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K Randerath

Publications and source records attributed to K Randerath.

At least 127 records · Page 7Linked to original sources
PubMed1987

32P-analysis of DNA adducts in somatic and reproductive tissues of rats treated with the anticancer antibiotic, mitomycin C.

Mitomycin C (MMC) is a clinically used drug with mutagenic and antitumor activities, presumably elicited through its covalent binding to DNA, however, little is known about MMC binding to DNA in vivo. A 32P-postlabeling method that does not require radiolabeled test compounds was employed here to study the formation of DNA adducts in somatic and reproductive tissues of rats 24 h after an i.p. dose of 9 mg/kg MMC. Among 14 tissues studied in female rats, MMC-DNA adduct levels were within a 2-fold range in 11 tissues, i.e. bladder, colon, esophagus, heart, kidney, liver, lung, ovary, pancreas, small intestine and stomach (minimum levels of 9.6-21.9 adducts per 10(7) N). Three other tissues, i.e. brain, spleen and thymus, exhibited lower adduct levels (0.2 5.4 and 1.4 adducts, respectively, per 10(7) N). Liver DNA adduct levels were 32% lower in male than in female rats. Testicular DNA contained 2.5 adducts per 10(7) N, i.e. 5.3 times less than ovarian DNA. 32P-labeled adduct patterns were qualitatively similar among the different tissues and consisted of 10 adducts, one of which comprised 71 (+/- 5)% of the total. All these adducts were chromatographically identical to adducts formed by the reaction of chemically reduced MMC with DNA in vitro, demonstrating that metabolic activation of MMC occurred via reduction. Using homopolydeoxyribonucleotides modified with MMC, in vivo adducts were shown to be mostly (greater than 90%) guanine derivatives and small amounts of adenine, cytosine and thymine products. Most of the adducts appeared to be monofunctional derivatives of DNA nucleotides. Dose-dependent MMC-DNA adduct formation was determined in rat liver over an 82-fold range of MMC administered (0.11-9.0 mg/kg). The lowest dose level studied was 4.5 times lower than the recommended single dose for human cancer chemotherapy (20 mg/m2). Thus, these results predict that 32P-postlabeling methodology is suitable to monitor and quantify DNA adducts in tissue biopsies of patients receiving MMC chemotherapy.

Animals↗
PubMed1987

N-methylation reduces the DNA-binding activity of 7H-dibenzo[c,g]carbazole approximately 300-fold in mouse liver but only approximately 2-fold in skin: possible correlation with carcinogenic activity.

N-methyl-dibenzo[c,g]carbazole (MeDBC) lacks the potent hepatocarcinogenic activity in mice characteristic for 7H-dibenzo[c,g]carbazole (DBC), while both compounds are local carcinogens, leading to papilloma and carcinoma formation in skin after topical application. Because DNA binding is considered an essential step in the initiation of chemical carcinogenesis, the DNA adduction by MeDBC was compared with that by DBC in mouse liver and skin via a 32P-postlabeling technique. Both compounds elicited chromatographically similar adducts in liver; however, the extent of total DNA binding of DBC was 343- and 265-fold greater than that of MeDBC 24 h after topical and i.p. administration, respectively, of a 37 mumol/kg dose. In skin, the adduct pattern elicited by either compound after topical application was different from that seen in liver, and three of four adducts derived from MeDBC were chromatographically distinct from those produced by DBC. Quantitative analysis revealed that total adduction in skin by DBC was 2.3-fold higher than by MeDBC. When the adduct levels were compared between liver and skin, topically applied MeDBC bound preferentially to skin versus liver DNA by a factor of 10, while the opposite was true for DBC. These data are in agreement with the carcinogenicity reported for DBC and MeDBC and support the hypothesis that the extent of covalent DNA modification by these compounds is associated with their biological activity. We conclude that an unsubstituted nitrogen is essential for the genotoxic activity of DBC in liver but not skin. The results also demonstrate the potential of the 32P-postlabeling assay in predicting the organotropism of closely related carcinogens.

Administration, Topical↗
PubMed1987

The correlation between DNA adducts and chromosomal aberrations in the target organ of benzidine exposed, partially-hepatectomized mice.

