James W. Flesher, Ph.D.
Professor;
Loyola University, 1958.
Office: MS305 William R. Willard Medical Education Building
Tel: (859) 323-6209
The aim of the research program conducted in our laboratory is to discover how polynuclear hydrocarbons and their known or potential metabolites induce mammary cancer, lung cancer, sarcoma and leukemia in experimental animals and humans. Polynuclear hydrocarbons and many derivatives are found in cigarette smoke, auto and diesel engine exhaust, coal tar, and charcoal-broiled steaks. Some compounds, that produce metabolites with pronounced carcinogenic properties in experimental animals, have also been implicated as a cause of cancer in humans.
Our earlier investigations, using the methods of synthetic and biological chemistry, aided by the necessary tests for carcinogenic activity, showed that hydroxylation by oxidases of the 7-methyl-group of 7,12-dimethylbenz[a]anthracene (DMBA) produced a metabolite with pronounced carcinogenic activity, whereas hydroxylation of the terminal ring at the 4-position did not. This was evidence for two different and independent reactions. There was also evidence that metabolism of DMBA, in rats, formed covalent bonds with nucleic acids and proteins in mannary gland and liver, suggesting the occurrence of a definite chemical reaction. The implications of this evidence made it mandatory that if a highly reactive and carcinogenic metabolite of DMBA is formed, in vivo, it must be isolated, identified and synthesized. It seemed likely that if the 7-hydroxymethyl-derivative is an intermediary carcinogenic metabolite, further metabolism would be required to produce a highly reactive electrophilic and carcinogenic form.
Methods for the synthesis of known or potential metabolites of DMBA, 3-methylcholanthrene, benz[a]anthracene, 7-methylbenz[a]anthracene, dibenz[a,h]anthracene, benzo[a]pyrene and other potent hydrocarbons were devised in our laboratory. The carcinogenic activity of synthetic hydrocarbons and derivatives were determined in studies conducted in collaboration with Dr. Katherine L. Sydnor, MD, Professor of Medicine. The results of these investigations led Flesher and Sydnor to conclude that sulfuric acid esters, formed by PAPS-dependent sulfotransferases and capable of generating benzylic carbocations, were potential ultimate electrophilic and carcinogenic forms of methyl-substituted hydrocarbons, with aromatic structural type ArCH2X. The carcinogenic activity of all polynuclear hydrocarbons and their known or potential metabolites must be for or against some hypothesis or theory. The unified hypothesis or Meso-region theory predicts 1) that hydrocarbons lacking methyl-substitution must undergo methyl-substitution by SAM-dependent methyl-transferases, if it is to be carcinogenic 2) that model compounds capable of generating benzylic carbocations possess strong carcinogenic properties whereas compounds capable of generating ring-oxidized radical-cations, diol-epoxides, o-quinones, phenols or K-region epoxides do not. In recent publications, others have proposed, radical-cations together with terminal ring diol-epoxides and o-quinones that form part of a "bay or fjord region" to be major ultimate electrophilic and carcinogenic forms of hydrocarbons with or without methyl-substitution. Tests for carcinogenic activity of synthetic sulfuric acid esters and other model compounds capable of generating benzylic carbocations strongly support the Meso-region theory. Many laboratories have published observations for or against the unified hypothesis without discussing the hypothesis or our investigations for it.
Recent studies are aimed at obtaining more evidence for known or potential metabolites of polynuclear hydrocarbons with aromatic structural type ArX or ArCH2X. Our research has led us to propose that metabolites with structural type ArCH2X and pronounced carcinogenic activity can be incorporated in a very satisfactory manner into mechanistic pathways of the most potent polynuclear hydrocarbons known. Studies of the metabolites of hydrocarbons in human bone marrow cells using a sensitive HPLC analytical method showed that these cells produce metabolites with aromatic structural type ArCH2X. There is, therefore, reason to believe that carcinogenic metabolites with aromatic structural type ArCH2X will be found in other human cells and tissues. Our publications record the contributing members of our team.
In conclusion, the appearance of carcinogenic properties in polynuclear hydrocarbons and their known or potential metabolites of fundamentally different aromatic structural types ArX or ArCH2X are mutually exclusive. The appearance of strong carcinogenic properties in one aromatic structural type, ArCH2X, excludes the appearance of carcinogenic properties in the other. Furthermore, the known or potential ring-oxidized metabolites with only aromatic sturctural type ArX are not mutually exclusive, and it has been proposed that at least three known or potential metabolites must be produced in the target tissue, if the hydrocarbon is to be carcinogenic. The comparative carcinogenicity of many oxidized metabolites of unmethylated and methylated polynuclear hydrocarbons is not known. Our conclusions are based on discoveries of chemical-biochemical relationships between carcinogenic activity and molecular structure of polynuclear hydrocarbons and their known or potential metabolites. Clearly, the unified hypothesis could be disproved if it could be shown that compounds capable of producing ring-oxidized radical cations, and terminal ring diol-epoxides and o-quinones, together with reactive oxygen species, possess pronounced carcinogenic properties whereas meso-substituted compounds capable of producing benzylic carbocations do not.
Selected Abstracts and Publications
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Abstracts
Flesher, J.W., Horn, J., and Lehner, A.F.
Comparative Carcinogenicity of Oxidized Metabolites of Preprocarcinogens and Procarcinogens with Aromatic Structural Types ArX and ArCH2X
Southeastern Regional Meeting American Chemical Society (SRMACS), 2007. Abstract
Flesher, J.W., Horn, J., and Lehner, A.F.
