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Electron paramagnetic resonance study of iodine-induced radicals of benzo( ) pyrene and other polycyclic hydrocarbons
Abstract
Free radicals have been postulated previously as intermediates in the chemical linkage of the environmental carcinogen benzo[a]pyrene to nucleic acids when activated by iodine. Electron paramagnetic resonance studies indicate the presence of benzo[a]pyrene radicals in benzene, methanol and cyclohexane solution induced by iodine. These radicals are quenched by pyrimidine, purine, nucleosides, imidazole, and other nitrogenous compounds but not by alcohol, aldehyde, or water. These results strongly support the proposal that radicals of benzo[a]pyrene are involved in the chemical reaction between the hydrocarbon and nucleic acids in the presence of iodine. The electron paramagnetic resonance studies on the steady-state radical concentration of 14 polycyclic hydrocarbons formed in the presence of iodine indicate that, in general, the carcinogenic compounds such as benzo[a]pyrene, 7,12-dimethylbenzanthracene, 3-methylcholanthrene, etc., have a much higher concentration of radicals than the non-carcinogenic compounds such as benzo[e]pyrene, benzanthrene, pyrene, naphthacene, etc. There are one or two exceptions. The steady-state radical concentrations of these compounds do not correlate well with their ionization potentials, though the compounds having low ionization potentials do tend to yield higher concentration of radicals.
... The greatest reactivity of BP with electrophiles occurs at C-6, followed by C-1 and C-3 (Cavalieri andCalvin, 1971, 1972). In BP radical cation (BP +. ), it is again C-6 that displays by far the major reactivity with nucleophiles (Blackburn et al., 1974;Caspary et al., 1973;Cavalieri and Auerbach, 1974;Jeftic and Adams, 1970;Johnson and Calvin, 1973;Menger et al., 1976;Rochlitz, 1967;Stack et al., 1995;Wilk et al., 1966;Wilke and Girke, 1972). C-1 and C-3 follow in decreasing order (Cavalieri andCalvin, 1971, 1972;Cremonesi et al, 1989). ...
Elucidation of estrogen carcinogenesis required a few fundamental discoveries made by studying the mechanism of carcinogenesis of polycyclic aromatic hydrocarbons (PAH). The two major mechanisms of metabolic activation of PAH involve formation of radical cations and diol epoxides as ultimate carcinogenic metabolites. These intermediates react with DNA to yield two types of adducts: stable adducts that remain in DNA unless removed by repair and depurinating adducts that are lost from DNA by cleavage of the glycosyl bond between the purine base and deoxyribose. The potent carcinogenic PAH benzo[a]pyrene, dibenzo[a,l]pyrene, 7,12-dimethylbenz[a]anthracene and 3-methylcholanthrene predominantly form depurinating DNA adducts, leaving apurinic sites in the DNA that generate cancer-initiating mutations. This was discovered by correlation between the depurinating adducts formed in mouse skin by treatment with benzo[a]pyrene, dibenzo[a,l]pyrene or 7,12-dimethylbenz[a]anthracene and the site of mutations in the Harvey-ras oncogene in mouse skin papillomas initiated by one of these PAH.
... The strategy of double-labeling experiments can be applied because one-electron oxidation involves a substitution reaction and tritium is lost from the position participating in the covalent bond between BP and DNA. Nucleophilic trapping in chemical experiments proceeds almost exclusively at C-6 in the BP radical cation (Tables 1 and 2) (37,39,41,43,44,71,72) BP is bound to DNA in mouse skin and 94% from C-6 in the HRP/H202-catalyzed binding of BP to DNA. Although these results suggest that C-6 of BP is involved in the covalent bond to DNA, determination of the structure of BP-DNA adducts is necessary to substantiate this evidence. ...
Carcinogenic activation of polycyclic aromatic hydrocarbons (PAH) involves two main pathways: one-electron oxidation and monooxygenation. One-electron oxidation produces PAH radical cations, which can react with cellular nucleophiles. Results from biochemical and biological experiments indicate that only PAH with ionization potentials below ca. 7.35 eV can be metabolically activated by one-electron oxidation. In addition, the radical cations of carcinogenic PAH must have relatively high charge localization to react effectively with macromolecules in target cells. Metabolic formation of PAH quinones proceeds through radical cation intermediates. Binding of benzo[a]pyrene (BP) to mouse skin DNA occurs predominantly at C-6, the position of highest charge localization in the BP radical cation, and binding of 6-methyl BP to DNA in mouse skin yields a major adduct with the 6-methyl group bound to the 2-amino group of deoxyguanosine. Studies of carcinogenicity by direct application of PAH to rat mammary gland indicate that only PAH with ionization potentials low enough for activation by one-electron oxidation produce tumors in this target tissue. These constitute some of the results which provide evidence for the involvement of one-electron oxidation in PAH carcinogenesis.
