James W. Flesher, Ph.D.
Professor Emeritus;
Loyola University, 1958.
Office: MS305 William R. Willard Medical Education Building
Tel: (859) 323-6209
Although there is no a priori reason that cancer-producing properties are not dependent solely on the physical properties of hydrocarbons, it is also possible that the biological actions of hydrocarbons depend upon the occurrence of a definite chemical reaction. All observation must be either for or against some hypothesis or theory, if your aim is to advance science. Over the years many hypotheses or theories have been devised, which lend themselves to specific predictions, offering a means of testing their validity. The advantage of many different hypotheses is that all sides of the question will be tested by experiments and the results discussed before reaching a definite conclusion. A further advantage is that the empty theories must eventually be disproved leaving only one possible theory. No satisfactory theory has yet arrived until all alternatives have been disproved. Science advances only by disproofs. A testable hypothesis is like a roadmap - it predicts what you will find at the next checkpoint, but it does not predict how long it will take you to get there.
In 1967, our group reported the synthesis of a series of compounds related to 7,12-dimethylbenz[a]anthracene (DMBA). In collaboration with Dr. Katherine L. Sydnor, MD, Professor of Medicine, these were tested for cancer-producing properties. In 1970, we devised the Hypothesis that the first step in carcinogenesis by DMBA is hydroxylation of the 7-methyl group which forms a proximate carcinogen. The 7-hydroxymethyl derivative was shown to be a metabolite of DMBA with cancer-producing properties. The next hypothetical step was esterification by PAPS-dependent sulfotransferases to form an ultimate carcinogen, bearing a good leaving group, capable of generating a benzylic carbocation. Our conclusions for DMBA did not receive an enthusiastic reception and hence, a one-electron transfer pathway for DMBA (Fried, 1974) and a diol-epoxide pathway for benzo[a]pyrene (Sims,1974) were strongly advocated. However, we were in perfect agreement with the Miller Hypothesis (1970) that most, if not all, chemical carcinogens are either themselves electrophiles or they are converted to electrophiles by metabolism. On the other hand, our conclusion that benzylic carbocations react with DNA as rapidly as with proteins challenged Heidelberger’s Protein Deletion Hypothesis, which excluded DNA reactions, and Fieser’s thesis (1962) in which he concluded that dibenzo[a,i]pyrene (DBP), lacking methyl groups, was itself carcinogenic without the intervening process of metabolism.
We next turned our attention to the synthesis of derivatives of benzo[a]pyrene (BP), a cancer-producing hydrocarbon lacking a methyl group. Boyland’s Hypothesis (1950) suggested K-region epoxides with molecular structure type ArX could theoretically explain all of the cancer-producing metabolites but by 1973 this hypothesis was disproved. The K-region Electronic Calculation Theory devised by the Pullmans (1955), the Millers K-region 5-Hydroxymethyl-DMBA activation Hypothesis (1967) and Newman’s modified K-region Hypothesis (1972) were disproved. We predicted that either biological or chemical methylation of BP and DBP was required for metabolism to an active electrophile. Evidence that a methylation pathway is a necessary first step for BP carcinogenesis would exclude all alternative pathways. If its not methylated, its not activated.
In 1972, Flesher and Sydnor were the first to devise the Unified Hypothesis, based on the meso-region methylation pathway, that predicted 6-hydroxymethyl sulfate ester, bearing a good leaving group and capable of generating a highly reactive benzylic carbocation, was the major ultimate electrophilic and cancer-producing form of BP, 6-methylBP, 6-hydroxymethylBP (proximate carcinogen) and the meso-aldehyde. Therefore, the ultimate electrophilic and cancer-producing forms of both meso-methyl-substituted polycyclic aromatic hydrocarbons (MPAH) and hydrocarbons lacking methyl groups (PAH) must have molecular structure type ArCH2X. A second essential prediction of the Hypothesis is that detoxified metabolites of PAH must have molecular structure type ArX, X= O, OH, etc. with low or no cancer-producing properties. The lack of cancer-producing properties of most, if not all, derivatives or metabolites of PAH with molecular structure type ArX confirms this prediction. The meaning and significance of this prediction is that most, if not all, cancer-producing MPAH and PAH must either themselves be methylated or be methylated in the first metabolic step, most favorably at the 9,10-carbons of an anthracene nucleus in PAH, the active sites of electrophilic substitution.
