Tissue and subcellular distribution of 3H-dioxane in the rat and apparent lack of microsome-catalyzed covalent binding in the target tissue

Using ³H-dioxane, the distribution of dioxane among a number of tissues and various subcellular fractions of rat liver was studied. At various times after i.p. injection, dioxane was found to distribute more or less uniformly among various tissues (liver, kidney, spleen, lung, colon and skeletal muscle), consistent with its polar/nonpolar nature. Studies of the nature of dioxane binding, however, revealed that the extent of “covalent” binding (as measured by incorporation into lipid-free, acid-insoluble tissue residues) was significantly higher in the liver (the main carcinogenesis target tissue), spleen and colon than that in other tissues. Investigations of the subcellular distribution in liver indicated that most of the radioactivity was in the cytosol, followed by the microsomal, mitochondrial and nuclear fractions. The binding of dioxane to the macromolecules in the cytosol was mainly noncovalent. The percent covalent binding was highest in the nuclear fraction, followed by mitochondrial and microsomal fractions and the whole homogenate. Pretreatment of rats with inducers of microsomal mixed-function oxidases had no significant effect on the covalent binding of dioxane to the various subcellular fractions of the liver. There was no microsome-catalyzed $\text{in}$$\text{vitro}$ binding of ³H- or ¹⁴C-dioxane to DNA under conditions which brought about substantial binding of ³H-benzo[a]pyrene.     

Alkylation of deoxyribonucleic acid in vivo in various organs of C57BL mice by the carcinogens N-methyl-N-nitrosourea, N-ethyl-N-nitrosourea and ethyl methanesulphonate in relation to induction of thymic lymphoma. Some applications of high-pressure liquid chromatography.

1. Methods were developed for analysis of alkylpurines, O2-alkylcytosines, and representative phosphotriesters [alkyl derivatives of thymidylyl(3'-5')thymidine], in DNA alkylated in vivo, using high-pressure liquid chromatography. 2. The patterns of alkylation products in DNA in vivo at short times were closely similar to those found for reactions in vitro. Alkylation by the nitrosoureas was complete in vivo within 1 h, but with ethyl methanesulphonate was maximal at 2--4h. 3. The time course of persistence of alkylation products in vivo was determined for several tissues. In addition to the rapid loss of 3- and 7-alkyladenines reported previously for all tissues, a relatively rapid loss of O6-alkylguanines from DNA of liver was found which was more rapid at lower doses. In brain, lung and kidney, excision of O6-alkylguanine was much less marked, but was not entirely excluded by the data. In thymus, bone marrow and small bowel, all alkylated bases were lost with half-lives of 12--24h, at non-cytotoxic doses of alkylation. 4. No evidence for any marked excision of other minor products from alkylated DNA in vivo was found; thus 1-methyladenine, O2-ethylcytosine (found in appreciable amount only with N-ethyl-N-nitrosourea), 3-methylguanine, and dTp(Alk)dT persisted in alkylated DNA, including DNA of liver. 5. The induction of thymic lymphoma was determined over the range of single doses by intraperitoneal injection up to about 60% of the LD50 values, and related to the extent of alkylation of target tissues thymus and bone marrow. With N-methyl-N-nitrosourea over 90% tumour yield was attained at 60 mg/kg, and with N-ethyl-N-nitrosourea up to 52% at 240 mg/kg, but with ethyl methanesulphonate at up to 400 mg/kg only a few per cent of tumours were obtained. 6. The carcinogenic effectiveness of the agents was positively correlated with the extents of alkylation of guanine in DNA of target tissues at the O-6 atom. On the basis that at doses giving equal carcinogenic response these extents of alkylation would be equal, the chemical analyses showed that the ratio of equipotent doses to that for N-methyl-N-nitrosourea would be, for N-ethyl-N-nitrosourea, 5.3 for ethyl methanesulphonate about 21, and for methyl methanesulphonate [Frei & Lawley (1976) Chem.-Biol. Interact. 13, 215--222] about 144. These predictions were in reasonably good agreement with the observed dose-response data for these agents.     

