User:Drbogdan/BogdanDennis-PhD-Dissertation-1973

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Dr. Dennis Bogdan, PhD
"Master Editor IV".
(Editor at Wikipedia/1n3, Wiktionary, WikiSpecies) (User ID: 7809325).

"INTERACTION OF BENZO (α) PYRENE AND NUCLEIC ACIDS IN THE PRESENCE OF A MIXED-FUNCTION OXIDASE SYSTEM"
CLICK HERE => PhD Diploma and Complete Dissertation (177 pages, includes 46 pictures) (doc,epub,odt,pdf,txt) (05/19/2021; ZIP-File)
USA – STATE UNIVERSITY OF NEW YORK AT BUFFALO (SUNYAB) – 1973 (ProQuest 302781013; ISBN 979-8661021359)
(HomePage; DissertationPage; Archive; ArchiveST; FreezePage; GhostArchive; WayBack)

CLICK HERE FOR MY COMPLETE DISSERTATION ON WIKIPEDIA

By Dr. Dennis Paul Bogdan, Ph.D. (References; Publications)

SUMMARY: My PhD Dissertation (SUNYAB; 1973) involved the chemical interactions of the carcinogen benzo(a)pyrene (found in barbequed meats/cigarette smoke/automobile exhausts/polluted city air/and more) with DNA and related nucleic acids, a chemical interaction that can potentially initiate cancer processes (carcinogenesis) at the nucleic acid-level within living cells - hope this helps in some way - in any case - Stay Safe and Healthy !! - Drbogdan (talk) 22:00, 1 March 2021 (UTC)


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TITLE PAGE

INTERACTION OF BENZO (A) PYRENE AND NUCLEIC ACIDS IN THE

PRESENCE OF A MIXED-FUNCTION OXIDASE SYSTEM

by

Dennis Paul Bogdan

A dissertation submitted to the

Faculty of the Graduate School of

State University of New York at Buffalo

in partial fulfillment of the requirements

for the degree of

Doctor of Philosophy

February, 1973


I ABSTRACT

The exact mechanism of chemical carcinogenesis remains unclear, although it is generally agreed that modification of genetic expression constitutes an essential step in the process. The possibility that DNA itself represents the crucial biological target of chemical carcinogens is supported by in vivo studies, involving various polycyclic hydrocarbons, demonstrating a positive correlation between carcinogenicity and covalent binding to DNA. Polycyclic hydrocarbons are relatively unreactive chemically and, apparently, require biological activation for covalent bond formation with DNA in vivo. The overall objectives of this research are: first, to study the biotransformation of the polycyclic hydrocarbon carcinogen benzo(a)pyrene in mammalian systems; also, to study in the presence of such biotransformation the interaction between benzo(a)pyrene and polynucleotides, including mammalian DNA; and finally, to study the biological significance of the above interactions.

A comparison of characteristics of benzo(a)pyrene biotransformation processes in various tissues of the human and rat fetal-placental unit was attempted and was based on known features of aryl hydrocarbon hydroxylase (a NADPH-dependent mixed-function oxidase system able to transform polycyclic hydrocarbons) including inducibility, subcellular localization, requirements for optimal activity and the effect of various agents. Although differences were

II

observed between enzyme systems of the various tissues studied, the similarities appeared more striking and provided a basis for choosing the adult rat liver system as a model suitable for studies dealing with the remaining objectives of the present research.

Inclusion of calf thymus DNA in incubation mixtures containing benzo(a)pyrene and the model biotransformation system substantially reduced the recovery of BaP products as compared to controls. Similar decreases were observed with Poly G and, to lesser extents, with Poly I and the alternating copolymer Poly (I-C). No effects were observed with the double-stranded Poly I-Poly C, Poly A, Poly U nor with DNA modified with nitrogen mustard; increases were observed with yeast RNA and denatured DNA. The decrease in recoverable benzo(a)pyrene products was directly correlated with covalent binding of benzo(a)pyrene and polynucleotides as determined by sedimentation studies. Neither DNA nor Poly G enhanced the enzymatic disappearance of recovered benzo(a)pyrene products. The above findings suggest that benzo(a)pyrene, as a result of biotransformation, interacts chemically and specifically with base residues on the native DNA molecule in a manner which, apparently, involves a reactive BaP intermediate product.

DNA treated with benzo(a)pyrene or with the noncarcinogenic benzo(e)pyrene in the presence of the rat liver aryl hydrocarbon hydroxylase system exhibited decreases in template abilities as measured in an E. Coli DNA-dependent

III

RNA polymerase system when compared to template abilities of DNA controls. These findings suggest that DNA, treated enzymatically with benzopyrenes, is biologically modified but in a manner which does not appear to be carcinogen-specific.