An experimental system was developed to test the association between benzidine--DNA adduct levels and chromosome aberrations in the target organ, the liver, of mice. A 2/3 partial hepatectomy was performed (0 h), then the animals were treated with benzidine (0, 7.8, 19.5, 38.2 or 97.8 mg/kg, i.p.) and an agar-coated 50 mg 5-bromodeoxyuridine tablet was implanted subcutaneously (58 h). Colcemid was given at 4 mg/kg i.p. (70 h), and the animals were sacrificed 2 h later. The liver from each animal was divided, with portions allocated for cytogenetics and DNA adduct analysis. DNA adducts were analyzed with the 32P-postlabeling technique. DNA adduct and chromosomal aberration data were available on a total of 43 animals. Benzidine was shown to be a potent clastogen in liver, the target organ, as opposed to its reported weak activity in the bone marrow. A linear dose response was demonstrated for benzidine--DNA adducts found in the liver. The correlation between adduct levels and aberrations in individual animals was 0.43 (P less than 0.05). However, most of the residual variance was due to four outlying cases. When these cases were removed from the data set and the analysis repeated, the linear correlation coefficient increased to 0.74. When the data were analyzed by dose groups, the correlation was 0.91. These data support the hypothesis that carcinogen-DNA adducts are responsible for the induction of chromosomal aberrations, and perhaps other genotoxic events, including neoplasia.

Animals↗
PubMed1987

32P-postlabeling analysis of DNA adduction in mice by synthetic metabolites of the environmental carcinogen, 7H-dibenzo[c,g]carbazole: chromatographic evidence for 3-hydroxy-7H-dibenzo[c,g]carbazole being a proximate genotoxicant in liver but not skin.

The DNA adduction by the environmental carcinogen 7H-dibenzo[c,g]carbazole (DBC) and chemically synthesized 2-OH, 3-OH, and 4-OH metabolites of DBC was investigated in liver and skin of female CD-1 mice. After topical application to the skin of 37 mumol/kg of DBC or the phenolic metabolites, DNA adducts were measured by a 32P-post-labeling assay employing carrier-free [gamma-32P]ATP and ATP-deficient conditions. In liver, DBC produced four major and several minor chromatographically distinct adducts of as yet undetermined chemical structure. The adduct pattern elicited by 3-OH-DBC was qualitatively similar to the DBC adduct pattern, while this was not the case for 2-OH-DBC and 4-OH-DBC. On the basis of co-chromatography experiments under various conditions, the DBC and 3-OH-DBC adducts appeared identical, and the total of adduction elicited by these compounds in liver was substantial. Similar results were observed when DBC or 3-OH-DBC were administered i.p. As a major difference between the two compounds, one 3-OH-DBC adduct (no. 3) was 4.4- and 7.0-fold lower than the corresponding DBC adduct after i.p. and topical dosing, respectively. In skin, DBC produced two major adduct fractions after topical application, one of which could be chromatographically resolved into three subcomponents. Prominent adducts produced in skin DNA by each of the three metabolites were different from those elicited by DBC, and the level of adduction by the metabolites was significantly lower than that by DBC. Comparison of the skin and liver DBC-DNA adduct patterns after topical application of DBC showed that only one of the four major chromatographically resolved skin adducts corresponded to a major liver adduct (no. 3), and that total adduction in liver was 13.5-fold higher than in skin. These results suggested that activation of DBC to DNA-binding compounds in liver occurs through at least two pathways with 3-OH-DBC being a proximate carcinogen involved in the formation of most of the adducts; 3-OH-DBC and the other two phenolic metabolites investigated play a minor role, if any, in the formation of DBC-DNA adducts in skin; metabolic activation of DBC to DNA-binding compounds in liver and skin appears to follow pathways that are different in terms of both the chemical nature and the amount of the adducts formed; and DBC and 3-OH-DBC exhibit a strong preference for liver versus skin DNA.

Animals↗
PubMed1987

32P-postlabeling assay for carcinogen-DNA adducts: nuclease P1-mediated enhancement of its sensitivity and applications.