Two main pathways are involved in the carcinogenic activation of polycyclic aromatic hydrocarbons (PAH): one-electron oxidation and monooxygenation. One-electron oxidation produces PAH radical cations, which can react with cellular nucleophiles. Biochemical and biological data indicate that only PAH with relatively low ionization potentials (below ca. 7.35 eV) can be activated by one-electron oxidation. Furthermore, a carcinogenic PAH must have a relatively high charge localization in its radical cation to react effectively with target cellular macromolecules. Binding of benzo[a]pyrene (BP) to DNA in vitro and in vivo occurs predominantly at C-6, the position of highest charge density in the BP radical cation, and binding of 6-methylBP to mouse skin DNA yields a major adduct in which the 6-methyl is bound to the 2-amino of deoxyguanosine. PAH radical cations are also involved in the metabolic conversion of PAH to PAH diones. Carcinogenicity studies of PAH in rat mammary gland indicate that only PAH with ionization potential low enough for activation by one-electron oxidation induce tumors in this target organ. These results and others indicate that one-electron oxidation of PAH is involved in their tumor initiation process.
The role of charge transfer complexes in bioelectrochemical processes is discussed. It is shown that their formation as intermediates prior to a chemical reaction proper affects the probability of such reaction. It is suggested that the percolation mode of electron transfer proposed for amorphous solids be applicable to biological systems and that such electron transfer not only imparts conductivity to otherwise non-conducting bio-compounds such as proteins, but also facilitates linkage by charge transfer complexation between electron donating and electron accepting regions. Surface and micellar charge transfer complexes are shown to enter into electrodic bio-processes especially on membranes by virtue of surface electric fields generated, resulting in the formation of localized excited surface states on the substrate. It is proposed also that protonic complexes, especially in non-aqueous systems, play a role in biological charge and energy transfer.
Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous contaminants of the human environment, and some PAHs are relatively potent carcinogens. This chapter reviews the chemistry of the PAHs and their heterocyclic analogs and surveys their most important types of reactions from the viewpoint of their potential environmental significance. PAHs in the environment are subject to various chemical and photochemical processes some of which result in degradation to less toxic products, while others result in formation of compounds, such as nitrosubstituted PAHs, which exhibit greater carcinogenicity. While the major focus is on the reactions of the more intensively investigated alternant hydrocarbons, the nonalternant PAHs and the heterocyclic aza- and thio-PAHs are also discussed. Current concepts concerning the mechanisms of carcinogenesis of the PAHs and their heterocyclic analogs are also reviewed.
Chemical carcinogens comprise a large and structurally diverse group of synthetic and naturally occurring compounds. It appears almost axiomatic that such compounds must react with tissue components in order to induce neoplastic transformation. With the exception of the carcinogenic alkylating agents, most chemical carcinogens are not reactive per se and must be converted to reactive forms either chemically or metabolically. Data are emerging to indicate that electrophilic reactants are the ultimate form of most, if not all, chemical carcinogens (Miller, 1970). The proximate forms of a number of chemical carcinogens might be convertible to free radicals, i.e., electrophilic reactants, and suggest a possible role for the free radical in carcinogenesis. The presence of free radicals in tobacco smoke has been demonstrated (Lyons and Spence, 1960; Bluhm et al., 1971) and the increased incidence of lung cancer in cigarette smokers based on epidemiological studies is well known. The chemical and metabolic conversion of a number of chemical carcinogens to free radicals and the interaction of these radicals with DNA will be described in this communication.
Detailed mechanisms of electrochemical reductions of seven polynuclear aromatic hydrocarbons (PAHs) including benzo[a]pyrene (BaP), benzo[e]pyrene (BeP), dibenz[a,h]anthracene, dibenz[a,c]anthracene, 3-methylcholanthrene, 7,12-dimethylbenz[a]anthracene, and benz[a]anthracene were studied using cyclic voltammetry, single potential step chronoamperometry, voltammetry at rotating disk electrodes, and controlled potential coulometry in tetrahydrofuran solution using 0.1 M tetra-n-butylammonium perchlorate as a supporting electrolyte. Results indicate that these compounds undergo ECE reactions to a final product reduced at L-regions of each PAH with the exception of BaP and BeP. For these two PAHs, unreduced hydrocarbons were identified after exhaustive electrolysis. Heterogeneous electron-transfer rate constants, as well as homogeneous reaction rate constants, for the addition of the proton to electrogenerated anion radicals are also reported.