Apparently, as a result of the described studies, benzo(a)pyrene can biologically and chemically modify mammalian nucleic acid in the presence of a mixed-function oxidase system found in rat and human tissues in a reaction which appears to involve an enzyme generated benzo(a)pyrene intermediate product, to be dependent on the secondary or tertiary structure of nucleic acids and to be base-specific. These in vitro findings appear to be consistent with the general hypothesis that initiation of carcinogenic processes in mammalian systems in vivo can be a result of chemical reactions between biologically activated chemical carcinogens and critical sites on cellular macromolecules.


IV

ACKNOWLEDGEMENT

I wish to express my deep appreciation to Dr. Zdzislaw F. Chmielewicz, Dr. Lorne K. Garrettson, Dr. Peter M. Hebborn, and Dr. Thomas I. Kalman for their help, criticism and encouragement during the course of this research, without which this thesis could not have been completed in its present form.

V

For my wife, Jan, without whom . . .


TABLE OF CONTENTS

INTRODUCTION---------------------------------------------------------------------------------------1-19

MATERIALS--------------------------------------------------------------------------------------------20-22

METHODS

PREPARATION OF HOMOGENATES IN FETAL-

PLACENTAL UNIT STUDIES---------------------------------------------------------------------23-25

PREPARATION OF RAT LIVER MICROSOMES--------------------------------------------25-26

ENZYME ASSAYS IN FETAL-PLACENTAL UNIT STUDIES-----------------------------26-27

ASSAY FOR ARYL HYDROOCARBON HYDROXYLASE--------------------------------28-30

PREPARATION OF FLUORESCENT BaP

HYDROXYLATED PRODUCTS-----------------------------------------------------------------30-31

SUCROSE GRADIENT SEDIMENTATION STUDIES-------------------------------------31-32

PREPARATION OF DNA MODIFIED WITH

NITROGEN MUSTARD---------------------------------------------------------------------------32-33

PREPARATION OF DNA FOR TEMPLATE STUDIES------------------------------------33-35

ASSAY FOR DNA TEMPLATE ACTIVITY----------------------------------------------------35-36

PREPARATION OF RNA POLYMERASE----------------------------------------------------36-40

DETERMINATION OF DNA----------------------------------------------------------------------40

DETERMINATION OF RNA----------------------------------------------------------------------40-41

DETERMINATION OF PROTEIN----------------------------------------------------------------41

RESULTS

BIOTRANSFORMATION OF BaP IN VARIOUS

TISSUES OF THE HUMAN AND RAT FETAL-

PLACENTAL UNIT---------------------------------------------------------------------------------42-50

EFFECT OF CALF THYMUS DNA ON THE APPEARANCE

OF FLUORESCENT PRODUCTS OF BaP BIOTRANSFORMATION----------------50-57

EFFECT OF VARIOUS MONONUCLEOTIDES AND SYNTHETIC

POLYNUCLEOTIDES ON THE APPEARANCE OF FLUORESCENT PRODUCTS

OF BaP BIOTRANSFORMATION-------------------------------------------------------------57-60

EFFECT OF CALF THYMUS DNA MODIFIED

BY NITROGEN MUSTARD ON THE APPEARANCE

OF FLUORESCENT PRODUCTS OF BaP BIOTRANSFORMATION---------------60-61

EFFECT OF YEAST RNA AND DENATURED

CALF THYMUS DNA ON THE APPEARANCE

OF FLUORESCENT PRODUCTS OF BaP

BIOTRANSFORMATION-----------------------------------------------------------------------61-63

BIOLOGICAL ACTIVITY OF CALF THYMUS

DNA CHEMICALLY MODIFIED BY BaP

BIOTRANSFORMATION----------------------------------------------------------------------63-65

DISCUSSION---------------------------------------------------------------------------------154-167

BIBLIOGRAPHY-----------------------------------------------------------------------------168-177


CLICK HERE FOR MY COMPLETE DISSERTATION ON WIKIPEDIA


"INTERACTION OF BENZO (α) PYRENE AND NUCLEIC ACIDS IN THE PRESENCE OF A MIXED-FUNCTION OXIDASE SYSTEM"
CLICK HERE => PhD Diploma and Complete Dissertation (177 pages, includes 46 images) (doc,epub,odt,pdf,txt) (05/19/2021; ZIP-File)
USA – STATE UNIVERSITY OF NEW YORK AT BUFFALO (SUNYAB) – 1973 (ProQuest 302781013; ISBN 979-8661021359)

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