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Biomedical subjects

E Randerath

Publications and source records attributed to E Randerath.

At least 73 records · Page 4Linked to original sources
PubMed1987

Correlation of aromatic hydroxylation of 11 beta-substituted estrogens with morphological transformation in vitro but not with in vivo tumor induction by these hormones.

In estrogen-induced cancer, catechol formation from administered steroids has been postulated to be a necessary event for estrogen activation and subsequent damage to cellular macromolecules. In the present study, this hypothesis has been tested using two homologous series of structurally related estrogens: estradiol, 11 beta-methylestradiol, 11 beta-ethylestradiol, 11 beta-methyl-17 alpha-ethinylestradiol, 11 beta-ethyl-17 alpha-ethinylestradiol, 11 beta-methoxy-17 alpha-ethinylestradiol, and 17 alpha-ethinylestradiol. In the Syrian hamster renal carcinoma model, only 11 beta-methylestradiol and 17 alpha-ethinylestradiol were weak carcinogens (2 of 20 and 2 of 24 hamsters with tumors, respectively). The other estrogens tested induced renal carcinoma within 6 to 9 months with an incidence in the 80-100% range. The tumor incidence in vivo did not correlate with the rates of catechol formation by hamster kidney microsomes in vitro. Compared to estradiol (relative rate, 100), catechol formation by the substituted estrogens was significantly lower, ranging from 48 (11 beta-methylestradiol) to 2 (11 beta-methoxy-17 alpha-ethinylestradiol). Kidney DNA of hamsters treated with the four 17 alpha-ethinyl estrogens, when analyzed by 32P postlabeling assay, contained the same set of covalently modified nucleotides the formation of which had previously been found to precede estrogen-induced renal carcinogenesis in vivo. In contrast, relative rates of catechol estrogen formation by BALB/c 3T3 microsomes correlated with induction of morphological transformation of BALB/c 3T3 cells and decreased in the following order: 11 beta-methylestradiol greater than 17 alpha-ethinylestradiol greater than or equal to estradiol greater than 11 beta-ethylestradiol greater than 11 beta-methoxy-17 alpha-ethinylestradiol. The hormonal potencies of several estrogen derivatives studied by various assays did not correlate with in vivo carcinogenic or in vitro cell-transforming activities. It is concluded from these experiments that in cell culture catechol formation and morphological transformation are directly related. In vivo, aromatic hydroxylation of administered estrogens did not correlate with the incidence of estrogen-induced renal carcinoma in Syrian hamsters.

Animals↗
PubMed1987

Localization of estrogen-induced DNA adducts and cytochrome P-450 activity at the site of renal carcinogenesis in the hamster kidney.

Renal carcinoma in male Syrian hamsters, induced by chronic administration of estradiol for 5-7 months, is known to arise in the cortex at the cortico-medullary junction. In this in vivo model for hormonal carcinogenesis, estrogen-induced covalent DNA adducts have previously been observed in whole kidney and have been postulated to be involved in tumor induction. In the present study, the intrarenal distribution of estrogen-induced DNA modification and estrogen metabolizing enzymes were investigated in male Syrian hamsters to ascertain a role of metabolism and adduct formation in estrogen-induced carcinogenesis. The highest estrogen-induced DNA adduct concentrations as measured by 32P-postlabeling analysis were found in the renal cortex of hamsters treated with estradiol for 7 months. Total adduct levels in medullary DNA were approximately one-half of those found in cortex. Cytochrome P-450 enzymes were detected only in microsomes of kidney cortex (approximately 0.8 +/- 0.6 nmol P-450/mg protein) but not medulla of untreated male Syrian hamsters. Prostaglandin endoperoxide synthase activity in kidney cortical microsomes was 1/5 of the activity found in medullary microsomes. Thus, microsomal cytochrome P-450 levels and estrogen-induced DNA adduct formation were highest in hamster kidney cortex, the origin of renal tumorigenesis. It is postulated that estrogen metabolism by cytochrome P-450 enzymes leading to covalent DNA modification plays a role in hormonal carcinogenesis in the hamster kidney.

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

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 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

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↗
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

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↗
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

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

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↗