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K Randerath

Publications and source records attributed to K Randerath.

At least 145 records · Page 8Linked to original sources
PubMed1985

32P-postlabeling analysis of DNA adducts persisting for up to 42 weeks in the skin, epidermis and dermis of mice treated topically with 7,12-dimethylbenz[a]anthracene.

The initial and persistent levels of 7,12-dimethylbenz[a]-anthracene (DMBA)-DNA adducts in mouse skin, epidermis and dermis after topical carcinogen application were studied by 32P-postlabeling assay. In the major experiment, a single dose of 1.2 mumol of the carcinogen was applied to the shaved backs of adult female BALB/cANN mice, and DNA was isolated from epidermis and dermis, respectively, 24 h and 1, 2, 3, 4, 8, 16, 24, 36 and 42 weeks later. Total binding at 24 h was approximately 34 and approximately 28 adducts in 10(7) normal nucleotides for epidermal and dermal DNA, respectively. (One adduct in 10(7) nucleotides equals 0.3 fmol adduct/microgram DNA.) While initial binding was higher in epidermal DNA, the adducts were approximately 10 times more persistent in dermal DNA: at 42 weeks, total binding levels were approximately 0.17 and approximately 1.7 adducts in 10(7) nucleotides for epidermis and dermis, respectively. To quantitate low levels of DMBA-DNA adducts, 32P-postlabeling assays were run in the presence of a limiting amount of carrier-free [gamma-32P]ATP; this was found to favor labeling of the adducts, thereby leading to a 20- to 100-fold enhancement of the method's sensitivity for individual adducts. One of the three major DMBA-DNA adducts was more persistent than were the others; the level of this adduct remained constant at approximately 60% of the total in epidermal and dermal DNA during the last 18 weeks of the 42-week observation period. Since a [3H]thymidine-labeling experiment showed a normal epidermal DNA turnover 40 weeks after DMBA treatment, it was concluded that the bulk of the persistent adducts was present in subpopulations of dormant cells. We have hypothesized that such cells, in the absence of a promoting stimulus, are incapable of division because of the adduction and/or mutation of genes critical for growth (proto-oncogenes), and may thus correspond to the 'latent tumor cells', as defined by Berenblum and Shubik in their classical analysis of the attributes of tumor initiation and promotion.

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

Tissue-specific DNA adduct formation in mice treated with the environmental carcinogen, 7H-dibenzo[c,g]carbazole.

Covalent adduction of DNA by chemical agents is commonly thought to be an essential part of the initiation of chemical carcinogenesis. Until recently, assays of DNA damage by covalent binding of chemicals have been restricted mostly to substances that are available in radiolabeled form, which excludes many environmental compounds with carcinogenic potential. In this paper, the binding of non-radioactive 7H-dibenzo[c,g]carbazole (DBC), a known environmental carcinogen, to DNA in female CD-1 mice after s.c. injection of 44 mumol/kg of the compound has been investigated using a 32P-postlabeling assay. DBC showed strong hepatic specificity with a mean total level of 107 adducts per 10(7) nucleotides at 24 h, while much lower levels of binding were seen in kidney, lung, spleen, skin and brain with 4.3, 2.1, 1.3, 0.4 and 0.04 adducts, respectively, per 10(7) nucleotides. Proportions of individual DBC adducts also varied considerably between tissues. The degree of hepatic preference displayed by DBC is not seen with other polycyclic aromatic carcinogens such as benzo[a]pyrene and 2-acetylaminofluorene. The DNA-binding data, together with other hepatotoxic effects of the compound, may be causally related to the known hepatocarcinogenicity of DBC.

2-Acetylaminofluorene↗
PubMed1985

Postlabeling methods for carcinogen-DNA adduct analysis.