Exceedingly sensitive assays are required for the detection of DNA adducts formed in humans exposed to low levels of environmental genotoxicants and therapeutic drugs. A 32P-postlabeling procedure for detection and quantitation of aromatic carcinogen-DNA lesions with a sensitivity limit of 1 adduct in 10(7) to 10(8) nucleotides has been described previously. In the standard procedure, DNA is enzymatically digested to 3'-phosphorylated normal and adducted mononucleotides, which are 32P-labeled at 5'-hydroxyl groups by T4 polynucleotide kinase-catalyzed [32P]phosphate transfer from [gamma-32P]ATP. 32P-labeled derivatives are resolved by TLC, detected by autoradiography, and quantitated by counting. This assay has been recently utilized for the determination and partial characterization of DNA adducts formed in somatic and reproductive tissues of rats given the clinically used anticancer drug, mitomycin C. The drug exhibits similar levels of covalent binding to DNA in most tissues. Further studies have revealed that adducted nucleotides are primarily guanine derivatives that are resistant to 3'-dephosphorylation by Penicillium citrinum nuclease P1. The latter observation has been utilized to enhance the 32P-assay's sensitivity to 1 adduct in 10(10) nucleotides for a 10-micrograms DNA sample by postincubation of DNA digests with nuclease P1 before 32P-labeling. The enzyme dephosphorylates the normal nucleotides but not most aromatic and bulky nonaromatic adducts, so that only the latter serve as substrates for the kinase-catalyzed labeling reaction. The new assay has also shown utility in the analysis of very low levels of age- and tissue-related DNA modifications, which might arise from dietary or endogenous compounds, in untreated rats and in humans.

Animals↗
PubMed1986

Detection of smoking-related covalent DNA adducts in human placenta.

The presence of covalent DNA chemical addition products (adducts) in human term placentas was investigated by recently developed immunologic and 32P-postlabeling assays. DNA from placental specimens of smokers showed a small but not statistically significant increase in adduct levels when tested by antibodies to DNA modified with a benzo[a]pyrene dihydrodiol epoxide (BPDE-I), the ultimate carcinogenic derivative of benzo[a]pyrene. The postlabeling assay detected several modified nucleotides, one of which (adduct 1) strongly related to maternal smoking during pregnancy. This adduct was present in placental tissue from 16 of 17 smokers, but only 3 of 14 nonsmokers. Among smokers, levels of adduct 1 in general were only weakly related to questionnaire and biochemical measures of the intensity of smoking exposures, which suggests modulation by individual susceptibility factors. The adduct seemed to be derived from an aromatic carcinogen, but it may not result from several of the most intensely studied polycyclic aromatic hydrocarbons or aromatic amines in tobacco smoke. The data show the association of cigarette smoking with covalent damage to human DNA in vivo.

Carcinogens↗
PubMed1986

Differences in the covalent binding of benzo[a]pyrene, safrole, 1'-hydroxysafrole, and 4-aminobiphenyl to DNA of pregnant and non-pregnant mice.

The effects of pregnancy on the covalent binding of several carcinogens to DNA were investigated in mice. Non-pregnant or timed-pregnant (18th day of gestation) ICR mice of similar age were treated with benzo[a]pyrene (BP, 200 mumol/kg), safrole (600 mumol/kg), 1'-hydroxysafrole (400 mumol/kg), 4-aminobiphenyl (4-ABP, 800 mumol/kg), or trioctanoin (4 ml/kg) per os. Tissue DNA adduct levels at 24 h after carcinogen treatment were analyzed via a 32P-postlabeling assay. Pregnancy lowered the binding of the ultimate carcinogenic metabolite of BP, 7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE I), to liver and lung DNA by 29-41%, but not the binding of other metabolites. The binding of safrole and its proximate carcinogen, 1'-hydroxysafrole, to liver and kidney DNA was increased 2.3-3.5 fold. Pregnancy decreased the binding of 4-ABP to liver DNA by approximately 18% but increased its binding to kidney DNA by 67%. The results suggest that exposure to some genotoxic compounds, especially those requiring conjugation reactions for metabolic activation, may be more hazardous during pregnancy than in the non-pregnant state.

Aminobiphenyl Compounds↗
PubMed1986

Estrogen-induced endogenous DNA adduction: possible mechanism of hormonal cancer.

