Sensitivity of DNA and nucleotides to oxidation by permanganate and hydrogen peroxide

no abstract availabl

The relevance of covalent binding to mouse liver DNA to the carcinogenic action of hexachlorocyclohexane isomers

[$^3$H]Hexachlorocyclohexane (HCH) was synthesized by chlorination of [$^3$H]benzene prepared by catalytic tritiation of benzene with tritiated water. The isomers of HCH were separated by adsorption chromatography on silica gel. In order to determine the covalent binding to DNA, [$^3$H]HCH was administered to male mice by oral gavage, and liver DNA was isolated via cbromatin. The specific radioactivity of the DNA was nonnalized by the dose administered and expressed in the molar units of the Covalent binding index, CBI = DNA damage/dose = ($\mu$mol bound HCH/mol DNA nucleotide)/(mmol HCH administered/kg body weight). CBI values of - 0.2 were found 10 h after the administration of alpha- and gamma-HCH. Enzymatic digestion of the DNA to the nucleosides and h.p.l.c. analysis revealed that - 40% of the radioactivity co-migrated with the natural nucleosides. At elution volumes known to contain the more lipophilic carcinogen-nucleoside adducts, - 10% of the radioactivity could be detected. The remaining 50% of th,e radioactivity eluted with the front, representing a mixture of oligonucleotide- HCH adducts and/or hydrophilic degradation products which were strongly bot not covalently associated with intact DNA. Therefore, a true CBI of 0.02-0.1 must be expected both for alpha- and gamma-HCH. This CBI is by a factor of 10$^5$ -10$^6$ below the value found with the strongest DNAbinding carcinogens like aflatoxin B1 or dimethylnitrosamine and is unlikely to be decisive for the liver tumor induction in mice because of the foUowing additional findings: (i) both isomers gave rise to similar Ievels of DNA darnage although the alpha-isomer is a much morepotent tumor inducer. This similarity was seen not only at the time of mäximum binding but up to 10 days after oral administration; (ii) three mouse strains with apparently different susceptibility to tumor induction by gamma-HCH could not be distinguished with respect to DNA binding; (iii) the level of DNA binding of alpha-HCH (CBI = 0.02-0.1) is more than three orders of magnitude lower than would be expected if the mechanism of tumor induction was by genotoxicity mediated by DNAbinding. For a preliminary investigation on a potential stimulatory effect on liver DN A replication and ceU division, [$^{14}$]thymidine was admlnistered i.p. 3.5 h before sacrifice of the [$^3$H]HCH-treated mice. The alpha-isomer was found to be more potent than the gamma-isomer in this respect. Taken together, our data allow the conclusion that the non- mutational processes must be more important for the carcinogenicity of HCH

DNA methylation in rat liver by daminozide, 1,1-dimethylhydrazine, and dimethylnitrosamine

DNA Methylation in Rat Li ver by Daminozide, 1, 1-Dimethylhydrazine, and Dimethylnitrosamine. SAGELSDORFF, P., LUTZ, W. K., AND ScHLAITER C. (1988). Fundam. Appl. Toxico/. 11, 723-730. [methyP4C]Daminozide (succinic acid 2',2'-dimethylhydrazide; 37 mgjkg), l,l( 14C]dimethylhydrazine (UDMH; 19 mgtkg), and (14C]dimethylnitrosamine (DMNA; 0.1 mg/ kg) were administered by oral gavage to male Sprague-Dawley rats. After 24 hr, the animals were killed and DNA was purified from the livers to constant specific radioactivity. After enzymatic degradation of the DNA to the 3'-deoxynucleotides the Ievel of DNA methylation was determined by HPLC analysis. Radiolabeled 7-methylguanine (7mG) was identified by cochromatography with unlabeled 7mG added as standard after acidic depurination of DNA and HPLC analysis ofpurines and apurinic acid. All three compounds were found to methylate DNA. The relative potencies were 1:47:4900 for daminozide:UDMH:DMNA. With [methyPH]UDMH, the formation of7mG was investigated as a function of dose administered, at 20, 2, and 0.2 mgj kg. The methylation ofDNA was strictly proportional to the dose. The data were used to compare the Ievel of DNA alkylation derived from residues of daminozide and UDMH in treated apple with the genotoxicity of the intake of N-nitroso compounds in Germany and Japan. It is estimated that these residues could Iead to a DNA methylation in the Ii ver of about 6% of an average exposure to DMN