Radioactive carcinogens have provided most of our present knowledge about the chemistry of interactions between carcinogens and biological systems. The requirement of radioactive carcinogens has restricted carcinogen-DNA binding studies to chemicals that are readily available in isotopically labeled form, i.e., a minute fraction of all potentially mutagenic or carcinogenic chemicals. To extend the scope of carcinogen-DNA binding studies, an alternative method, which does not require radioactive test chemicals, has been developed. In this approach, radioactivity (32P) is being incorporated into DNA constituents by polynucleotide kinase-catalyzed [32P]phosphate transfer from [gamma-32P]ATP after exposure of the DNA in vitro or in vivo to a nonradioactive, covalently binding chemical, and evidence for the alteration of DNA nucleotides is provided by the appearance of extra spots on autoradiograms of thin-layer chromatograms of digests of the chemically modified DNA. Quantitation of adduct levels is accomplished by scintillation counting. The sensitivity of the technique depends on the experimental conditions for 32P-labeling and on the chemical structure of the adducts. Greater sensitivity may be achieved if adducts can be separated as a class from the normal nucleotides. This is the case for an estimated 80% of all carcinogens, giving rise to bulky and/or aromatic substituents in DNA. Under the present conditions, one such adduct in 10(9) to 10(10) normal nucleotides can be detected. A total of approximately 80 compounds has been studied thus far Binding to DNA of rodent tissues was readily detected by the 32P-postlabeling assay for all known carcinogens among these compounds, and adducts were detected in DNA from human placenta of smokers.

Animals↗
PubMed1985

Formation and persistence of sterigmatocystin--DNA adducts in rat liver determined via 32P-postlabeling analysis.

A 32P-postlabeling method has been employed to detect the in vitro and in vivo modification of DNA by the mycotoxin sterigmatocystin (ST). ST-modified DNA was initially incubated under buffered alkaline conditions to convert unstable ST-N7-guanine moieties to stable, putative ST-formamidopyrimidine derivatives. DNA was subsequently digested with micrococcal nuclease and spleen phosphodiesterase, and the resulting ST-modified nucleotides, purified by reverse-phase thin-layer chromatography (TLC), were labeled at the 5' position via incubation with [gamma-32P]ATP and T4 polynucleotide kinase. 32P-labeled ST-nucleotides were separated by reverse-phase and anion-exchange TLC. Cerenkov quantitation of excised TLC fractions indicated that ST-DNA moieties could be detected with a sensitivity of 1 ST adduct in 3-5 X 10(7) nucleotides. Initial enzymatic digestion of ST-modified DNA was found to yield ST-modified di- and trinucleotides which, upon 32P-labeling followed by incubation with nuclease P1, liberated unmodified 5'-terminal nucleotides suggesting that ST-formamidopyrimidine-modified DNA was a poor substrate for micrococcal nuclease and spleen phosphodiesterase. Dose-dependent ST-DNA adduct formation was detected in the liver of male Fischer 344 rats over a 27-fold range of ST administered (0.33-9 mg/kg). In addition, ST-DNA adducts, formed in rats given a 9 mg/kg dose, were found to persist up to 105 days after treatment at a level of 0.5% of the 2-h value. Loss of these adducts from liver DNA was observed to exhibit a triphasic profile: rapid loss during the first 24 h (t 1/2 = 12 h) followed by a slower decline from 1 to 14 days post dosing (t 1/2 = 7 days) and an extremely slow decline from days 14 to 105 post treatment (t 1/2 = 109 days). This experimental approach to the study of mycotoxin-DNA interactions permits the quantitative description of DNA modification in ST-treated animals. Further refinement of this approach may be useful in defining the precise relationship between ST exposure and tumorigenesis in ST-exposed human populations.

Animals↗
PubMed1984

32P-post-labelling analysis of DNA adducts formed in the livers of animals treated with safrole, estragole and other naturally-occurring alkenylbenzenes. I. Adult female CD-1 mice.

