TY - CHAP A1 - Shephard, S. E. A1 - Meier, I. A1 - Lutz, Werner K. T1 - Alkylating potency of nitrosated amino acids and peptides N2 - Tbe alkylating potency of unstable N-nitrosamino acids and N-nitrosopeptides was investigated in vitro using 4-(para-nitrobenzyl)pyridine (NBP) as nucleophile. Of the amino acids, Met and those with an aromatic side chain were the most potent. The relative overall alkylating potency was 23:10:5:4:2:1: for Trp, Met, His, 1)rr, Phe and Gly, respectively. The homo-dipeptides were much more potent than the amino acids, with relative potencies of 400:110:100:8:3:1, for Trp-Trp, l)T-'I)T, Met-Met, Asp-Asp, Phe-Phe and Gly, respectively. In the one-phase reaction system (in which NBP is already present durlog the nitrosation reaction at acidic pH), all amino acids tested showed a second-order reaction for nitrite. In the two-phase system (in which NBP is added only after bringing the nitrosation reaction mixture to neutrality), all amino acids tested except one again showed a second-order reaction for nitrite (Phe, His, Asp and the dipeptide artiticial sweetener aspartame); only Met under these conditions bad a reaction order of one for nitrite. This could mean that nitrosation of the side chain of Metproduces a second N-nitroso product which is relatively stable in acid but reacts with NBP under neutral conditions. In the human stomach, this side-chain nitrosation might become more important than the reactions at the primary amino group, firstly because of the greater stability of the product(s) in acid and secondly because of the tirst-order reaction rate for nitrite. A decrease in nitrite concentration from the millimolar concentrations ofthe in-vitro assay to the micromolar concentrations in the stomach reduces the reaction rate by a factor of 1000 for the side-chain nitrosation, whereas a million-fold reduction will be observed for nitrosation of the amino group. KW - Aminosäuren Y1 - 1991 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-86320 ER - TY - CHAP A1 - Cantoreggi, S. A1 - Gupta, R. C. A1 - Lutz, Werner K. T1 - An improved 32P-postlabelling assay for detection and quantitation of styrene 7,8-oxide-DNA adducts N2 - Using DNA modified with [7-3H]styrene 7,8-oxide (SO) in vitro we have standardized the 32P-postlabelling assay for detecting SO-DNA adducts. Nuclease P 1-enriched adducts were 32P-labelled and purified by high-salt ( 4.0 M ammonium formate, pH 6.1} C1s reverse-phase TLC. After elution from the layer with 2-butoxyethanol:H20 (4:6), adducts were separated by two-dimensional PEI cellulose TLC in non-urea solvents (2.0 M ammonium formate, pH 3.5, and 2.7 M sodium phosphate, pH 5.6). One major, three minor and several trace adducts were detected. The efficiency of the kinase reaction depended on the ATP concentration. Use of standard labelling conditions (['Y· 32P]ATP, <3000 Ci/mmol; <2 Mikromol) resulted in poor ( 4-7%) adduct recovery. An ATP concentration of 40 Mikromol, however, increased the labeJling efficiency by a factor of 5-8 (35-55% based on 3H-SO labelied DNA). The results indicate that the new separation technique is suitable for the relatively polar SO-DNA adducts and that high labelling efficiency can be achieved. KW - Medizin Y1 - 1993 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-86305 ER - TY - CHAP A1 - Shephard, S. E. A1 - Hegi, M. E. A1 - Lutz, Werner K. T1 - In-vitro assays to detect alkylating and mutagenic activities of dietary components nitrosated in situ N2 - Nitrosation of dietary components has been combined with the 4-(para-nitrobenzyl)pyridine (NBP) colorimetric test for screening alkylating agents and with the Ames test for the detection of mutagenic activity. This allowed the investigation of short-hved nitrosation products of dietary components which generate electrophilic degradation products requiring no metabolic activation (natural amino acids and some derivatives, ureas, guanidines, primary alkyl and aryl amines). In a first system, precursor, nitrous acid and NBP were present simultaneously. All amino acids tested, except glutamic acid and glutamine, gave positive results. The reactivities spanned more than three orders of magnitude, with the aromatic amino acids and methionine the most active; two primary amines, tryptamine and histamine, were also strongly reactive. All guanidines tested, except the amino acid arginine, gave negative results. A second system consisted of two phases: NBP was added only after destruction of residual nitrite and adjustment of the pH to neutrality. This system was useful for the study of ureas, which are stable in acid but not in neutral media. The range of responses covered more than two orders of magnitude. Most amino acids and primary amines also gave positive results, but could be assessed only after analysing the kinetics of the competing reactions and choosing appropriate reaction times. In a third system, Salmonella typhimurium strain TA1OO replaced NBP. Representatives of the class of amino acids, ureas, the primary amine tryptamine, and aniline became higbly mutagenic upon nitrosation. Methylguanidine was only weakly mutagenic under the present assay conditions. The results indicate that further studies with unstable nitrosation products of dietary components are required to understand more thoroughly the role of endogenous nitrosation in gastric cancer. KW - Medizin Y1 - 1987 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-86194 ER - TY - CHAP A1 - Shephard, S. E. A1 - Schlatter, C. A1 - Lutz, Werner K. T1 - Model risk analysis of nitrosatable compounds in the diet as precursors of potential endogenous carcinogens N2 - The potential health risk posed by the endogenous formation of N-nitroso compounds (NOC) from nitrosation of dietary ureas, guanidines, amides, amino