Linda M. Reik

PCBs: Structure-Function Relationships and Mechanism of Action

Numerous reports have illustrated the versatility of polychlorinated biphenyls (PCBs) and related halogenated aromatics as inducers of drug-metabolizing enzymes and the activity of individual compounds are remarkably dependent on structure. The most active PCB congeners, 3,4,4′,5-tetra-, 3,3′,4,4′-tetra-, 3,3′,4,4′,5-penta- and 3,3′,4,4′,5,5′-hexachlorobiphenyl, are substituted at both para and at two or more meta positions. The four coplanar PCBs resembled 3-methylcholanthrene (3-MC) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) in their mode of induction of the hepatic drug-metabolizing enzymes. These compounds induced rat hepatic microsomal benzo(a)pyrene hydroxylase (aryl hydrocarbon hydroxylase, AHH) and cytochromes P-450a, P-450c and P-450d. 3,4,4′,5-Tetrachlorobiphenyl, the least active coplanar PCB, also induced dimethylaminoantipyrine N-demethylase and cytochromes P-450b+e and resembled Aroclor 1254 as an inducer of the mixed-function oxidase system. Like Aroclor 1254, all the mono-ortho- and at least eight di-ortho-chloro analogs of the coplanar PCBs exhibited a “mixed-type” induction pattern and induced microsomal AHH, dimethylaminoantipyrine NM-demethylase and cytochromes P-450a–P-450e. Quantative structure–activity relationships (QSARs) within this series of PCBs were determined by comparing their AHH induction potencies (EC50) in rat hepatoma H-4-II-E cells and their binding affinities (ED50) for the 2,3,7,8-TCDD cytosolic receptor protein. The results showed that there was an excellent correlation between AHH induction potencies and receptor binding avidities of these compounds and the order of activity was coplanar PCBs (3,3′,4,4′-tetra-, 3,3′,4,4′,5-penta- and 3,3′,4,4′,5,5′-hexachlorobiphenyls) > 3,4,4′,5-tetrachlorobiphenyl ~ mono-ortho coplanar PCBs > di-ortho coplanar PCBs. It was also apparent that the relative toxicities of this group of PCBs paralleled their biological potencies. The coplanar and mono-ortho coplanar PCBs also exhibit differential effects in the inbred C57BL/6J and DBA/2J mice. These compounds induce AHH and cause thymic atrophy in the former “responsive” mice whereas at comparable or higher doses none of these effects are observed in the nonresponsive DBD/2J mice. Since the responsiveness of these two mice strains is due to the presence of the Ah receptor protein in the C57BL/6J mice and its relatively low concentration in the DBA/2J mice, the results for the PCB cogeners support the proposed receptor-mediated mechanism of action. Although the precise structural requirements for ligand binding to the receptor have not been delineated, the halogenated aromatic hydrocarbons which exhibit the highest binding affinities for the receptor protein are approximate isostereomers of 2,3,7,8-TCDD. 2,3,4,4′,5-Pentachlorobiphenyl elicits effects which are qualitatively similar to that of TCDD and the presence of the lateral 4′-substituent is required for this activity. Thus the 4′-substituted 2,3,4,5-tetrachlorobiphenyls have been used as probes for determining the substituent characteristics which favor binding to the receptor protein. Multiple regression analysis of the competitive binding EC50 values for 13 substituents gave the following equation: log (1/EC50) = 1.53σ + 1.47π + 1.09 HB + 4.08 where σ is electronegativity, π is hydrophobicity, HB is hydrogen bonding and r is the correlation coefficient (r = 0.978). The utility of this equation in describing ligand:receptor interactions and correlations with toxicity are being studied with other halogenated hydrocarbons and PAHs.

