Ramesh C. Gupta

Postlabeling analysis of carcinogen-DNA adducts in mussel, Mytilus galloprovincialis

Abstract The 32P-postlabeling analysis of DNA adducts formed in mussel digestive gland homogenates incubated with 2-aminofluorene (32 μ m ) showed one major and one minor adduct in the range of 1 adduct per 1–4 × 108 nucleotides. The predominant adduct was found to be chromatographically similar to the known C8-aminofluorene-substituted guanine adduct. The metabolic activation of 2-aminofluorene was blocked in the presence of methimazole (470 μ m ), a competitive inhibitor of FAD-containing monooxygenase. In contrast to 2-aminofluorene, benzo(a)pyrene-modified DNA either did not show any adduct or a very weak adduct spot (1 adduct per 1–4 × 109 nucleotides). The samples of DNA from mussel, carp and bream, which had no obvious exposure to pollutants, showed one to several weak ‘natural adducts’. The origin of these adducts is as yet undetermined. Since postlabeling assay enables measurement of carcinogen-DNA adducts formed with any, even unknown, chemicals from the composite environmental mixture of genotoxic pollutants, it may become a valuable tool for risk assessment also in marine environments.

Sequence studies on tRNAPhe from human placenta: Comparison with known sequences of tRNAPhe from other normal mammalian tissues

Abstract Phenylalanine-specific tRNA was isolated from human placenta and degraded to mixtures of oligonucleotides. Tritium sequence analysis of the digestion products indicates that the sequence of human placenta tRNA Phe is identical to that of calf liver tRNA Phe and differs only slightly from that of rabbit liver tRNA Phe .

DNA adducts as biomarkers in genotoxic risk assessment in the aquatic environment

Abstract The presence of ‘natural’ (‘pre-existing’) DNA adducts in indicator organisms may complicate the use of pollution-related DNA-adduct measurements as a biomarker in the assessment of both the biologically relevant exposure to carcinogens and the pathobiological consequences of that exposure. Here, we present data demonstrating that detection and identification of exposure-related DNA adducts in marine and freshwater organisms (sponge, mussel and fish) is possible despite the presence, sometimes, of great numbers and significant levels of natural DNA adducts.

Comparative metabolism of benzo[a]pyrene and (−)benzo[a]pyrene-7,8-dihydrodiol by hepatocytes isolated from two species of bottom-dwelling fish

Abstract In order to elucidate the biochemical bases of species differences in susceptibility to hepatotumorigenesis induced by polycyclic aromatic hydrocarbons (PAHs) such as benzo[a]pyrene (BP), we investigated the metabolism of [ 3 H]BP and its proximate carcinogenic metabolite (−)BP-7,8-dihydrodiol (BP-7,8-diol) by two benthic fish: brown bullheads ( Ictalurus nebulosus ) (a susceptible species) and mirror carp ( Cyprinus carpio ) (not known to be susceptible). Freshly isolated hepatocytes prepared from both species efficiently converted both BP and BP-7,8-diol (the proximate carcinogenic metabolite of BP) to glutathione conjugates, glucuronides and sulfates and polyhydroxylated compounds. Nevertheless, carp hepatocytes (which metabolized both BP and BP-7,8-diol about 2-fold faster than did bullhead hepatocytes) produced several-fold larger amounts of DNA adducts from both substrates, suggesting the formation by carp liver of significantly larger amounts of reactive intermediates that bind to DNA. The apparently greater susceptibility of brown bullheads to PAH-induced carcinogenesis thus awaits further clarification.

32P-adduct assay: Short- and long-term persistence of 2-acetylaminofluorene-DNA adducts and other applications of the assay

32P-Postlabeling techniques have been developed to detect and measure adducts formed by covalent binding of carcinogens of Known or unknown origin with DNA (and RNA). The assay is applicable to various classes of chemical carcinogens and permits detection of many adducts at attomole (10−18 mol) level using microgram amounts of DNA. Here, we demonstrate the application of the assay for the analysis of short- and long-term persistence of 2-acetylaminofluorene-DNA adducts in rat liver in vivo and also outline examples illustrating the applicability of the procedure to different experimental problems.

Sequence analysis of small amounts of nonradioactive oligoribonucleotides containing ribose-methylated nucleosides by a combination of 3H- and 32P-labeling techniques.

Abstract The oligonucleotides A-G-A-Cm-U and Gm-A-A-Y-A-ψ were used as model compounds to demonstrate how the complete nucleotide sequence of small amounts of nonradioactive oligoribonucleotides (0.2–0.3 nmol) can be derived by a combination of 3 H-labeling procedures previously published and a new method for the characterization of 2′- O -methylated nucleosides based on enzymatic 32 P labeling. The newly developed method for the identification of ribose-methylated nucleosides entails 32 P labeling by [γ- 32 P]ATP/polynucleotide kinase of the 5′-terminus of a ribonuclease T 2 -stable 2′- O -methylated dinucleotide derived from the polyribonucleotide, conversion of the labeled dinucleotide to the 32 P-labeled 2′- O -methylated nucleoside 5′-monophosphate, and identification of the monophosphate by its chromatographic properties on a polyethyleneimine-cellulose thin layer. The novel method is simple, fast, and sensitive and, at present, represents the only way by which ribose-methylated nucleosides can be analyzed in small amounts (0.01 nmol) of nonradioactive oligonculeotides or RNA.

Nucleotide sequence of a reiterated rat DNA fragment

A reiterated component of rat DNA was isolated by restriction with HindIII endonuclease and polyacrylamide gel electrophoresis. Sequence analysis revealed that the fragment was 179 nucleotides long. Unlike the known 370N reiterated rat DNA fragment which contained a high m5C content (2.7 mole%), this repetitive element contained a rather low m5C content (0.5 mole%). The present rat repetitive sequence appeared to be of α‐type as shown by its significant homologies with α DNA sequenes of African green monkey and human. The possibility of sequence heterogeneity is discussed.