STZ markedly increased the activation, by liver microsomes in vitro, of Trp-P-1 and Trp-P-2 to mutagens, the effect being totally preventable by nicotinamide and successfully antagonised with insulin therapy. The diabetic animals were similarly more efficient in activating MeIQ but the effect was not preventable by nicotinamide or reversed by insulin. In contrast no changes were seen in the activation of IQ and only a modest increase in the case of MeIQx. It is concluded that diabetes may modulate the metabolic activation of some chemical carcinogens, presumably by changing the ratio of the various cytochrome P-450 isoenzymes.     

Dietary constituents altering the responses to chemical carcinogens.

This paper deals with two categories of compounds having the capacity to inhibit the neoplastic effects of chemical carcinogens on the host. The first are inducers of increased microsomal mixed function oxidase activity. An increasing number of these inducers are being found in natural products. Cruciferous vegetables including brussels sprouts, cabbage, and cauliflower contain such compounds. Recently indole-3-acetonitrile, indole-3-carbinol and 3,3'-diindolylmethane have been identified as inducers in these three plants. Other naturally occurring inducers include flavones, safrole, isosafrole, beta-ionone, and oxidized sterols. Since previous work has shown that synthetic inducers may protect against chemical carcinogens, the composition of the diet could play a role in inhibiting the neoplastic response to these carcinogenic agents. The second category of inhibitors comprises the antioxidants. Several of these compounds have been found to inhibit the carcinogenic effects of a variety of chemical carcinogens. Considerable work of this nature has been done with butylated hydroxyanisole and butylated hydroxytoluene two antioxidants extensively used as food additives. Other antioxidants having carcinogen inhibiting capacities include ethoxyquin, disulfiram, and dimethyldithiocarbamate.     

In vitro transformation assays for chemical carcinogens

A variety of in vitro mammalian cell assays, designed specifically for the identification of carcinogenic compounds, have been in operation for more than a decade. Although no individual transformation system has won universal acceptance during this time, recent advances have led to the improved reliability and sensitivity of a number of these short-term tests. The underlying problems associated with the most widely used assays are identified and new developments in this rapidly expanding field are noted and discussed.     

A mathematical model of the equilibrium distribution of chemical complexes and the biological effects of chemical binding

Summary A general equation is derived describing the concentration of all possible complexes of a central molecule with a set of ligands bound to the central molecule. This deduction allows the reaction rate constants for the binding of a given molecule to the central molecule to depend on the species of molecules already bound and the location of the molecules already bound. The model thus allows for structural alteration of the central molecule by binding. Functions describing the concentration dependence of any effect whatever depending on the distribution of complexes are deduced. Possible applications and methods of application are indicated.

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Zusammenfassung Eine allgemeine Vergleichung hat man bekommen welche die Konzentration aller möglichen Komplexen eines zentralen Moleküles mit einer Reihe von Ligands verbunden mit dem zentralen Molekül beschreibt. Diese Folgerung gestattet dass die Reaktiongeschwindigkeit Konstanten für die Bindung eines gegebenen Moleküles mit dem zentralen Molekül abhÄngt von den Arten schon gegebenen Molekülen und die Ortsbestimmung der schon gebundenen Molekülen. Das Model zeigt die strukturelle Änderungen des zentralen Moleküls durch Bindung. Funktionen, die die Konzentrationband jedes Effektes beschreiben dass abhÄngt von der Distribution der Komplexen, sind abgeleitet.Mögliche Anwendungen und Methoden von Anwendung sind dargelegt.

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Résumé II est dérivé une équation qui décrit la concentration des toutes les complexes d'une molécule centrale avec une collection de ligands connectés à la molécule centrale. La déduction montre comme les coefficients de la rapidité de reaction entre la molécule centrale et les molécules de la collection dépend de l'espèce et de la location des molécules déjà connectées.La modèle dérivée de l'équation montre les changements structurelles par la connection. Les functions qui décrivent la dependence de chaque effet à la concentration, sont dérivés. Ces effects dépendent aussi à la distribution des complexes. Des applications et des méthodes sont indiqués.