Purification, characterization and regulation of five rat hepatic microsomal cytochrome P-450 isozymes

1. Five hepatic microsomal cytochrome P-450 isozymes (cytochromes P-450a, P-450b, P-450c, P-450d, P-450e) have been purified to apparent homogeneity from immature male rats treated with various xenobiotics. 2. The unique electrophoretic properties, substrate specificities and spectral characteristics of these haemoproteins have been compared and contrasted. 3. Structural studies of these cytochrome P-450 isozymes have included NH2-terminal amino acid sequence analyses, as well as electrophoretic profiles of limited proteolytic digests and cyanogen bromide fragments of the haemoproteins. 4. Specific antibodies have been prepared against four of the isozymes and used to evaluate immunochemical relationships among these cytochrome P-450s by Ouchterlony double-diffusion analyses. 5. The levels of some of these cytochrome P-450 isozymes have been quantified immunologically in hepatic microsomal preparations from untreated rats and following induction by phenobarbital, 3-methylcholanthrene or Aroclor 1254. 6. Antibodies directed against cytochromes P-450a and P-450b have been used to establish the presence of more than one 7 alpha- and 16 alpha-testosterone hydroxylase in rat hepatic microsomes. The relative contributions of cytochromes P-450c and P-450d to the overall microsomal metabolism of benzo[a]pyrene have been evaluated using antibodies to these haemoproteins.

A Selective Phosphatase of Regenerating Liver Phosphatase Inhibitor Suppresses Tumor Cell Anchorage-Independent Growth by a Novel Mechanism Involving p130Cas Cleavage

The phosphatase of regenerating liver (PRL) family, a unique class of oncogenic phosphatases, consists of three members: PRL-1, PRL-2, and PRL-3. Aberrant overexpression of PRL-3 has been found in multiple solid tumor types. Ectopic expression of PRLs in cells induces transformation, increases mobility and invasiveness, and forms experimental metastases in mice. We have now shown that small interfering RNA-mediated depletion of PRL expression in cancer cells results in the down-regulation of p130Cas phosphorylation and expression and prevents tumor cell anchorage-independent growth in soft agar. We have also identified a small molecule, 7-amino-2-phenyl-5H-thieno[3,2-c]pyridin-4-one (thienopyridone), which potently and selectively inhibits all three PRLs but not other phosphatases in vitro. The thienopyridone showed significant inhibition of tumor cell anchorage-independent growth in soft agar, induction of the p130Cas cleavage, and anoikis, a type of apoptosis that can be induced by anticancer agents via disruption of cell-matrix interaction. Unlike etoposide, thienopyridone-induced p130Cas cleavage and apoptosis were not associated with increased levels of p53 and phospho-p53 (Ser(15)), a hallmark of genotoxic drug-induced p53 pathway activation. This is the first report of a potent selective PRL inhibitor that suppresses tumor cell three-dimensional growth by a novel mechanism involving p130Cas cleavage. This study reveals a new insight into the role of PRL-3 in priming tumor progression and shows that PRL may represent an attractive target for therapeutic intervention in cancer.

Human group II phospholipase A2: Characterization of monoclonal antibodies and immunochemical quantitation of the protein in synovial fluid

Murine monoclonal and rabbit polyclonal antibodies were generated against human group II phospholipase A2 (PLA2) in order to study the role of this enzyme in inflammatory disease, the source of its synthesis, and the interaction of PLA2 with its substrate. Monoclonal antibody PLA187 exhibits potent inhibitory activity toward human PLA2 using autoclaved E. coli membranes as the substrate. Three other monoclonal antibodies (PLA184, PLA185, and PLA186) also inhibit enzyme activity, but with about 50-fold less potency. Based on the results of double-antibody competition experiments and enzyme inhibition profiles, PLA184 and PLA185 appear to recognize the same epitope. Monoclonal antibody PLA186 recognizes an epitope which is spatially distinct from that recognized by PLA184/185. The results also suggest that the epitope recognized by PLA187 may overlap with both epitopes recognized by PLA186 and PLA184/185. A double-antibody sandwich ELISA was developed using a combination of PLA185 and rabbit polyclonal antibody against PLA2. The ELISA provides a sensitive and quantitative method for monitoring specifically group II PLA2 in various biological sources, independent of factors which may affect enzyme activity. We have utilized this assay to quantitate PLA2 levels in synovial fluid from the joints of individuals with rheumatoid arthritis as well as from non-arthritic joints. Our results indicate that elevated levels of group II PLA2 in synovial fluid are not necessarily associated with arthritis.

The crystal structure of human muscle glycogen phosphorylase a with bound glucose and AMP: An intermediate conformation with T-state and R-state features.