In animals and humans, estrogens are able to induce cancer in susceptible target organs, but the mechanism(s) of estrogen-induced carcinogenesis has not been elucidated. A well-known animal model is the development of renal carcinoma in estrogen-treated Syrian hamsters. Previous work demonstrated the presence of covalent DNA addition products (adducts) in premalignant kidneys of hamsters exposed to the synthetic estrogen, diethylstilbestrol, a known human carcinogen. In the present study, the natural hormone, 17 beta-estradiol, and several synthetic steroid and stilbene estrogens were examined by a 32P-postlabeling assay for their capacity to cause covalent DNA alterations in hamster kidney. Chronic exposure to each of the estrogens tested led to the gradual formation of five chromatographically distinct unusual nucleotides specifically in kidney DNA. Irrespective of the estrogen used, chromatograms exhibited identical mobilities of each of these adducts in seven different systems on PEI-cellulose anion-exchange TLC, in three different conditions on reversed-phase TLC, and in one system on silica gel partition TLC. Therefore, the DNA adducts observed did not contain moieties derived from the structurally diverse estrogens. It is concluded that each of the estrogens induced the binding of the same unknown endogenous compound (or compounds) to target tissue DNA. This novel property of estrogens is postulated to play a key role in hormone-induced malignancy.

Animals↗
PubMed1986

Comparative DNA binding of 7,12-dimethylbenz[a]anthracene and some of its metabolites in mouse epidermis in vivo as revealed by the 32P-postlabeling technique.

The binding of some mouse skin metabolites and related derivatives of the tumor initiator 7,12-dimethylbenz[a]anthracene (DMBA) was investigated by 32P-postlabeling analysis after its topical administration. DMBA and trans-3,4-dihydro-3,4-dihydroxy-DMBA (DMBA-3,4-dihydrodiol) both led to the formation of four DNA adducts, which showed a very similar pattern of spots on thin-layer chromatograms. With trans-8,9-dihydro-8,9-dihydroxy-7,12-dimethylbenz[a]anthracene (DMBA-8,9-dihydrodiol) one major adduct was obtained which was chromatographically indistinguishable from one of the DMBA adducts. In contrast, 7-hydroxymethyl-12-methylbenz[a]anthracene (7-OHM-12-MBA) gave rise to two major adducts which were separable from DMBA adducts. 3-hydroxy-7,12-dimethylbenz[a]anthracene (3-OH-DMBA) and 7,12-dimethylbenz[a]anthracene-7,12-epoxide (DMBA-O2) did not lead to detectable amounts of adducts. Quantitative determination of DNA binding showed that an initiating dose (i = 100 nmol) of DMBA yielded approximately 12 adducts/10(7) normal nucleotides. Adduct formation with the same dose of DMBA-3,4-dihydrodiol was 7-8 times higher. At a 4-fold higher dose level, DMBA-8,9-dihydrodiol exhibited a 3- to 6-times weaker binding and 7-OHM-12-MBA a slightly stronger binding than DMBA. Chromatography of the DMBA and DMBA-3,4-dihydrodiol adducts with a solvent containing borate showed a decreased mobility of two out of four adducts in each case. These adducts were also sensitive to oxidation by periodate. The results suggest that two DMBA adducts carried vicinal cis-hydroxyl groups and thus were probably derived from the anti-3,4-dihydrodiol-1,2-oxide(s) of DMBA. The other two adducts were probably derived from the syn-stereoisomer(s). When the DNA-modifying capabilities and initiating activities of the more prominent mouse-skin metabolites are considered in relation to DMBA, DMBA-3,4-dihydrodiol is postulated to be a proximate and DMBA-3,4-dihydrodiol-1,2-oxide(s) to be ultimate initiators.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1986

Nuclease P1-mediated enhancement of sensitivity of 32P-postlabeling test for structurally diverse DNA adducts.