Theories of structure and mechanism of PAH carcinogenicity.
The 230th American Chemical Society National Meeting, Washington, DC, Aug. 28-Sept. 1, 2005.
Flesher, J.W., Horn, J., and Lehner, A.F.
Formation of benzylic alcohols, by two-electron and oxygen transfer, a model for the first step in the metabolic activation of 7, 12-dimethylbenz[a]anthracene, 3-methylcholanthrene, and 6-methylbenzo[a]pyrene.
American Association for Cancer Research (AACR), Volume 45, 2004.
Horn, J., Lehner, A.F., and Flesher, J.W.
Synthesis and complete carcinogenicity of (+/-)-7, 12-dimethylbenz[a]anthracene-trans-3, 4-dihydrodial and its trans-3, 4-dihydrodiacetate derivative.
American Association for Cancer Research Meeting (AACR), 2002.
Lehner, A.F., Horn, J., Neill, D., and Flesher, J.W.
Mass spectral analysis of unstable N7-aralkyl DNA adducts resulting from reaction of 7-sulfooxymethyl-12-methylbenz[a]anthracene (SMBA) with DNA.
18th International Symposium on Polycyclic Aromatic Compounds. Sept. 9-13, 2001, Cincinnati, O.H.
Flesher, J.W., Horn, J., and Lehner, A.F.
Role of the bay and L-regions in the metabolic activation and carcinogenicity of picene and dibenz[a,h]anthracene.
18th International Syposium on Polycyclic Aromatic Compounds. Sept. 9-13, 2001, Cincinnati, O.H.
Flesher, J.W., Horn, J., and Lehner, A.F.
Mass spectral analysis of novel aralkyl DNA adducts derived from the reaction of 7-sulfooxymethyl-12-methylbenz(a)anthracene(SMBA) with deoxynucleoside-3'-monophosphates.
91st Annual Meeting of the American Association for Cancer Research. Apr. 1-5, 2000, San Francisco, C.A.
Vadhanam, M.V., Horn, J., Arif, J.M., Flesher, J.W., and Gupta, R.C.
Detection of aralkyl DNA adducts resulting from 7-hydroxymethyl sulfate ester of 7,12-dimethylbenz(a)anthcene(DMBA)in vitro.
91st Annual Meeting of the American Association for Cancer Research. Apr. 1-5, 2000, San Francisco, C.A.
Publications
Horn, J., Lehner, A.F., and Flesher, J.W. (2005)
Rapid induction of mammary cancer by repeated subcutaneous injection of the trans-3,4-dihydrodiol of 7,12-dimethylbenz["]anthracene in the female Sprague-Dawley rat. Cancer Letters 220:(2) 155-160.
Kumar, R., Vadhanam, M.V., Horn, J., Flesher, J.W., and Gupta, R.C. (2005)
Formation of Benzylic-DNA Adducts Resulting from 7,12-Dimethylbenz["]anthracene in Vivo. Chemical Research in Toxicology 18:(4) 686-691.
Flesher, J.W., Horn, J. and Lehner, A.F. (2004)
Formation of benzylic alcohols and meso-aldehydes by one-electron oxidation of DMBA: A model for the first metabolic step in methylated carcinogenic hydrocarbon activation. Polycyclic Aromatic Compounds 24:501-511.
Lehner, A.F., Horn, J., and Flesher, J.W. (2004)
Mass spectrometric analysis of 7-sulfoxymethyl-12-methylbenz[a]anthracene and related electrophilic polycyclic aromatic hydrocarbon metabolites. J. Of Mass Spectrometry 39:(11)1366-1378.
Lehner, A.F., Horn, J., and Flesher J.W. (2004)
Formation of radical cations in a model for the metabolism of aromatic hydrocarbons. Biochem. Biophys. Res. Commun. 322:1018-1023.
Horn, J., Flesher, J.W., and Lehner, A.F. (2003)
The metabolism of formyl-substituted benzanthracenes to hydroxymethyl metabolites in rat liver in vitro and in vivo. Chem Biol Interact. 145:17-32.
Vadhanam MV, Horn J, Flesher JW, Gupta RC. (2003)
Detection of benzylic adducts in DNA and nucleotides from 7-sulfooxymethyl-12-methylbenz[a]anthracene and related compounds by 32P-postlabeling using
new TLC systems.
Chem Biol Interact. 146:81-7.
Flesher JW, Horn J, Lehner AF. (2002)
Comparative carcinogenicity of picene and dibenz[a,h]anthracene in the rat.
Biochem Biophys Res Comm 290:275-9.
Flesher, J.W., Horn, J., and Lehner, A.F. (2002)
Role of the bay- and L-regions in the metabolic activation and carcinogenicity of picene and dibenz[a,h]anthracene. Polycyclic Aromatic Compounds. 22: 737-745.
Flesher, J.W., Horn, J., and Lehner, A.F. (2002)
The meso-region theory of aromatic hydrocarbon carcinogenesis. Polycyclic Aromatic Compounds. 22: 379-393.
Lehner, A.F., Horn, J., Neill, D., and Flesher, J.W. (2002)
Mass spectral analysis of unstable N7-aralkyl DNA adducts resulting from reaction of 7-sulfooxymethyl-12-methylbenz[a]anthracene (SMBA) with DNA and deoxynucleotides. Polycyclic Aromatic Compounds. 22: 415-432.
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