The Crystal Structure of Human Muscle Glycogen Phosphorylase a with Bound Glucose and AMP: An Intermediate Conformation with T-State and R-State Features Christine M. Lukacs,,* Nikos G. Oikonomakos, Robert L. Crowther, Li-Na Hong, R. Ursula Kammlott, Wayne Levin, Shirley Li, Chao-Min Liu, Debra Lucas-McGady, Sherrie Pietranico, and Linda Reik Roche Pharmaceuticals, Discovery Chemistry, F. Hoffmann-La Roche, Nutley, New Jersey Roche Pharmaceuticals, Roche Discovery Technologies, F. Hoffmann-La Roche, Nutley, New Jersey Institute of Organic and Pharmaceutical Chemistry, The National Hellenic Research Foundation, Athens, Greece

Multiplicity and functional diversity of rat hepatic microsomal cytochrome P450 isozymes

Three potential fates await lipophilic foreign chemicals that enter a living organism: the compounds remain unchanged, undergo spontaneous breakdown, or enzymatic metabolism. The biotransformation products are, in general, more water soluble and are more easily eliminated from the organism than the parent compounds. Hence, both the duration and intensity of action of many biologically active xenobiotics, including drugs, are determined largely by their rate of conversion to more polar products. Although the biotransformation of xenobiotics occurs in many tissues and organs of higher animals, the highest metabolic activity resides in the liver. The foundations for modern research in xenobiotic metabolism were established through the pioneering efforts of several investigators (Mueller & Miller, 1953; Axelrod, 1955; Brodie et al., 1955; Williams, 1959) during the 1950s.

Antibodies as Probes of Cytochrome P450 Isozymes

Cytochrome P450*, an integral membrane protein, is widely distributed in many mammalian tissues, but is present in the highest concentration in hepatic endoplasmic reticulum. It functions as the terminal oxidase of an electron transport system that is involved in the metabolism of a large number of xenobiotics as well as endogenous substrates such as steroids, bile acids, fatty acids and prostaglandins (1). Although this enzyme system formerly was believed to function principally in detoxification, it is now known to metabolically activate many compounds to reactive metabolites that initiate toxic and carcinogenic events (2). Even more interesting is the observation that only certain optical isomers of bay-region diol epoxides of polycyclic aromatic hydrocarbons are carcinogenic, and that the cytochrome P450-dependent mixed function oxidase system preferentially catalyzes the formation of the optical isomer with the highest carcinogenic activity (3).

Characterization and regulation of rat hepatic microsomal cytochrome P-450 isozymes.

In mammalian liver, the oxidative metabolism of a wide variety of exogenous and endogenous compounds is catalyzed by the microsomal mixed-function oxidase system.’ Cytochrome P-450, which functions as the terminal oxidase of this electron transport system, refers to a family of integral membrane hemoproteins that exhibit different but overlapping substrate selectivity.* Both the total amount of cytochrome P-450 and the relative levels of certain of these hemoproteins in endoplasmic reticulum are known to be dramatically altered by treatment of an animal with various inducers such as structurally diverse drugs, pesticides, and polycyclic and polyhalogenated hydrocarbons.’,’ Several inducible hepatic microsomal cytochromes P-450 have been purified from rats treated with phenobarbital, 3-methylcholanthrene, Aroclor 1254, isosafrole, clofibrate, 0-naphthoflavone, and pregnenolone 1 6 a ~ a r b o n i t r i l e . ~ ’ ~ Five hepatic microsomal cytochrome P-450 isozymesb (P-450a, P-450b, P-4%. P-450d, and P-450e) have been purified in this laboratory from rats treated with various xenobiotics, and these hemoproteins have been shown to be separate gene produ~ts .~.~~’”’’ Three additional cytochromes P-450 (P-45Of. P-450g, and P-450h) have recently been purified to electrophoretic homogeneity from both untreated and ethanol-treated rats.” The eight hemoproteins have been designated in a nondescriptive manner as they are purified because of the lack of a single, sufficiently specific criterion to differentiate them. This report first outlines several properties of these purified cytochrome P-450 isozymes, illustrating problems associated with spectral, catalytic, and minimum molecular weight determinations to identify an individual cytochrome P-450. Results are presented of two complementary approaches, that is, immunoquantitation and two-dimensional isoelectric focusing-sodium dodecyl sulfate (IF-SDS) gel electropho-