acids and amanes (primary, secondary and aromatic) was estimated according to the model: Risk = ( daily intake of precursor] X (gastric concentration of nitrite ]n X [nitrosatability rate constant] X [cilrcinogenicity of derivative]. The daily intakes ofthese compound classes span five orders ofmagnitude (100 g/day amides, top; 1-10 mg/day secondary amines, ureas, bottom); the nitrosation rate constants span seven orders of magnitude (aryl amines, ureas, top; amides, secondary amines, bottom); and the carcinogenicity estimates span a 10 000-fold range from 'very strong' to 'virtually noncarcinogenic'. The resulting risk estimates likewise span an enormous range (nine orders of magnitude ): dietary ureas and aromatic amines combined with high nitrite concentration could pose as great a risk as the intake of preformed N-nitrosodimethylamine in the diet. In contrast, the risk posed by the in-vivo nitrosation of primary and secondary amines is probably negligible. The risk contributed by amides (including protein), guanidines and primary amino acids is intermediate between these two extremes. KW - Risikoanalyse KW - Carcinogen KW - Ernährung Y1 - 1987 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-86188 ER - TY - CHAP A1 - Lutz, Werner K. T1 - Dose-response relationships in chemical carcinogenesis: from DNA adducts to tumor incidence N2 - Mechanistic possibilitles responsible for nonlinear shapes of the dose-response relationship in chemical carcinogenesis are discussed. (i) Induction and saturation of enzymatic activation and detoxification processes and of DNA repair affect the relationship between dose and steady-state DNA adduct Ievel; (ii) The fixation of DNA adducts in the form of mutations is accelerated by stimulation of the cell division, for Jnstance due to regenerative hyperplasia at cytotoxic dose Ievels; (iii) The rate of tumor formation results from a superposition of the rates of the individual steps. It can become exponential with dose if more than one step is accelerated by the DNA damage exerted by the genotoxic carcinogen. The strongly sigmoidal shapes often observed for dose-tumor incidence relationships in animal bioassays supports this analysis. A power of four for the dose in the su~linear part of the curve is the maximum observed (formaldehyde). In contrast to animal experiments, epidemiological data ln humans rarely show a slgnificant deviation from linearity. The discrepancy might be explained by the fact that a I arge nu mber of genes contribute to the overall sensitivity of an individual and to the respective heterogeneity within the human population. Mechanistic nonlinearities are flattened out in the presence of genetic and life-style factors which affect the sensitivity for the development of cancer. For a risk assessment, linear extrapolation from the high-dose lncidence to the spontaneaus rate can therefore be approprlate in a heterogeneous population even if the mechanism of action would result in a nonlinear shape of the dose-response curve in a homogeneaus population. KW - aflatoxin B1 KW - 2-acetylaminofluorene KW - DNA KW - adduct KW - covalent KW - binding KW - carcinogen KW - dose KW - extrapolation KW - individual KW - susceptibility KW - heterogeneous population KW - risk KW - tumour Y1 - 1991 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-71625 ER - TY - CHAP A1 - Lutz, Werner K. A1 - Cantoreggi, S. A1 - Velic, I. T1 - DNA binding and stimulation of cell division in the carcinogenicity of styrene 7,8-oxide N2 - [7-3H)Styrene 7,8-oxide was administered by oral gavage to male CD rats at a dose of 1.3 mg/kg. After 4 h, the forestomach was excised, DNA was isolated, purified to constant specific radioactivity and degraded nzymatically to the 3 '-nucleotides. Highperformance liquid chromatography fractions with the normal nucleotides contained most of the radiolabel, but a minute level of adduct label was also detccted. Using the units of the covalent binding index (micromoles adduct per mole DNA nucleotide)/(millimole chemical administered per kilogram body weight), a DNA binding potency of 1.0 was derived. A comparison of the covalent binding indices and carcinogenic potencies of other genotoxic forestarnach carcinogens showed that the tumorigenic activity of styrene oxide is unlikely to be purely genotoxic. Therefore, styrene oxide was compared with 3-tbutylhydroxyanisole (BHA) with respect to stimulation of cell proliferation in the forestomach. Male Fischer 344 rats were treated for four weeks at three dose levels of styrene oxide (0, 137, 275 and 550 mg/kg, three times per week by oral gavage) and BHA (0, 0.5, 1 and 2% in the diet); the highest doses had been reported to result in 84% and 22% carcinomas in the forestomach, respectively. Cell proliferation was assessed by incorporation of bromodeoxyuridine into DNA and immunohistochemical analysis. An increase in the lablling indexwas found in a11 treated animals. In the prefundic region of the forestomach, the labeHing index increased significantly, from 42% (controls) to 54% with styrene oxide and from 41 to 55% with BHA. Rats treated with BHA also had severe hyperplastic lesions in the prefundic region, i.e., at the location of BHA-induced forestomach carcinomas. The number of cells per millimetre of section length was increased up to 19 fold. Hyperplastic lesions were not seen with styrene oxide, despite the higher tumour incidence reported with this compound. We conclude that the carcinogenicity of styrene oxide to the forestomach most probably involves a mechanism in which marginal genotoxicity is combined with promotion by increased cell proliferation. KW - Styrol KW - DNS-Bindung KW - Zellteilung KW - Carcinogenität Y1 - 1993 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-71597 ER -