Exceedingly sensitive procedures are required to detect the presence of covalent DNA adducts in humans exposed to environmental genotoxicants because of low levels of such derivatives (1 adduct in 10(8)-10(10) DNA nucleotides). A 32P-postlabeling assay for detection and quantitation of carcinogen--DNA adducts with a sensitivity limit of 1 adduct in 10(7)-10(8) nucleotides has been described previously. In the standard procedure, DNA is enzymatically digested to 3'-phosphorylated normal and adducted mononucleotides, which are quantitatively 32P-labeled at their 5'-hydroxyl groups by T4 polynucleotide kinase-catalyzed [32P]phosphate transfer from [gamma-32P]ATP. 32P-labeled derivatives are resolved by t.l.c., detected by autoradiography and quantitated by counting. We now report that a minor modification of this procedure, entailing the postincubation of DNA digests with Penicillium citrinum nuclease P1 before 32P-labeling, enhanced the technique's sensitivity to 1 adduct in approximately 10(10) nucleotides for a 10-micrograms DNA sample. Nuclease P1 cleaves deoxyribonucleoside 3'-monophosphates of normal nucleotides to deoxyribonucleosides which do not serve as substrates for polynucleotide kinase, while most adducted nucleotides were found to be totally or partially resistant to the 3'-dephosphorylating action of nuclease P1. The additional enzymatic step enabled specific labeling of adducts in up to 20 micrograms of DNA with excess carrier-free [gamma-32P]ATP. The enzymatic digestion conditions were standardized to afford optimal adduct recovery. The new procedure was found to be simple, highly reproducible, and applicable to the detection and measurement of aromatic or bulky non-aromatic DNA adducts formed with such structurally diverse carcinogens as benzo[a]pyrene, 7,12-dimethyl-benz[a]anthracene, dibenzo[c,g]carbazole, 4-aminobiphenyl, safrole and mitomycin C; most adducts were recovered quantitatively with a 500- to 1000-fold increase in 32P-count rates as compared with the standard procedure.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1986

Age- and tissue-related DNA modifications in untreated rats: detection by 32P-postlabeling assay and possible significance for spontaneous tumor induction and aging.

When liver, kidney, lung and heart DNA preparations of untreated Sprague-Dawley rats of different ages (3 days-10 months) were analyzed for the possible presence of covalent modifications by a 32P-postlabeling assay, characteristic tissue-specific patterns of 32P-labeled spots (termed I-spots) were observed on thin-layer chromatograms. Amounts of these DNA derivatives (termed I-compounds), which were not detected in newborn rat DNA, markedly increased with age. This novel type of DNA modification could be due to environmental (e.g. dietary) factors or to endogenous DNA-reactive metabolites and may conceivably play a role in the initiation of spontaneous cancers or other adverse health effects related to aging.

Aging↗
PubMed1986

32P-postlabeling assay in mice of transplacental DNA damage induced by the environmental carcinogens safrole, 4-aminobiphenyl, and benzo(a)pyrene.

Transplacental exposure of fetuses to carcinogens is known to induce tumors in the offspring, often with a high incidence and short latency. While covalent adduction of DNA appears to be essential for tumor initiation, little is known about the binding of carcinogens to the DNA of fetal tissues. A sensitive 32P-postlabeling method enabled us to study the binding of the environmental carcinogens safrole (600 mumol/kg p.o.), 4-aminobiphenyl (800 mumol/kg), and benzo(a)pyrene (200 mumol/kg) to the DNA of various maternal and fetal tissues after administration of test carcinogens to pregnant ICR mice on day 18 of gestation. The results show that these carcinogens bound to the DNA of maternal and fetal liver, lung, kidney, heart, brain, intestine, skin, maternal uterus, and placenta, with organ-specific quantitative and qualitative differences. It was possible for the first time to analyze DNA adduct patterns in minute amounts of tissue, for example those available from fetal heart. The covalent binding index (mumol adducted nucleotides per mol of DNA nucleotides/mumol carcinogen administered per g body weight) 24 h after safrole treatment was estimated for the different organs and ranged from 0.1 to 247 and 0.1 to 5.8 for maternal and fetal DNA, respectively. Covalent binding index values of 0.2 to 13 and 0.1 to 0.3 for maternal and fetal DNA, respectively, were found for 4-aminobiphenyl. Benzo(a)pyrene treatment yielded covalent binding index values of 0.6 to 6.5 and 0.3 to 0.7 for maternal and fetal DNA, respectively. In both maternal and fetal tissues, safrole exhibited preferential binding to liver DNA. 4-Aminobiphenyl bound preferentially to DNA of maternal liver and kidney but showed no preference among fetal tissues. Benzo(a)pyrene exhibited weak tissue preference in both maternal and fetal organs. For all of the compounds studied, the fetal adduct levels were generally lower than the corresponding maternal adduct levels, especially when the level of maternal adduction was high. The major finding was that several carcinogens of diverse structure or their metabolites readily crossed the placenta and gave rise to DNA adducts in fetal organs. The resulting DNA damage in rapidly proliferating tissues may play a critical role in transplacental carcinogenesis.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1986