1. The role of vitamin K1 in the hydroxylation of benzo(α)pyrene (BP) at the 6-position is correlated with the oxidative formation of vitamin K1-hydroperoxide and its subsequent reaction with BP in the presence of soluble rat lung hydroperoxidase.
2. Vitamin K1-hydroperoxide was synthesized from vitamin K1 and its structure verified by mass spectral analysis and enzymic studies.
3. The major product (the 3,6-BP dione) that results from the non-enzymic oxidation of 6-hydroxy-BP was isolated from the enzymic reaction and characterized by spectroscopic studies.
4. Thiodione (the menadione-glutathione adduct) inhibits both aryl hydrocarbon hydroxylase activity and vitamin K1-hydroperoxide/hydroperoxidase activity.
All twelve monomethylbenzo[a]pyrenes were studied using cyclic voltammetry and single‐ and double‐potential‐step chronoamperometry at a platinum electrode in acetonitrile, in order to determine the kinetic stability of the cation radicals, and to obtain information on the decay reaction mechanisms. For most of the compounds there may be more than one mechanism operating in parallel, but single‐potential‐step chronoamperometric data were amenable to calculation of rate constants for the reaction of cation radical with water. With some exceptions, larger rate constants correlate with lower calculated localization energies. There are some parallels between the kinetic results and animal carcinogenicity data reported eleswhere.
Radical cations of benzo[a]pyrene (BP) and 6-substituted derivatives were generated by one-electron oxidation with 2 equiv of Mn(OAc)3·2H2O. Some of the properties of these radical cations were investigated by nucleophilic trapping with acetate ion. BP produced predominantly 6-OAcBP and small amounts of BP 1,6-, 3,6-, and 6,12-dione. 6-FBP yielded 6-OAcBP, a mixture of 1,6-(OAc)2BP and 3,6-(OAc)2BP, and BP diones. In the case of 6-ClBP and 6-BrBP the major products obtained were a mixture of the 1-OAc and 3-OAc derivatives, and BP diones, while substantial starting material remained unreacted. 6-CH3BP afforded mostly 6-OAcCH2BP, a mixture of 1-OAc and 3-OAc derivatives of 6-CH3BP, and a mixture of 1-OAc and 3-OAc derivatives of 6-OAcCH2BP. These results indicate that nucleophilic substitution of BP•+ and 6-FBP•+ occurs exclusively at C-6. For 6-ClBP•+ and 6-BrBP•+ substitution at C-1 and C-3, which are the positions of second highest charge density in their radical cations after C-6, compete successfully for nucleophilic substitution. For 6-CH3BP•+ charge localization at C-6 activates the methyl group rendering it the most reactive toward nucleophilic attack. Competitive acetoxylation of 6-CH3BP•+ also occurs to a minor extent at C-1 and C-3. These mechanistic studies have been useful in clarifying some aspects of the metabolism of BP and its halogeno derivatives by cytochrome P-450 and peroxidases. Furthermore, this chemistry can provide some guidance in understanding the mechanism of tumor initiation by these compounds.
Upon incubation of benzo[a]pyrene (B[a]P) at 37$sup 0$ in rat liver
homogenates fortified with NADPH generating cofactors, a metabolite was formed
which gave rise spontaneously to an electron paramagnetic resonance (EPR) signal.
The radical and its metabolic precursor were extracted into benzene for
measurement, in which they are relatively stable. The EPR signal was identical
with that extracted after incubating synthetic 6-hydroxybenzo[a]pyrene (6-OH-
B[a]P) in rat liver homogenates and has been identified as the 6-
oxobenzo[a]pyrene radical by its characteristic hyperfine structure. No EPR
signal was detected when benzo[a]pyrene or cofactors were eliminated from the
incubation mixture or when the homogenate was heated. The radical concentration
reached a peak after 14-15 min of incubation and declined rapidly to a low level
at 20 min. The decay of 6-hydroxybenzo[a]pyrene in rat liver homogenate did not
appear to be enzymic and showed a first-order rate constant of 0.29 min$sup -1$
and a half-life of 2.4 min. The only significant products formed by oxidation of
6-hydroxybenzo[a]pyrene in rat liver homogenates were: 6,12-B[a]P dione (15
percent), 1,6-B[a]P dione (41 percent), and 3,6-B[a]P dione (44 percent).