     

Fumonisins: Isolation,chemical characterization and biological effects

The fumonisin B mycotoxins (FB₁ and FB₂) have been purified and characterized from corn cultures of Fusarium moniliforme strain MRC 826. Fumonisin B₁ (FB₁, the major fumonisin produced in culture, has been shown to be responsible for the major toxicological effects of the fungus in rats, horses and pigs. Recent investigations on the purification of compounds with chromatographic characteristics similar to FB₁ have led to the identification of two new fumonisins, FB₃ and FB₄. Fumonisins A₁ and A₂, the N-acetyl derivatives of FB₁ and FB₂ respectively, were also purified and shown to be secondary metabolites of the fungus. Short-term carcinogenesis studies in a rat liver bioassay indicated that over a period of 15 to 20 days, at dietary levels of 0.05–0.1%, FB₂ and FB₃ closely mimic the toxicological and cancer initiating activity of FB₁ and thus could contribute to the toxicological effects of the fungus in animals. In contrast, no biological activity could be detected for FA₁ under identical experimental conditions. These studies and others have indicated that the fumonisin B mycotoxins, although lacking mutagenicity in the Salmonella test or genotoxicity in the DNA repair assays in primary hepatocytes, appear to induce resistant hepatocytes similar to many known hepatocarcinogens.     

Immunostimulation effects of yellowtail heart extracts in vitro and in vivo

The immunostimulation effects of yellowtail heart extracts were examined. Screening various parts of the yellowtail viscera, we found that extracts from the yellowtail heart enhanced IgM production by human hybridoma HB4C5 cells. Yellowtail heart extracts heated at 121°C for 20 min and dialyzed showed the highest IgM production-stimulating activity toward HB4C5 cells. Also, immunoglobulin production by mouse spleen lymphocytes was stimulated by yellowtail heart extracts in vitro, and lymphocytes derived from mice administered the extract for 20 d were activated in vivo. Yellowtail heart extracts were partially purified by anion-exchange chromatography, and fractions containing a 33 kDa-protein exhibited immunostimulating activity. LC-MS/MS analysis revealed that the 33 kDa-protein was most similar to tropomyosin-4 from various fishes. Purified tropomyosin from porcine muscle enhanced IgM production by HB4C5 cells. This means that tropomyosin-4 is one of the immunostimulating substances in the yellowtail heart.     

Inhibition of DNA methylation by chemical carcinogens in vitro

A diverse range of ultimate chemical carcinogens inhibited the transfer of methyl groups from S-adenosylmethionine to hemimethylated DNA in a reaction catalyzed by mouse spleen methyltransferase. The formation of alkali-labile sites in DNA lessened its ability to accept methyl groups in vitro, but the methylation reaction was much less sensitive to thymine dimers or double-strand breaks. Carcinogens induced the formation of alkali-labile DNA lesions, but the degree of methyltransferase inhibition observed was greater than that expected for this damage alone. Certain carcinogens were also capable of direct modification and inactivation of the methyltransferase enzyme. Benzo(a)pyrene treatment of living BALB/3T3 A31 clone 1-13 but not C3H/10T1/2 clone 8 cells resulted in a 12% decrease in total 5-methylcytosine content of cellular DNA. Carcinogenic agents may therefore cause heritable changes in 5-methylcytosine patterns in certain cell types by a variety of mechanisms, including adduct formation, induction of apurinic sites and single-strand breaks and direct inactivation of DNA methyltransferase.     

Analysis of DNA-protein complexes induced by chemical carcinogens.

DNA-protein complexes induced in intact cells by chromate have been isolated and compared with those formed by other agents such as cis-platinum. Actin has been identified as one of the major proteins that is complexed to the DNA by chromate based upon a number of criteria including, a molecular weight and isoelectric point identical to actin, positive reaction with actin polyclonal antibody, and proteolytic mapping. Chromate and cis-platinum both complex proteins of very similar molecular weight and isoelectric points and these complexes can be disrupted by exposure to chelating or reducing agents. These results suggest that the metal itself is participating in rather than catalyzing the formation of a DNA-protein complex. An antiserum which was raised to chromate-induced DNA-protein complexes reacted primarily with a 97,000 protein that could not be detected by silver staining. Western blots and slot blots were utilized to detect p97 DNA-protein complexes formed by cis-platinum, UV, formaldehyde, and chromate. Other work in this area, involving studying whether DNA-protein complexes are formed in actively transcribed DNA compared with genetically inactive DNA, is discussed. Methods to detect DNA-protein complexes, the stability and repair of these lesions, and characterization of DNA-protein complexes are reviewed. Nuclear matrix proteins have been identified as a major substrate for the formation of DNA-protein complexes and these findings are also reviewed.