Human cell-mediated cytotoxicity, mutagenicity, and DNA adduct formation of 7H-dibenzo(c,g)carbazole and its N-methyl derivative in diploid human fibroblasts.

The aromatic N-heterocyclic 7H-dibenzo(c,g)carbazole (DBC), like polynuclear aromatic hydrocarbons, is a potent inducer of local sarcomas, papillomas, and respiratory tumors, but unlike such compounds it also induces hepatomas. The N-methyl derivative of DBC, N-methyl-dibenzo(c,g)carbazole (MeDBC), also induces sarcomas, papillomas, and respiratory tumors but at a lower frequency than DBC. However, MeDBC lacks hepatocarcinogenic potential, suggesting that DBC undergoes metabolic activation at its carbon atoms and also at the nitrogen position and that the N-7 position plays a role in liver carcinogenesis by DBC. We compared the cytotoxic and mutagenic potential of DBC and MeDBC using a human epithelial cell-mediated activation assay. Repair-deficient, diploid human fibroblasts derived from a xeroderma pigmentosum (XP) patient were used as target cells, and a human hepatoma cell line, Hs703T, was used as a source of exogenous metabolism. Resistance to 6-thioguanine served as the genetic marker for mutations. The Hs703T cells, but not the target XP cells, activated DBC and MeDBC into forms capable of interacting with DNA. In the cell-mediated assay, both DBC and MeDBC induced cytotoxicity and mutations in the target XP cells in a dose-dependent manner. However, DBC was effective at 2-fold lower concentrations than MeDBC. DNA adduct analysis using a 32P-postlabeling assay showed that at biologically significant low doses DBC was 2.5 times more effective than MeDBC in covalent binding to DNA. At higher doses, the difference in ability to bind to DNA was 1.3-fold. In both XP and Hs703T cells, DBC produced three major adducts, while MeDBC produced two major adducts, one of which was the same as one detected in the DBC adduct pattern. The number of DBC- and MeDBC-induced DNA adducts corresponding to a particular level of cytotoxicity and mutagenicity in the XP cells was 10 times lower than that for (+/-)-7 beta, 8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene.

Animals↗
PubMed1986

Comparative 32P-analysis of cigarette smoke-induced DNA damage in human tissues and mouse skin.

Previous studies using a highly sensitive 32P-postlabeling assay for the analysis of carcinogen/mutagen-induced DNA damage have shown the presence of tobacco smoking-related DNA adducts in human placenta (Everson, R.B., Randerath, E., Santella, R.M., Cefalo, R.C., Avitts, T. A., and Randerath, K., Science (Wash. DC), 231: 54-57, 1986). The occurrence of such adducts in smokers' bronchus and larynx is reported here. Since the chemical nature of these adducts could not be characterized by direct methods due to the extremely low levels of individual adducts (less than 0.03 fmol per microgram DNA), we have sought an experimental animal model for studying the formation of tobacco-related DNA adducts. Because cigarette smoke condensate is known to initiate tumors in mouse skin, ICR mice were treated topically with cigarette tar equivalent to 1.5, 3, 6, and 9 cigarettes for 0.4, 3, 5, and 7 days, respectively, and skin DNA was isolated 1 day after the last treatment. When DNA from exposed mice was analyzed by the 32P-postlabeling assay, 12 distinct 32P-labeled DNA adduct spots, as well as a diagonal radioactive zone, which presumably reflected the presence of incompletely resolved adducts, were noted on polyethyleneimine-cellulose TLC fingerprints. One derivative in particular (adduct 1) was seen to increase rapidly during the early treatment phase and also to persist to 8 days after treatment. The prominent adduct 1 was observed in the same location on the fingerprints of DNA samples from smokers. Cochromatography experiments suggested identity of human and mouse DNA adduct 1. Similarly, several other human and mouse adducts (adducts 3, 5, 6, and 9) appeared identical, and the diagonal radioactive zone was also present on DNA adduct maps from smokers. While absolute levels of individual human adducts were too low to be accurately quantitated, semiquantitative estimation of total tobacco-related aromatic DNA adducts in the human specimens gave values of 1 adduct in (1.7-2.9) X 10(7) nucleotides (0.10-0.18 fmol per micrograms DNA), with adduct 1 constituting 8.5-14% of the total. On the basis of these results, it appears now feasible to determine the chemical origin of smoking-induced DNA adducts in human tissues by preparation of authentic 32P-labeled reference adducts from animals treated with characterized subfractions of cigarette tar, 32P-postlabeling, and cochromatography of 32P-labeled human and animal adducts.

