Betty K. Mansfield

Benzo[a]pyrene metabolism in human T 47D mammary tumor cells: evidence for sulfate conjugation and translocation of reactive metabolites across cell membranes.

Human mammary tumor T 47D cells were examined for their capacity to metabolize benzo[a]pyrene (BaP) to gain insight into potential metabolic pathways for BaP in human epithelial tissue. Confluent cultures metabolized 95% of 4 microM BaP after 24 h incubation. BaP metabolites were analyzed from culture medium since only residual amounts remained in cells. Tetraols/triols, dihydrodiols, quinones and phenols were either unconjugated or existed as sulfate conjugates. Glucuronide conjugation was minor. Remaining water-soluble (WS) metabolites could be extracted with butanol or removed from culture medium protein with methanol/water and suggestive evidence indicates these may be glutathione conjugates. A portion of BaP-WS metabolites were covalently bound to medium protein. This latter phenomenon is attributed to translocation of reactive BaP metabolites across cell membranes which could potentially occur in vivo during cellular processing of BaP.

Simultaneous preparation of nuclear DNA, RNA, and protein from carcinogen treated-hamster embryo fibroblasts☆☆☆

Nuclei from hamster embryo fibroblasts treated with radioactive benzo(a)pyrene were lysed in 6 M guanidine, and nuclear macromolecules were separated by isopycnic centrifugation in Cs/sub 2/SO/sub 4/. Control experiments showed that cross-contamination of the RNA, DNA, and protein fractions was less than 2% of the total recovery of each macromolecular class. When compared to previous techniques utilizing phenol extraction, similar specific activities of bound hydrocarbon (pmol benzo(a)pyrene/mg protein or nucleic acid) were obtained. However, overall recoveries of macromolecular components were higher with the present method. In addition, recovery of undegraded histones in the density gradient preparation of nuclear protein was demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and recovery of native DNA was demonstrated by thermal denaturation studies. Although developed specifically for work with carcinogenic hydrocarbons, the Cs/sub 2/SO/sub 4/ technique should be generally useful in cases where it is necessary to prepare all three classes of macromolecules from one batch of nuclei.

AUTOMATED ANALYSIS OF DIGITAL IMAGES GENERATED BY TWO-DIMENSIONAL GEL ELECTROPHORESIS

Two-dimensional gel electrophoresis is a method to separate proteins according to their molecular weights and mobilities in an electric field at a certain pH value. The presence of a radioactively or fluorescently labeled protein in a preparation is indicated by a spot that is produced on a film with the intensity of the spot indicative of the amount of protein. This article presents algorithms that accomplish automated quantitative analysis of digitized gel images. The limits of currently available analysis systems are discussed. The methods are implemented by software on a commercially available digital image processor that uses a minicomputer host. 13 refs., 4 figs., 3 tabs.

Species Heterogeneity in the Metabolic Processing of Benzo[a]pyrene

The detoxification response of the organism toward chemical carcinogens is to transform these potentially toxic compounds into more polar, less lipid soluble substances that are readily excretable and therefore harmless. However, it would appear that nature has made a serious mistake in the case of chemical carcinogens. This concept can be stylized by superimposing the steps in metabolic activation upon a chemical energy activation diagram (see Figure 1). It is generally assumed that the parent molecules of an environmentally prevalent chemical carcinogen are structurally stable and relatively inactive metabolically. This assumption is not unreasonable from a teleological point of view since one would expect labile chemical substances to be rapidly degraded or oxidized, due to sunlight and weather, if released in the open environment. Synthetically prepared activated carcinogens, such as polyaromatic epoxides and nitrosamines, have been shown to possess very short half-lives under physiological conditions. Therefore, the parent compound undergoes a decrease in entropy to increase its potential energy for subsequent metabolic degradation. This change requires enzymatic transformation into a reactive intermediate antecedent to further catabolism. Current evidence shows that all known carcinogenic chemicals are electrophilic reagents that seek out nucleophilic sites inside the cells (1). The peak of the curve in Figure 1 is the zone where the electron-deficient reactive metabolite is thought to interact with nucleophilic target sites hypothesized to begin the process of malignant transformation. If no such interaction takes place, the most common reaction is hydroxylation to form a metabolically inactive polar structure that is more hydrophilic and can be readily excreted. Therefore, the major thrust of the detoxification process is to render the parent compound into a structure of greater entropy and consequently less potential to exert a toxic effect.

Methods for computer analysis and comparison of two-dimensional protein patterns obtained by electrophoresis

Mammalian cells contain thousands of proteins each representing an important function for maintaining the homeostasis of the cell at the biochemical, genetic, or structural level. Two-dimensional gel electrophoresis is uniquely sensitive in its ability to separate these proteins in the first dimension by isoelectric focusing as a function of net charge, and in the second dimension as a function of molecular weight ([13]). The presence of a certain radioactively labeled protein in a preparation is indicated by a spot that is produced on a film. The intensity of the spot is indicative of the amount of protein. Usually several hundred, even up to a few thousand spots, i.e., proteins, can be distinguished in a gel image, which makes rigorous quantitative analysis by simple visual inspection impossible. Analysis of the voluminous information inherent in a gel image requires localization, quantitation and possibly identification of each protein. In addition, and equally importantly, it is necessary to make local and global comparisons between multiple gels that reflect potential changes in protein patterns from various disease states or after carcinogen or mutagen treatment, in order to demonstrate subtle changes that occur in the progression to the diseased state.

Specificity of Interaction between Carcinogenic Polynuclear Aromatic Hydrocarbons and Nuclear Proteins: Widespread Occurrence of a Restricted Pattern of Histone Binding in Intact Cells

Publisher Summary The first steps in the induction of malignancy by chemical carcinogens involve covalent interactions with cellular macromolecules. For the widespread environmental contaminant benzo[a] pyrene (B[a]P), metabolic activation by cellular enzymes produces a number of potentially reactive metabolites. The end products of one metabolic pathway, 7,8-dihydroxy-9,10-oxy-7,8,9,10 tetrahydro-B[a]P (BPDE), are responsible for essentially all DNA adduct formation in animal cells treated with B[a]P, and a particular stereoisomer, designated (+)-anti-BPDE is thought to be the ultimate carcinogenic derivative of B[a]P. It has been shown that in hamster embryo cell nuclei treated with (+)-anti-BPDE, two of the histones of nucleosomal core, H3 and H2A are covalently modified, while the remaining core histones—H4 and H2B—are essentially unmodified. All four purified core histones, however, serve as the targets for (+)-anti-BPDE in vitro .

Time course of metabolism of benzo[e]pyrene by hamster embryo cells and the effect of chemical modifiers☆

In cultures of hamster embryo cells, benzo[a]pyrene (B[a]P) is metabolized primarily in the bay region. In contrast, little or no bay region metabolism of the noncarcinogenic isomer benzo[e]pyrene (B[e]P) could be detected during 12--96-h incubations of hamster embryo cells with 4 microM [3H]B[e]P. The upper limit to 9,10-dihydro-9,10-dihydroxy-B[e]P formation is about 0.2% of the ethyl acetate-soluble metabolites (less than 0.1% of the total metabolites). The major identified metabolites of B[e]P were 4,5-dihydro-4,5-dihydroxy B[e]P and the glucuronide conjugates of 3-OH-B[e]P and 4,5-dihydro-4,5-dihydroxy B[e]P. Simultaneous treatment of cells with either B[a]P or 7,8-benzoflavone (BF) did not induce bay region metabolism of [3H]B[e]P.