The binding of a series of alkenylbenzenes to liver DNA of adult female CD-1 mice, isolated 24 h after i.p. administration of non-radioactive test compound (2 or 10 mg/mouse), was investigated by a modified 32P-post-labelling assay. The known hepatocarcinogens, safrole, estragole and methyleugenol, exhibited the strongest binding to mouse-liver DNA (1 adduct in 10 000 - 15 000 DNA nucleotides or 200 - 300 pmol adduct/mg DNA after administration of a 10 mg dose), while several related compounds, which have not been shown thus far to be carcinogenic in rodent bioassays, bound to mouse-liver DNA at 3 - 200x lower levels. The latter compounds included allylbenzene, anethole, myristicin, parsley apiol, dill apiol and elemicin. Eugenol did not bind. Low binding to mouse-liver DNA was also observed for the weak hepatocarcinogen, isosafrole. Two main 32P-labelled adducts, which appeared to be guanine derivatives, were detected for each of the binding chemicals on thin-layer chromatograms. The loss of safrole adducts from liver DNA was biphasic: a rapid loss during the first week (t 1/2 approximately 3 days) was followed by a much slower decline up to 20 weeks after treatment (t 1/2 approximately 2.5 months). Adducts formed by reaction of 1'-acetoxysafrole, a model ultimate carcinogen, with mouse-liver DNA in vitro were chromatographically identical to safrole-DNA adducts formed in vivo. Pretreatment with pentachlorophenol, a known inhibitor of sulphotransferases, inhibited the binding of safrole to mouse-liver DNA, providing further evidence that the metabolic activation of the allylbenzenes proceeds by the formation of 1'-hydroxy derivatives as proximate carcinogens and 1'-sulphoöxy derivatives as ultimate carcinogens.

Allylbenzene Derivatives↗
PubMed1984

32P-post-labelling analysis of DNA adducts formed in the livers of animals treated with safrole, estragole and other naturally-occurring alkenylbenzenes. II. Newborn male B6C3F1 mice.

When a series of nine alkenylbenzenes were administered to preweanling male mice, safrole, estragole and methyleugenol induced a significant incidence of hepatic carcinomas, while eugenol, anethole, elemicin, myristicin, dill apiol and parsley apiol did not (Miller et al., Cancer Res., 43, 1124-1134, 1983). Following the protocol used to test seven of these compounds, male C57Bl X C3H/He F1 mice were injected with 0.25, 0.5, 1.0 and 3.0 mumol of a compound on days 1, 8, 15 and 22 after birth, respectively. Groups of mice were killed and their liver DNA isolated on days 23, 29 and 43, and analysed by a modified 32P-post-labelling procedure. Highest levels of adducts were detected with methyleugenol (72.7 pmol/mg DNA), estragole (30.0) and safrole (17.5). After correction for liver growth it was estimated that most of these adducts were still present at 43 days. Significant levels of DNA binding by myristicin (7.8 pmol/mg DNA) and elemicin (3.7) were also found but in the former case the adducts were less persistent. Only low levels of adducts were detected with anethole, dill apiol and parsley apiol (less than 1.4 pmol/mg DNA); no DNA binding was detected with eugenol. Thus, all but one of the alkenylbenzenes studied became bound to newborn mouse-liver DNA, but the levels and the persistence of adducts formed by the carcinogenic compounds were greater.

Allylbenzene Derivatives↗
PubMed1984

32P-postlabeling test for covalent DNA binding of chemicals in vivo: application to a variety of aromatic carcinogens and methylating agents.

Carcinogen--DNA adducts were detected and determined by 32P-postlabeling assay after exposure of mouse or rat tissues in vivo to a total of 28 compounds comprising 7 arylamines and derivatives, 3 azo compounds, 2 nitroaromatics, 12 polycyclic aromatic hydrocarbons, and 4 methylating agents. DNA was isolated from mouse skin, mouse liver, and rat liver after treatment with the individual carcinogens, then digested enzymatically to deoxyribonucleoside 3'-monophosphates, which were converted to 5'-32P-labeled deoxyribonucleoside 3',5'-bisphosphates by T4 polynucleotide kinase-catalyzed [32P]phosphate transfer from [gamma-32P]ATP. The nucleotides were resolved by anion-exchange t.l.c. on polyethyleneimine-cellulose and detected by autoradiography. The determination of low levels of DNA binding of the aromatic carcinogens entailed the removal of normal nucleotides prior to the resolution of adduct nucleotides. For this purpose, an alternative procedure employing reversed-phase t.l.c. was devised which offered advantages for the detection of quantitatively minor adducts. The procedures described enabled the detection of 1 aromatic DNA adduct in approximately 10(8) normal nucleotides, while the limit of detection of methylated adducts was 1 adduct in approximately 6 X 10(5) nucleotides. The results show that a great number of carcinogen-DNA adducts of diverse structure are substrates for 32P-labeling by polynucleotide kinase-catalyzed phosphorylation. Because covalent DNA adduct formation in vivo appears to be an essential property of the majority of chemical carcinogens, 32P-postlabeling analysis of carcinogen--DNA adducts in mammalian tissues may serve as a test for the screening of chemicals for potential carcinogenicity.