Animals↗
PubMed1985

12-O-tetradecanoylphorbol-13-acetate-induced rapid loss of persistent 7,12-dimethylbenz[a]anthracene-DNA adducts in mouse epidermis and dermis.

Twice-weekly application to mouse skin of 10 nmol of the potent tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), beginning at 3 weeks after topical application of 1.2 mumol of 7,12-dimethylbenz[a]anthracene (DMBA), was found to cause a rapid loss of persistent DMBA-DNA adducts from both epidermal and dermal DNA. This effect is thought to reflect TPA-induced proliferation of quiescent initiated skin cells containing persistent adducts and may be causally related to the irreversibility of the initial phase of tumor promotion, which is known to require cell proliferation.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1985

Distribution of initial and persistent 2-acetylaminofluorene-induced DNA adducts within DNA loops.

The intranuclear distribution of initial and persistent DNA adducts induced in vivo after four weekly injections of the hepatocarcinogen 2-acetylaminofluorene was examined in rat liver by using a protocol that fractionates chromatin from various regions of each of the multiple nuclear DNA loops. Ten hours after the initial dose, two acetylated [(N-acetyl-N-(deoxyguanosin-8-yl)-2-aminofluorene and 3-(deoxyguanosin-N2-yl)-2-acetylaminofluorene] and one deacetylated [N-(deoxyguanosin-8-yl)-2-aminofluorene] adduct were detected by a 32P-labeling assay and were found to have a random genomic distribution, as evident by their relative concentrations in various chromatin fractions. These data suggest that all regions of the DNA loops are equally susceptible to adduct formation. A nonrandom persistence of the deacetylated adduct in the regions where the DNA loops are constrained by the nuclear matrix was evident by 6 days after the last dose and was markedly apparent by 60 days. In contrast, all chromatin fractions had equally inefficient removal of the N2-acetylated adduct by 6 days as well as 60 days but had complete removal of the C8-acetylated adduct. These findings suggest that pronounced regional differences in adduct repair along the DNA loops may play a role in chemically induced hepatocarcinogenesis.

2-Acetylaminofluorene↗
PubMed1985

Target organ-specific covalent DNA damage preceding diethylstilbestrol-induced carcinogenesis.

The synthetic estrogen diethylstilbestrol (DES), a known human carcinogen, induces renal carcinoma in male Syrian hamsters within 6 months after s.c. implantation. Tumor formation could be evoked by its hormonal properties or by a reactive genotoxic metabolite binding to DNA, but previous attempts to detect adducts have failed. In the present study, kidney DNA of male Syrian hamsters, treated with s.c. DES implants to induce renal carcinoma, was analyzed for the presence of DES-induced adducts using 32P-postlabeling assay. Covalently-modified DNA nucleotides were detected in the kidneys after chronic DES treatment, but not in kidneys of untreated hamsters, or in liver or tumor tissue of DES-treated animals. This report demonstrates for the first time the ability of an estrogen to give rise to covalent DNA modification in vivo specifically in the target organ of carcinogenesis. DES-induced covalent DNA adducts are taken as evidence for tumor initiation by DES via damage to cellular macromolecules, in addition to tumor-promotional effects described previously.

Adenosine Triphosphate↗