Formation of 6-OH-B[a]P was found to represent about 18 and 20 percent of total
metabolism for female Sprague-Dawley rats and female ACI rats, respectively. The
data indicate that 6-OH-B[a]P is a major metabolite of benzo[a]pyrene. This
metabolite is very labile, being readily oxidized to the 6-oxobenzo[a]pyrene
radical which is a transient intermediate in the further oxidation to quinones.
(auth)
A labile metabolite of the carcinogen benzo[a]pyrene, 6-hydroxybenzo[a]pyrene, is autoxidized in aqueous buffer-ethanol solutions to produce the three stable 6,12- 1,6-, and 3,6-benzo[a]pyrene diones and a small amount of an unidentified paramagnetic, violet-colored material. In this reaction, oxygen is reduced to hydrogen peroxide and probably to other reactive reduced oxygen species as well. A free radical derived from 6-hydroxybenzo[a]pyrene by one-electron oxidation, 6-oxobenzo[a]pyrene radical, is most likely an obligatory intermediate in this autoxidation as indicated by its kinetics of formation and decay. In addition, this free radical can be produced quantitatively in benzene by oxidation of 6-OH-B[a]P with aqueous K 3Fe(CN) 6 and isolated in the absence of oxygen. Like 6-hydroxybenzo[a]pyrene, this isolated free radical is autoxidized in aqueous buffer-ethanol solutions and yields identical products. 6-Hydroxybenzo[a]pyrene is also oxidized rapidly in rat liver homogenate to the three benzo[a]pyrene diones, indicating that it is a labile precursor of these oftenfound products of benzo[a]pyrene metabolism. The production of reactive hydrocarbon and reduced oxygen intermediates can explain the results of previous studies which demonstrated that 6-hydroxybenzo[a]pyrene binds covalently to, and causes strand breakage in, nucleic acid. This reactivity of 6-hydroxybenzo[a]pyrene may also be related to its high toxicity toward cells in culture and its ability to morphologically transform Syrian hamster embryo fibroblasts.
Molecular orbital electronic structure calculations for twelve polynuclear aromatic hydrocarbons were performed by the samo method. Results indicate that the carcinogenicity of such aromatic hydrocarbons is related to a K-region π-bond order greater than 0.340. There is no correlation with δ-bond order or overall charge density, perhaps accounting for the success of earlier theoretical treatments based on the π-electron model. Exceptions to a simple K-region treatment are discussed in terms of other models for carcinogenic activity.
The transformation rates of 25 polycyclic aromatic hydrocarbons (PAH) and 4 derivatives of anthracene are investigated in dilute solutions containing dinitrogen tetraoxide and nitric and nitrous acid under conditions which are relevant for determining the relative reactivities of PAH and their derivatives toward nitrogen compounds in the atmosphere. The reactivities of some of the PAH are several orders of magnitude higher than that of benzene. The reactions are apparently electrophilic, as electron-donating substituents, e.g., methyl, enhance the reactivity of anthracenes, and electron-withdrawing substituents, e.g., nitro, diminish it. The reactivities of PAH correlate with spectroscopical constants, and it seems possible to relate the differences in reactivity to differences in the chemical structure. A classification of the reactivity of PAH in electrophilic reactions in the environment under dark and weak lighting conditions is set up. 60 references.
At least four different free radicals can be formed from benzo[a]pyrene under different reaction conditions, namely the 6-oxybenzo[a]pyrene radical, the benzo[a]pyrene anion and cation radicals and a radical from heated benzo[a]pyrene. The formation and esr spectra of these radicals have been studied with the aim of clarifying the nature of the radical species involved under different reaction conditions. Additionally the reactivity of the 6-oxybenzo[a]pyrene and the benzo[a]pyrene cation radicals towards several phenolic antioxidants have also been investigated.
An extended summary is presented of current progress in research on the basic mechanism of chemical carcinogenesis conducted in the Division of Biophysics, School of Hygiene and Public Health, at the Johns Hopkins University. The first section concerns the involvement of free radicals in benzo[a]pyrene metabolism and carcinogenesis, the second the relationship between neoplastic transformation and somatic mutation, and the third the changes in DNA sequence organization and gene expression in neoplastic transformation.