Identification and quantitation of benzo[a]pyrene-derived DNA adducts formed at low adduction level in mice lung tissue

The two major metabolic pathways of benzo[a]pyrene (BP) that lead to DNA lesions are monooxygenation that results in diolepoxides (BPDE) and one-electron oxidation that yields a BP radical cation. These pathways result in formation of stable and depurinating DNA adducts, respectively. Most in vivo animal studies with BP, however, have employed dosage/DNA adduct levels several orders of magnitude higher than the DNA damage level expected from environmentally relevant exposures. Presented are results of experiments in which A/J strain mice were intraperitoneally exposed to 50-microg/g doses of BP. It is shown that non-line-narrowed fluorescence and fluorescence line-narrowing spectroscopies possess the selectivity and sensitivity to distinguish between helix-external, base-stacked, and intercalated conformations of DNA-BPDE adducts formed in lung tissue. Concentrations measured by 32P postlabeling 2 and 3 days after intraperitoneal injection were 420-430 and 600-830 amol BPDE-type adducts per microg DNA. The external and base-stacked conformations are attributed mainly to (+)-trans-anti-BPDE-N2dG and the intercalated conformations to (+)-cis-anti adducts. A stable adduct derived from 9-OH-BP-4,5-epoxide was also detected at a concentration about a factor of 10 lower than the above concentrations. The DNA supernatants were analyzed for the presence of depurinating BP-derived adducts by capillary electrophoresis laser-induced fluorescence and high-performance liquid chromatography mass spectrometry.     

Differential effects of nutritional folic acid deficiency and moderate hyperhomocysteinemia on aortic plaque formation and genome-wide DNA methylation in vascular tissue from ApoE-/- mice

Low folate intake is associated with vascular disease. Causality has been attributed to hyperhomocysteinemia. However, human intervention trials have failed to show the benefit of homocysteine-lowering therapies. Alternatively, low folate may promote vascular disease by deregulating DNA methylation. We investigated whether folate could alter DNA methylation and atherosclerosis in ApoE null mice. Mice were fed one of six diets (n?=?20 per group) for 16?weeks. Basal diets were either control (C; 4% lard) or high fat (HF; 21% lard and cholesterol, 0.15%) with different B-vitamin compositions: (1) folic acid and B-vitamin replete, (2) folic acid deficient (-F), (3) folic acid, B6 and B12 deficient (-F-B). -F diets decreased plasma (up to 85%; P?相似文献   

Comparative induction of gene mutations and chromosome damage by 1-methoxy-1,3,5-cycloheptatriene (MCHT), 2. Results using L5178Y mouse lymphoma cells to detect both gene and chromosome damage; validation with ionizing radiation, methyl methanesulphonate, ethyl methanesulphonate and benzo[a]pyrene

A variation of the mouse lymphoma (L5178Y TK+/(-)-3.7.2c) assay has been developed using a microtiter cloning technique instead of the standard agar method. The cell line has been used to detect both gene mutations (at the Na+/K+ ATPase and thymidine kinase loci) and chromosome damage (micronucleus induction) in the same experiment. The system was validated using gamma-irradiation (a known clastogen), 2 direct-acting mutagens, ethyl and methyl methanesulphonate and an indirect-acting mutagen, benzo[a]pyrene. Using the assay, 1-methoxy-1,3,5-cycloheptatriene was shown to be a clastogenic mutagen in the presence of S9, since a clear dose-dependent increase in micronuclei was observed, mainly small colony thymidine kinase mutants were observed, and no ouabain-resistant mutants were induced, a profile very similar to gamma-irradiation. The results suggest that metabolic activation potential explains the results in the accompanying paper (Asquith et al., 1990). The implications for mutagenicity testing are discussed.     

Drosophila mutations at the mei-9 and mus(2)201 loci which block excision of thymine dimers also block induction of unscheduled DNA synthesis by methyl methanesulfonate, ethyl methanesulfonate, N-methyl-N-nitrosourea, UV light and X-rays

The mei-9 and mus(2)201 mutants of Drosophila melanogaster were identified as mutagen-sensitive mutants on the basis of larval hypersensitivity to methyl methanesulfonate and characterized as excision repair-deficient on the basis of a greatly reduced capacity to excise thymine dimers from cellular DNA. The high degree of larval cytotoxicity observed with a variety of other chemical and physical agents indicated that these mutants may be unable to excise other important classes of DNA adducts. We have measured the ability of the single mutants and the double mutant combination mei-9;mus(2)201 to perform the resynthesis step in excision repair by means of an autoradiographic analysis of unscheduled DNA synthesis (UDS) induced in a mixed population of primary cells in culture. The 3 strains exhibit no detectable UDS activity in response to applied doses of 1.5-6.0 mM methyl methanesulfonate, 1.0-4.5 mM N-methyl-N-nitrosourea or 10-40 J/m2 254-nm UV light, dose ranges in which control cells exhibit a strong dose-dependent UDS response. The mei-9 and mei-9;mus(2)201 mutants also have no detectable UDS response to X-ray doses of 300-1800 rad, whereas the mus(2)201 mutant exhibits a reduced, but dose-dependent, response over this range. These data correlate well with the degree of larval hypersensitivity of the strains and suggest that mutations at both loci block the excision repair of a wide variety of DNA damage prior to the resynthesis step.