Alkylating Agents↗
PubMed1984

Long term instability and molecular mechanism of 5-azacytidine-induced DNA hypomethylation in normal and neoplastic tissues in vivo.

We have previously shown that treatment of normal and neoplastic cells with the antileukemic drug, 5-azacytidine, led to the rapid synthesis of a low molecular weight RNA containing 5-azacytosine. This fraudulent RNA inhibited tRNA (cytosine-5)-methyltransferase early after drug administration. The absence of tRNA (cytosine-5)-methyltransferase activity resulted in the synthesis of tRNA specifically deficient in 5-methylcytosine. Here, we show that treatment of L1210 cells, grown intraperitoneally in mice, with 5-azacytidine led to a rapid and prolonged inactivation of DNA (cytosine-5)-methyltransferase activity and to the synthesis of undermethylated DNA. DNA isolated from the treated tissue was found to inactivate the DNA methylase (decreased Vmax) in in vitro DNA (cytosine-5)-methyltransferase assays. Kinetic analysis showed noncompetitive inhibition of the substrate by the inhibitor. The persistence of DNA undermethylation after treatment with 5-azadeoxycytidine or 5-azacytidine in animals has not been measured directly; therefore, we have investigated this phenomenon in the intact animal. Prolonged treatment with 5-azacytidine was required to maintain a a fraction of undermethylated sites in DNA of L1210 cells in vivo for up to 4 months or longer after drug withdrawal. Such treatment led to instability of DNA methylation levels in L1210 cells in vivo. At least a partial restoration of DNA 5-methylcytosine levels was observed after acute and chronic 5-azacytidine treatment, respectively. 5-Azacytidine was also found to induce DNA hypomethylation in regenerating, but not in normal adult mouse liver cells. Our results show that: 1) it was extremely difficult to decrease the DNA methylation level to less than 50% of control; and 2) it was also difficult to maintain stable DNA methylation levels in vivo after exposure to the drug.

Animals↗
PubMed1984

Specific lack of the hypermodified nucleoside, queuosine, in hepatoma mitochondrial aspartate transfer RNA and its possible biological significance.

Tumor nucleic acids have frequently been found to be deficient in methylated and other modified nucleotides. In particular, cytoplasmic transfer RNAs (tRNAs) from various neoplasms partially lack the hypermodified nucleoside queuosine, a modification specific for anticodons of histidine-, tyrosine-, asparagine-, and aspartic acid-accepting tRNAs. Using aspartate tRNA as an example, we show here that liver mitochondria contain tRNA fully modified with respect to queuosine, while the corresponding tRNA from mitochondria of Morris hepatoma 5123D completely lacks this constituent. The sequences of these tRNAs, which were determined by a highly sensitive 32P-postlabeling procedure entailing the direct identification of each position of the polynucleotide chains, were found to be (sequence in text) Lack of queuosine in the hepatoma mitochondrial tRNA may be due to the inavailability of queuine in the hepatoma mitochondria for incorporation into tRNA or to inhibition of the modifying enzyme, tRNA (guanine)-transglycosylase, in the tumor. Taking into account results of others indicating a possible involvement of the queuosine modification in differentiation of eukaryotic cells, we hypothesize that the queuosine defect may develop at an early stage of carcinogenesis (i.e., during the promotion phase) and be directly involved in abnormalities of mitochondria which have been observed frequently in transformed cells and tumors.

Animals↗
PubMed1984

Biochemical (postlabelling) methods for analysis of carcinogen-DNA adducts.