The hard and soft acids and bases (HSAB) principle, which states that hard acids bind preferentially to hard bases and soft acids to soft bases, may serve to assess specific chemico-biological interactions. As living systems are composed mainly of "hard" elements, molecular events taking place within the cell are dominated by "hard-hard interactions". On this premise, it becomes likely that extraneous "soft" agents are particularly injurious to life. In the HSAB context a selected number of variegated phenomena are briefly discussed qualitatively; these include biocidal actions, heavy metal poisoning, chemical carcinogenesis, some enzymic reactions, and nucleic acid complexations. Although the HSAB principle cannot be used as a tool for mechanistic explanations of biochemical processes, it may provide clues to likely target molecules and the loci of action.
In the context of the bay region hypothesis for polycyclic aromatic hydrocarbon (PAH) carcinogenesis, molecular properties were calculated for seventeen polycyclic aromatic hydrocarbons related to (1) intrinsic substrate reactivities towards activating and detoxifying metabolism and (2) the stabilities of the putative carbocation ultimate carcinogens. All-valence electron methods were used, avoiding the inherent difficulties found in the pi-electron methods. The calculated substrate reactivities were found to predict major metabolites successfully, supporting the validity of their use in attempted correlations with observed carcinogenic potencies. Positive correlations were found between observed carcinogenic potencies and (1) the reactivities of the parent polycyclic aromatic hydrocarbons towards the initial distal bay region epoxidation and (2) the stabilities of the diol epoxide carbocations. The reactivities of the distal bay region diol epoxides, were high for both carcinogenic and non-carcinogenic compounds, implying that the second epoxidation does not determine relative carcinogenic activity. Support for a possible alternative hypothesis, that polycyclic aromatic hydrocarbons are activated by one electron oxidation, was also found.
Loss of tritium from specific positions in [3H,14C] aromatic hydrocarbons can elucidate their binding site(s) to DNA and RNA and indicate the mechanism of activation. Studies of tritium loss from [6-3H,14C]benzo[a]pyrene (B[a]P), [1,3-3H,14C]B[a]P, [1,3,6-3H,14C]B[a]P, [6,7-3H,14C]B[a]P, and [7-3H,14C]B[a]P were conducted in vitro using liver nuclei and microsomes from 3-methylcholanthrene-induced Sprague-Dawley rats and in vivo on the skin of Charles River CD-1 mice. The relative loss of tritium from [3H, 14C]B[a]P was measured after binding to skin DNA and RNA, to nuclear DNA, and to native and denatured calf thymus and rat liver DNA's and poly(G) by microsomal activation. In skin, nuclei, and microsomes plus native DNA, virtually all B[a]P binding occurred at positions 1,3 and 6; while with microsomes plus denatured DNA or poly(G), B[a]P showed no binding at the 6 position and a small amount at the 1 and 3 positions. In vivo and with nuclei, binding at the 6 position predominated. Little loss of tritium from the 7 position was seen; this was expected because binding at this position is not thought to occur. This confirms the interpretation of loss of tritium as an indication of binding at a given position. These results demonstrate that the use of microsomes to activate B[a]P is not a valid model system for delineating the in vivo mechanism of B[a]P activation, and support previous evidence for one-electron oxidation as the mechanism of activation of hydrocarbons in binding to nucleic acids.
A simple procedure for generation and trapping of polycyclic aromatic hydrocarbon radical cations in homoge-neous solutions of pyridine and iodine is described. Radical cations of benz[a]anthracene and its alkyl derivatives are trapped by nucleophilic attack of pyridine on the aromatic nucleus in the order C-7 > C-12 > C-5. When positions 7 or 7 and 12 are blocked by a methyl group, pyridine substitution on the alkyl group competes with ring substitution. Mechanisms for the two types of substitution are proposed and trapping specificity is discussed in terms of charge density and steric factors in the radical ions.
The field of chemical carcinogenesis is reviewed, with emphasis on three aspects: 1) environmental chemicals are a major cause of human cancer; 2) most chemical carcinogens require metabolic activation by mixed-function oxidases to electrophilic metabolites that form strong covalent chemical bonds with cellular macromolecules and thereby initiate the carcinogenic process; and 3) several systems are available in which normal cells can be transformed by chemical carcinogens into malignant cancer cells. Much has been learned about the cellular mechanisms of chemical carcinogenesis using these systems. They also have considerable potential to be used as prescreens for environmental carcinogens.