The prediction that biological methylation must be the first step in the metabolic activation of PAH was verified by showing that methylation is catalyzed by S-adenosyl-L-methionine dependent methyltransferases and the methylated products of BA, DBA, and BP in rat liver cytosol and in vivo were isolated and identified by comparison with authentic compounds prepared by synthesis. PAH structurally incapable of biological methylation must be carcinogenically inert. Accordingly, only MPAH, not PAH, can be transformed to active electrophiles with molecular structure type ArCH2X. The comparative carcinogenicity of either activated or detoxified metabolites of MPAH and PAH, with mutually exclusive structure types of either ArCH2X or ArX, must be either for or against the Meso-Region Methylation Theory, offering a means of testing its validity. A failure to agree, after 80 years of chemical and biological experiments, is a failure to disprove.
In a 2005 review, it was concluded that cancer-producing properties of hydrocarbons no longer depend on a single bay-region diol-epoxide pathway, a benzylic alcohol sulfate ester pathway, nor on any other pathway. With so many competing minor pathways for both MPAH and PAH, the reviewers concluded that the appearance of cancer-producing properties in MPAH depends partly, but not completely, on a benzylic alcohol sulfate ester pathway capable of generating benzylic carbocations. Cancer-producing properties depend either only on this pathway for MPAH or additional pathways are required. On the other hand, multiple bay-region or fjord-region diol-epoxides, M-region quinones, meso-region radical-cations, and ROS, etc. pathways, are necessary for PAH lacking methyl groups. The Unified Hypothesis (1972) or Meso-Region Methylation Theory (2002) which predicted the pathways to either activated or detoxified metabolites for both MPAH and PAH lacking methyl groups was not reviewed.
The pronounced carcinogenicity of numerous MPAH derivatives or metabolites with molecular structure type ArCH2X has been firmly established. The lack of carcinogenicity of most, if not all, derivatives or metabolites of PAH with molecular structure type ArX is well known. Activated and detoxified metabolites must have molecular structure type of either ArCH2X or ArX. It is now clear that detoxified metabolites, such as epoxides, diols, phenols, and quinones, are derived from PAH lacking methyl groups, and from most, if not all, MPAH, whereas the most potent activated electrophilic and carcinogenic forms, bearing a good leaving group, with molecular structure type ArCH2X, are derived only from MPAH. Our observations exclude the alternatives that either the parent PAH or their chemical or metabolic products are themselves carcinogenic. Our conclusions might be invalid if it could be shown that PAH lacking methyl groups can be metabolized by cytochrome P450(1A1), epoxide hydrolase, aldo-keto reductase, or one-electron transfer oxidation by peroxidases to multiple type ArX metabolic products with cancer producing properties.
Science advances only by investigators who know how to disprove the empty theories, and are already working on it. Who has innovative ideas ? Scientists working on disproofs. There is no faster way to advance science. The trick is knowing how to tap into them. What’s the evidence for or against your hypothesis ? What experiment could disprove your hypothesis ? What hypothesis does your experiment disprove ? It is only by denying the validity and generality of our theory that our conclusions can be avoided.
Selected Abstracts and Publications
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Abstracts
Flesher, J.W.
Evidence That 6-Hydroxymethyl Sulfate Ester Is a Major Ultimate Carcinogen of 6-Methylbenzo[a]Pyrene and Benzo[a]Pyrene
Southeastern Regional Meeting American Chemical Society (SRMACS), 2008. Abstract
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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