Potency of carcinogens derived from covalent DNA binding and stimulation of DNA synthesis in rat liver

~n order to investigate the role of the stimu~ation of ceU division for the initiation (and possi:bly promotion) of live·r tumors by chemical carcinogens, the incorporation of radiolabeUed thymidine into liver DNA was dete:rmined in male rats. Single doses of various level!s of af.latoxin 81, benzidine and carbon tetrachloride (aU known to be genotoxic via DNA binding} did not affect cell division, whereas several hepatoca:rcinogens known not to bind to DNA (alphaHCH, dofibrate, and 2,3;7,8-t!etrachlorodiibenzo~p~dioxin) gave rise to a dosedependent stimulation of Ii ver DNA synthesis within 24 h. An equation combining the infl.uences of mitotic stimu:lation, expressed as dose required to double the contro~ Ievei of DNA synthesis, and DNA binding potency, exp:ressed as t.he Covalent Binding Index, correliated weil with the cardnogenk potency for both dasses of hepatocardnogens

In vivo covalent binding of aflatoxin B1_1 and aflatoxin M1_1 to liver DNA of rat, mouse and pig

[$^{14}$C] Aflatoxin B$_1$ (AFB$_1$) was isolated from cultures of Aspergillus parasiticus grown on [1-$^{114}$C] sodium acetate. Covalent binding of AFB1 to liver DNA of rat and mouse was determined 6-8 h afteroral administration. The effectiveness of covalent binding, expressedas DNA binding per dose in the units of a 'Covalent Binding Index' (CBI), ($\mu$mol aflatoxin/mol DNA nucleotides)/(mmol aflatoxin/kg animal), was found to be 10 400 for rats and 240 for mice. These CBI partly explain the different susceptibility of the two species for the incidence of hepatic tumors. The corresponding values for pig liver DN A, 24 and 48 h after oral administration, were found to be as high as 19 100 and 13 300. DNA-binding has not so far been reported for this species although it could represent an appropriate animal model for studies where a human-like gastrointestinal tract physiology is desirable. Aflatoxin M $_1$ ( AFM$_1$) is a metabolite found in the milk of cows that have been fed AFB$_1$-contaminated diet. [$^{14}$C] AFM$_1$ was also found to be produced by cultures of A. parasiticus giving a yield of about 0.3% of the total aflatoxins. A test for covalent binding to rat liver DN A revealed a CBI of 2100 shoWing that AFM$_1$ must also be regarded as a strong hepatocarcinogen. It is concluded that AFB$_1$ contaminations should be avoided in dairy feed

Detection by 32P-postlabeling of thymidine glycol in gamma-irradiated DNA

The 32P-postlabeling method has been adapted for the analysis of thymidine-cis-glycol-3'-phosphate (cis-dTGp,cis-5,6-dihydroxy-5,6-dihydrothymidine-3'-phosphat e). Cis-dTGp was isolated and purified from normal nucleotides by phenylboronate affinity chromatography and phosphorylated by T4 polynucleotide kinase in presence of 1 mM BeCl2 at pH 7.5. These modifications of the postlabeling method resulted in a 5'-phosphorylation of dTGp with a labeling efficiency of up to 20% whereas the natural nucleotides were almost completely dephosphorylated at the 3' position under these conditions. The reaction products, containing radio-labeled thymidine-cis-glycol-3',5'-bis-[5'-32P]phosphate (cis-*pdTGp), were separated by two-dimensional anion-exchange TLC on polyethyleneimine cellulose sheets. Boric acid was added in the second dimension in order to selectively retard cis-glycols. The method was applied to gamma-irradiated nucleotides and calf thymus DNA. In the nucleotide mixture, 330-99,000 thymine glycol (TG) moieties were detected per 10(6) thymines (T) in a dose range of 14-1000 Gy respectively. In DNA, these values ranged from 400 to 2700 TG/10(6) T. The data are in good agreement with methods using radiochemical and immunological techniques. Non-irradiated DNA showed a background level of 10TG/10(6) T. This practical limit of detection was higher than can be achieved with the postlabeling technique, indicating that the present method might be a sensitive alternative for a determination of oxidative DNA damage