3-Methylcholanthrene (MC) in chloroform and trifluoroacetic acid-d1 yielded 3-methylcholanthrene-d4 (MC-d4), a compound selectively deuterated at the 1-, 5-, and 6-carbon atoms. In turn, the protodedeuteration of the labeled hydrocarbon at the 6-carbon atom led to 3-methylcholanthrene-d3 (MC-d3). A comparative test for carcinogenicity between MC and MC-d3 by repeated skin painting on female Swiss mice showed a significantly lower tumorigenic activity of the latter. The result implies that the 1-carbon atom of the hydrocarbon is a critical binding-site to cellular targets in the tumor-initiating process.
Three classes of products are formed when benzo[a]pyrene (BP) is metabolized by cytochrome P-450: dihydrodiols, phenols and the quinones, BP 1,6-, 3,6- and 6,12-dione. These products have been thought to arise from attack of a catalytically-activated electrophilic oxygen atom. In this paper we report chemical and biochemical experiments which demonstrate that BP quinones arise from an initial one-electron oxidation of BP to form its radical cation. BP, 6-fluorobenzo[a]pyrene (6-FBP), 6-chlorobenzo[a]pyrene (6-ClBP), and 6-bromobenzo[a]pyrene (6-BrBP) were metabolized by uninduced and 3-methylcholanthrene-induced rat liver microsomes in the presence of NADPH or cumene hydroperoxide (CHP) as cofactor. BP and 6-FBP produced similar metabolic profiles with induced microsomes in the presence of NADPH or 2 mM CHP. With NADPH both compounds produced dihydrodiols, phenols and quinones, whereas with CHP, they yielded only quinones. Metabolism of BP and 6-FBP was also similar with uninduced microsomes and 2 mM CHP, yielding the same BP quinones. With uninduced microsomes in the presence of NADPH, BP produced all three classes of metabolites, whereas 6-FBP afforded only quinones. At a low concentration of CHP (0.10 mM), BP was metabolized to phenols and quinones, whereas 6-FBP gave only quinones. 6-ClBP and 6-BrBP were poor substrates, forming metabolites only with induced microsomes and NADPH. One-electron oxidation of BP by Mn(OAc)3 occurred exclusively at C-6 with predominant formation of 6-acetoxyBP and small amounts of BP quinones. In the one-electron oxidation of 6-FBP by Mn(OAc)3, the major products obtained were 6-acetoxyBP, a mixture of 1,6- and 3,6-diacetoxyBP, and BP quinones. Reaction of BP and 6-FBP radical cation perchlorates with water produced the same BP quinones. Conversely, electrophilic substitution of 6-FBP with bromine or deuterium ion afforded C-1 and/or C-3 derivatives with retention of the fluoro substituent at C-6. These results indicate that metabolic formation of BP quinones from BP and 6-FBP can only derive from their intermediate radical cation.
The evidence for biological involvement, the spectroscopic properties (especially EPR), and the reactions, of free radicals derived from benzo(a)pyrene and its methylated, hydroxylated, and fluorinated derivatives are reviewed.
The behavior of benzo[a]pyrene (B[a]P) during peroxidation of phosphatidylcholine (PC) liposomes initiated by an azo compound was investigated to examine the mechanism of quinone formation from carcinogenic B[a]P mediated by nonenzymatic lipid peroxidation occurring in vivo. B[a]P had a retarding effect on the peroxidation of polyunsaturated fatty acid moiety of PC. The major oxidation products which accumulated in the peroxidized liposomes were B[a]P 1,6-, 3,6-, and 6,12-quinone. Antioxidants acting as scavengers of chain-propagating lipid peroxy radicals effectively prevented not only lipid peroxidation but also B[a]P oxidation in the liposomal suspension. PC hydroperoxides, the primary products of PC oxidation, did not react with B[a]P in the absence of the azo compound, indicating that lipid peroxy radicals, not lipid hydroperoxides, are responsible for the formation of these quinones. The experiments using 18O2 gas and 18O-labeled methyl linoleate hydroperoxides demonstrated that B[a]P quinones are formed by incorporating molecular oxygen and their origin is partly due to the lipid peroxy radical. The mechanism proposed for the formation of B[a]P quinones mediated by peroxidation of membrane lipids involves a direct attack of the lipid peroxy radical on B[a]P and subsequent autocatalytic oxidation. Weak carcinogenic and noncarcinogenic pentacyclic aromatic hydrocarbons showed little reactivity to the lipid peroxy radical in the liposomes. Thus, the facility of the peroxidative attack on B[a]P may be related to the powerful carcinogenic activity of this substance.
