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Biomedical subjects

M V Reddy

Publications and source records attributed to M V Reddy.

At least 91 records · Page 5Linked to original sources
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

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

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

Detection of circulating antigen in bancroftian filariasis by sandwich ELISA using filarial serum IgG.

The utility of the IgG fraction of human filarial serum immunoglobulin in detecting circulating antigen by sandwich enzyme linked immunosorbent assay (ELISA) was studied. 27 of 33 sera from persons with microfilaraemia, 19 of 30 sera from clinical cases of filariasis, 4 of 30 sera from normal persons from a region endemic for filariasis showed the presence of circulating filarial antigen. All the 20 normal sera from the area where filariasis was not endemic gave negative reaction for filarial antigen. Those sera from persons with microfilaraemia that showed the presence of circulating antigen also showed an apparent positive correlation between the microfilarial density and the antigen titre.

Antigens, Helminth↗
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

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↗
PubMed1984

Girls with muscular dystrophy.

Clinical and genetic studies were made on progressive muscular dystrophy in eight young girls. No chromosomal abnormality was observed in these patients and their mothers. The pedigrees of six cases suggested an autosomal recessive inheritance and clinical features were identical with those of Duchenne muscular dystrophy. Two other cases were compatible with childhood muscular dystrophy with autosomal recessive inheritance.

Consanguinity↗
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↗