Radioactive carcinogens have provided most of our present knowledge about the interactions between carcinogens and components of biological systems. The requirement of radioactive carcinogens restricts carcinogen-DNA binding studies to chemicals that are readily available in isotopically labelled form, i.e., a minute fraction of all potentially mutagenic or carcinogenic chemicals. To extend the scope of carcinogen-DNA binding studies, an alternative method, which does not require radioactive test chemicals, has been developed. In this approach, radioactivity (32P) is incorporated into DNA constituents by polynucleotide kinase-catalysed (32P)-phosphate transfer from (gamma-32P)ATP after exposure of the DNA, in vitro or in vivo, to a nonradioactive, covalently binding chemical; alteration of DNA nucleotides is shown by the appearance of extra spots on autoradiograms from thin-layer chromatograms of digests of the chemically modified DNA. Adduct levels are quantitated by scintillation counting. The sensitivity of the technique depends, to some extent, on the chemical structure of the adducts, in that greater sensitivity is achieved if adducts can be separated, as a class, from the normal nucleotides. An estimated 80% of all carcinogens can be separated in this way, giving rise to bulky and/or aromatic substituents in DNA. Under present conditions, one such adduct in 10(9)-10(10) normal nucleotides can be detected. A total of 41 compounds has been studied, so far. Binding to DNA of rodent liver and skin was readily detected by the 32P-postlabelling assay for all known carcinogens among these compounds, and adducts were detected in DNA from tissues of smokers.

Aflatoxin B1↗
PubMed1983

Specific effects of 5-fluoropyrimidines and 5-azapyrimidines on modification of the 5 position of pyrimidines, in particular the synthesis of 5-methyluracil and 5-methylcytosine in nucleic acids.

5-Fluoropyrimidines and 5-azapyrimidines were found in our laboratory to be specific inhibitors of modification reactions taking place at the 5 position of pyrimidines in nucleic acids. Thus, 5-fluorouracil and 5-fluorouridine specifically inhibit the formation of 5-methyluracil, pseudouridine, and 5,6-dihydrouracil in tRNA. 5-Fluorocytidine, which is partially biotransformed to 5-fluorouracil derivatives in mammalian cells, inhibits the formation of 5-methyluracil, pseudouridine, 5,6-dihydrouracil, and 5-methylcytosine, and 5-azacytidine is a specific inhibitor of the formation of 5-methylcytosine in tRNA and DNA. Inhibitory effects on tRNA modifications require RNA synthesis, as shown by the observation that various inhibitors of RNA synthesis block the drug effects. An inhibitory low-molecular-weight (4-7S) RNA, consisting mainly of tRNA and pre-tRNA, was isolated from livers of mice after treatment with 5-azacytidine. This RNA, when added to an in vitro tRNA methyltransferase assay, specifically interfered with the formation of 5-methylcytosine in substrate tRNA. Similarly, a DNA inhibiting the synthesis of 5-methylcytosine in an in vitro DNA methylation assay was isolated from L1210 leukemic cells treated with a high dose of 5-azacytidine for a short time. Our data are consistent with the hypothesis that incorporation of 5-azacytosine into positions that are normally occupied by C residues destined to become methylated is required for the inhibition to occur, and a similar situation probably applies to the 5-fluoropyrimidine analogs. Analog base moieties occupying such sites are likely to bind strongly, perhaps irreversibly, to the active sites of the particular modifying enzymes. All our observations with the 5-fluoro- and 5-azapyrimidines are in accord with this hypothesis. It was also observed that administration of 5-azacytidine to mice led to strong inhibition of tRNA cytosine-5-methyltransferase, while at the same time the activities and capacities of purine-specific tRNA methyltransferases became strongly elevated after an initial lag period. We speculate that such increases may represent a response of the cell to the methylation defect induced by the drug. Undermodified tRNAs present in neoplastic cells may also trigger an increased synthesis of modifying enzymes. A scheme has been presented which explains increased tRNA turnover and increased activities of modifying enzymes in neoplastic cells as a consequence of a primary defect in tRNA modification.

5-Methylcytosine↗
PubMed1983

tRNA alterations in cancer.