Polycyclic hydrocarbons and other xenobiotics are cooxygenated during the oxidative metabolism of polyunsaturated fatty acids. The products of polycyclic hydrocarbon cooxygenation depend upon the hydrocarbon but they appear to be formed by radical (or electron transfer) mechanisms. The cooxygenations are hydroperoxide-dependent oxidations catalyzed by peroxidases and may be triggered by enzymes which biosynthesize fatty acid hydroperoxides. Prostaglandin endoperoxide synthetase is the principal hydroperoxide-generating system which has been studied to date. The extent to which polyunsaturated fatty acid-dependent cooxygenation operates in vivo is uncertain but preliminary studies suggest it does occur.
1. The role of vitamin K1 in the hydroxylation of benzo(alpha)pyrene (BP) at the 6-position is correlated with the oxidative formation of vitamin K1-hydroperoxide and its subsequent reaction with BP in the presence of soluble rat lung hydroperoxidase. 2. Vitamin K1-hydroperoxide was synthesized for vitamin K1 and its structure verified by mass spectral analysis and enzymic studies. 3. The major product (the 3,6-BP dione) that results from the non-enzymic oxidation of 6-hydroxy-BP was isolated from the enzymic reaction and characterized by spectroscopic studies. 4. Thiodione (the menadione-glutathione adduct) inhibits both aryl hydrocarbon hydroxylase activity and vitamin K1-hydroperoxide/hydroperoxidase activity.
Wistar rats were divided into 4 groups: control group, Cro group, B(a) p group and Cro plus B(a) p group. Samples of lung tissue were collected 90, 180, 270, 360 and 540 days after the third time of intratracheal. The superoxide dismutase (SOD) activity, the level of lipid peroxide (LPO) and the ratio of SOD/LPO were observed. The results indicated that there was a synergistic action of Cro. and B(a) p.
Fluoro substitution of benzo[a]pyrene (BP) has been very useful in determining the mechanism of cytochrome P450-catalyzed oxygen transfer in the formation of 6-hydroxyBP (6-OHBP) and its resulting BP 1,6-, 3,6-, and 6,12-diones. We report here the metabolism of 1-FBP and 3-FBP, and PM3 calculations of charge densities and bond orders in the neutral molecules and radical cations of BP, 1-FBP, 3-FBP, and 6-FBP, to determine the mechanism of oxygen transfer for the formation of BP metabolites. 1-FBP and 3-FBP were metabolized by rat liver microsomes. The products were analyzed by HPLC and identified by NMR. Formation of BP 1,6-dione and BP 3,6-dione from 1-FBP and 3-FBP, respectively, can only occur by removal of the fluoro ion from C-1 and C-3, respectively, via one-electron oxidation of the substrate. The combined metabolic and theoretical studies reveal the mechanism of oxygen transfer in the P450-catalyzed formation of BP metabolites. Initial abstraction of a pi electron from BP by the [Fe(4+)=O](+)(*) of cytochrome P450 affords BP(+)(*). This is followed by oxygen transfer to the most electropositive carbon atoms, C-6, C-1, and C-3, with formation of 6-OHBP (and its quinones), 1-OHBP, and 3-OHBP, respectively, or the most electropositive 4,5-, 7,8-, and 9,10- double bonds, with formation of BP 4,5-, 7,8-, or 9,10-oxide.
Derivatives of adenosine and uridine, which were substituted only at the sugar moiety and which were solublein CCI4, have been prepared. The concentration-dependencies of the IR-spectra of 2’.3’-Isopropylidene-5’-trityl-adenosine (ITA) and 2’, 3’tsopropylidene-5’-trityl-uridine (ITU) in CCI4 indicated that hydrogen-bonded, cyclic dimers were formed at low concentrations. In solutions of equimolar amounts of ITA and ITU the hetero-associate containing an adenine-uracil-pair was found to be more stable than the homo-associates.
The characteristic charge-transfer absorption frequencies of a number of I2-hydrocarbon complexes in carbon tetrachloride solution have been found to follow the relation hv = ID - EA - Δ, and the stabilization energy Δ was constant for the whole series and was equal to 3.256 eV. Except the calculated values of molar extinction coefficients of the complexes, most of the experimental results appeared to support the general conclusion derived from Mulliken's theory of intermolecular charge transfer interaction. The experimental data, however, suggest that the extinction coefficients may not be dependent on the nature of the hydrocarbon part of the complex.