No measurable increase in thymidine glycol or 8-hydroxydeoxyguanosine in liver DNA of rats treated with nafenopin or choline-devoid low-methionine diet

Male rats were treated for 2 months with 1000 ppm nafenopin in the diet or for 4 or 7 days with a choline-devoid low-methionine diet. DNA was isolated from the livers and analyzed for the presence of cis-thymidine glycol-3'-phosphate (cis-dTGp) by 32P-postlabeling and for the level of 8-hydroxy-deoxyguanosine (8-OH-dG) by electrochemical detection (ECD). In no DNA sample was the level of cis-dTGp above the limit of detection of 1 modified thymidine per 10(6) nucleotides. With 8-OH-dG, a background level of this modification of 20 8-OH-dG per 10(6) nucleosides was found in liver DNA of control rats, which was not affected by either treatment. It is postulated for thymidine glycol that a potential increase was below the limit of detection or was rapidly repaired in vivo and that the steady-state level of endogenous 8-hydroxydeoxyguanosine appears not to be influenced by the treatments chosen

Trenbolone growth promotant: covalent DNA binding in rat liver and in Salmonella typhimurium, and mutagenicity in the Ames test

DNA binding in vivo: (6,7-$^3$H]ß-trenbolone (ß-TBOH) was administered p.o. and i.p. to rats. After 8 or 16 h, DNA was isolated from the livers and purified to constant specific radioactivity. Enzymatic digestion to deoxyribonucleotides and separation by HPLC revealed about 90% ofthe DNA radioactivity eluting in the form of possible TBOH-nucleotide adducts. The extent of this genotoxicity, expressed in units of the Covalent Binding Index, CBI = (~mol TBOH bound per mol nucleotide)/(mmol TBOH administered per kg body weight) spanned from 8 t~ 17, i. e. was in the range found with weak genotoxic carcmogens. Ames test: low doses of ß-TBOH increased the number of revertants in Salmonella strain TAl 00 reproducibly and m a dose-dependent manner. The mutagenic potency was 0.2 revertants per nmol after preincubation of the bacteria (20 min at 37° C) with doses between 30 and 60 $\mu$g per plate (47 and 94 $\mu$g/ml preincubation mixture). Above this dose, the number of revertants decreased to control values, accompanied by a reduction in survival. The addition of rat liver S9 inhibited the mutagenicity. DNA binding in vitro: calf thymus DNA was incubated with tritiated ß-TBOH with and without rat liver S9 Highest DNA radioactivities were determined in the absence of the "activation" system. Addition of inactive S9 (without cofactors) reduced the DNA binding by a factor of up to 20. Intermediate results were found with active S9. DNA binding in Salmonella: ß-TBOH was irreversibly bound to DNA isolated from S. typhimurium TA100 after incubation of bacteria with [$^3$H]ß-TBOH. Conclusions: Covalent DNA binding appears to be the mechanism of an activation-independent ("direct") mutagenicity of TBOH which is not easily detected because of the bactericidal activity. The genotoxicity risk arising from exposure of humans to trenbolone residues in meat was estimated using the in vivo data and compared to that from the exposure to unavoidable genotoxins aflatoxin B1 and dimethylnitrosamine. It ts concluded that trenbolone residues represent only a low genotoxic risk