1. 3H-, 125I-, and 32P-labeling methods were developed for base composition and sequence analysis of minute amounts of nonradioactive nucleic acids containing modified constituents. 2. Base composition analysis showed tRNA from two "liver-like" minimal deviation hepatomas, Morris hepatomas 5123D and 7777, to exhibit typical alterations when compared with liver tRNA. Our observations, which were made for different transplant generations of the tumors, indicated a trend toward undermethylation and undermodification of tRNA. 3. Sequence analysis of several cytoplasmic and mitochondrial tRNAs from hepatoma 5123D showed partial lack of m2G and complete lack of Gm and Q. 4. Sequence analysis of mitochondrial tRNAs from hepatoma 5123D indicated several instances of alterations of primary structure, a phenomenon not previously observed for cytoplasmic tRNAs from neoplasms. 5. Biochemical mechanisms underlying these alterations, as well as their functional implications, have yet to be investigated. 6. Modification patterns, but not primary structures, of mitochondrial tRNAs have been highly conserved when compared to prokaryotic and eukaryotic cytoplasmic tRNAs. This implies that (a) post-transcriptional modifications must play a crucial role in tRNA function, and (b) alterations of post-transcriptional modifications in tumor tRNAs have to be regarded as highly significant deviations from the norm.

Animals↗
PubMed1983

DNA hypomethylation in Morris hepatomas.

The 5-methylcytosine (m5C) content of DNAs from Morris hepatomas of varying growth rates and from normal liver was analyzed. DNA methylation in all hepatomas studied was found to be 20-45% less than in normal liver. This result was confirmed independently by restriction endonuclease (Hpa II and Msp I) analysis. While these results agreed with recent literature data suggesting hypomethylation of DNA from some neoplastic sources, no correlation was observed between the extent of DNA hypomethylation and the growth rates of the tumors.

5-Methylcytosine↗
PubMed1983

Highly persistent polycyclic aromatic hydrocarbon-DNA adducts in mouse skin: detection by 32P-postlabeling analysis.

A 32P-postlabeling method for carcinogen-DNA adduct analysis recently developed in our laboratory was applied to skin DNA from mice treated topically with polycyclic aromatic hydrocarbons (PAHs). After application of 4 doses of 1.2 mumol each of benzo[alpha]pyrene (BP), 3-methylcholanthrene (MC) and 7,12-dimethylbenz[alpha]anthracene (DMBA), respectively, total covalent adduct binding in mouse skin DNA initially amounted to 1 adduct in 6.0 X 10(4) - 1.3 X 10(5) nucleotides. Four weeks after treatment, these levels had declined to 1 adduct in 1.4 X 10(6) - 2.7 X 10(6) nucleotides. Substantial removal of DNA adducts occurred during the first 2 weeks after carcinogen application while adducts remaining thereafter underwent little or no repair between 2 and 4 weeks after treatment. These results raise the possibility that the persistent adducts occupy specific genomic sites in quiescent cells where they may not be amenable to repair because of localized conformational alterations of DNA or shielding by associated proteins.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1982

32P-postlabeling analysis of non-radioactive aromatic carcinogen--DNA adducts.

A newly developed enzymatic 32P-postlabeling method was applied to the analysis of DNA's containing non-radioactive arylamine, arylamide, and polycyclic aromatic hydrocarbon adducts. DNA reacted in vitro with N-hydroxy-2-amino-fluorene, N-acetoxy-2-acetylaminofluorene, and 7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene, respectively, as well as DNA preparations from the liver of rats treated with N-hydroxy-2-acetylaminofluorene and benzo[a]pyrene, respectively, were enzymatically digested to deoxyribonucleoside 3'-monophosphates, which were then converted to [5'-32P]deoxyribonucleoside 3',5'-bisphosphates by T4 polynucleotide kinase-catalyzed [32P]phosphate transfer from [gamma-32P]ATP. The 32P-labeled nucleotides were resolved by anion-exchange t.l.c. on polyethyleneimine-cellulose and detected by autoradiography. Aromatic adduct nucleotides were found to be retained at the origin in aqueous electrolyte solutions, but to migrate as distinct spots in solvents containing 7-8.5 M urea. Advantage was taken of this observation to remove 32P-labeled normal DNA nucleotides from adduct nucleotides. This purification enabled the detection of a single adduct in 10(7)-10(8) normal nucleotides. The method appears applicable to the ultrasensitive detection of a large number of carcinogen--DNA adducts of diverse structure without requiring radioactive carcinogens or specific antibodies.

Animals↗