Nucleophilic agents such as pyridine yield pyridinium salts with radical cations of aromatic carcinogens. In this way the intermediate radical cations, produced by the oxidation of aromatic systems with iondine or oxygen, may be caught.Presumably, the generation of oligomer aromatic hydrocarbons as described,1 passes via “oxidomeres” (similar to the excimeres). The results support our mechanism of oligomerization of radical cations as well as their reaction with nucleophilic cell structures. The “pyridine-reaction” indicates a possibility of stopping the activity of these intermediate states, which we consider to be decisive in primary processes of chemical carcinogenesis.
Ever since the discovery of the carcinogenicity of certain aromatic polycyclic hydrocarbons, workers in the field have been confronted with the challenging question about the origin of the biological activity and specificity of these compounds. How can these relatively chemically inert polycyclic hydrocarbons have such a profound biological effect? This question must be related to the basic mechanism of chemical carcinogenesis.
The absorption spectra of solutions of iodine in aromatic and oxygenated solvents are interpreted on the assumption that they are spectra of 1:1 complexes of iodine with solvent molecules. (The correctness of this assumption at least for certain aromatic complexes has been shown by Benesi and Hildebrand.) The extent to which the λ5200 absorption region, highly characteristic of the iodine molecule in vapor and in inert solvents, is shifted toward the ultraviolet and altered in shape or intensity, is used as the principal basis for a division of these complexes into three classes. By valence-theoretical considerations a unique probable geometrical and electronic structure is obtained for each class. The probable structures are as follows. In general agreement with considerations advanced by Benesi and Hildebrand, and others, the electronic wave functions of all three classes are believed to contain resonance components of the general type A+I2- or A+I-I. In the Ar·I2 complexes (Ar = benzene or methylated benzene), the iodine molecule lies above the plane of the benzene ring with its axis parallel to the latter. In the R′RO·I2 complexes the iodine molecule stands against the oxygen atom, with its axis perpendicular to the R′RO plane. The probable acetone-iodine complex is similar except that the iodine axis is coplanar with the RR2 bonds on the lefthand side C=O skeleton. The polar forces, which are present in all three cases, are aided in the first class by partial C-I bonding and in the second and third classes by partial O+-I bonding, and further in RR′2 bonds on the lefthand side C=O 2 bonds on the righthand side II by conjugation between the C=O and the I-I π electrons. The indicated structures are suggestive as to reaction mechanisms for the halogens. The ultraviolet spectra, especially of the aromatic complexes, are discussed. The very intense absorption of the aromatic complexes near λ3000 is attributed to a transition in the aromatic part of the complex. It is suggested that this transition, although nearly forbidden in the aromatic molecule, is made strongly allowed by strong interaction between excited states of the two partners in the complex. As an alternative, it is suggested that this absorption, as also the color of other organic molecular complexes, may be due to an intermolecular charge transfer process during light absorption. Bromine in benzene and toluene solutions shows spectra which indicate that it forms complexes of the same kind as described above for iodine. Iodine chloride and bromide solution spectra in R′RO solvents also indicate the formation of complexes similar to those formed by iodine, but tighter. Brief comments are made on some other organic molecular complexes. The nature of the spectra of iodine and bromine in vapor and inert solvents is reviewed and it is hoped somewhat clarified.
Methods for the preparation of O5′-monomethoxytrityladenosine, O5′-monomethoxytritylcytidine, and O6′-monomethoxytritylguanosine are described. Using these derivatives and the previously known O5′-trityluridine, the following acylated derivatives of ribonucleosides bearing free 5′-hydroxyl groups were prepared: N,N′,O2′,O3′-tetrabenzoyladenosine, N,O2′,O3′-tribenzoylcytidine, O2′,O3′-diacetylguanosine, N,O2′,O3′-triacetylguunosine, O2′,O3′-dibenzoyluridine, and N,O2′,O3′-tribenzoyluridine. Methods for the large-scale preparation of cytidine 3′-phosphate and guanosine 3′-phosphate arc described. Condensations of the N,O2′,O5′-triacetyl derivatives of these ribonucleotides with the protected ribonucleosides gave generally satisfactory yields of the following compounds: cytidylyl-(3′→5′)-adenosine, cytidylyl-(3′→5′)-cytidine, cytidylyl-(3′→5′)-guanosine, cytidylyl-(3′→5′)-uridine, guanylyl-(3′→5′)-adenosine, guanylyl-(3′→5′)-cytidine, guanylyl-(3′→5′)-guanosine, and guanylyl-(3′→5′)-uridine.