Fluorescence properties of a benzo(a)pyrene 7,8 dihydrodiol 9,10-oxide-DNA adduct. Conformation and effects of intermolecular DNA interactions

The pyrene-like fluorescence of the covalent benzo(a)pyrene diol-epoxide-DNA complex prepared by reacting 7,8,-dihydrodiol 9,10-epoxy benzo(a)pyrene (BPDE) with DNA in aqueous solution $\text{in vitro}$, has been investigated. It is shown that this fluorescence is $\text{sensitive}$ to molecular oxygen, to the concentration of $\text{native}$ DNA and to the ionic strength (KCl concentration), but is $\text{insensitive}$ to the concentration of $\text{denatured}$ DNA. These effects are related to the conformation of the pyrene-like chromophore of BPDE. Most of the fluorescence of a $\text{dilute}$ solution of the DNA-bound benzo(a)pyrene derivative originates from binding sites in which the pyrene moiety is not intercalated between the DNA base pairs, but is located on the outside of the DNA double helix.     

Effects of estrogens on aryl hydrocarbon hydroxylase mediated binding of benzo(a) pyrene metabolites to DNA in vitro

$\text{In vivo}$ and $\text{in vitro}$ studies were carried out to determine the effects of estradiol and other steroid hormones on aryl hydrocarbon hydroxylase-mediated binding of benzo(a)pyrene metabolites to DNA. Injection of female $\text{C57}\text{B1}\text{6J}$ mice with 0.2 mg or 2 mg of estradiol 24 hours prior to, during and 24 hours after injection of 3-methylcholanthrene resulted in a significant decrease in the capacity of hepatic microsomes from these animals to mediate the binding of benzo(a)pyrene metabolites to DNA when compared to microsomes from animals receiving 3 methylcholanthrene treatment only. Binding of benzo(a) pyrene metabolites was inhibited between 22 and 50%, depending on the dose of estradiol used. The enzyme and cytochrome components of the aryl hydrocarbon hydroxylase multienzymic complex were not affected by either estradiol treatment. The data suggests that estradiol inhibits aryl hydrocarbon hydroxylase mediated binding of benzo(a)pyrene metabolites to DNA by activity as a non-competitive inhibitor of aryl hydrocarbon hydroxylase activity.     

Reactions of benzo]pa]pyrene diol-epoxides with DNA and nucleosomes in aqueous solutions

The rate of solvolysis of benzo[$\text{a}$]pyrene diol-epoxide in aqueous solutions can be followed by fluorescence spectroscopy. When DNA was present the rat of breakdown of benzo[$\text{a}$]pyrene diol-epoxide was substantially enhanced, while at the same time fluorescence intensity was decreased. This decrease, however, was due to noncovalently bound tetraols and does not seem to be a function of the covalent adducts formed. Nucleosomal core particles, reacted under identical conditions, showed very little quenching of the pyrene-like chromophore. When increasing amounts of cysteine were present the covalent binding could be prevented in both free DNA and nucleosomal DNA. Analysis of the distribution of the carcinogen to nucleosomal DNA showed that the covalently bound carcinogen was located at or within 10 bases of the 5′-OH region of the nucleosomal DNA.     

Covalent binding of benzo[a]pyrene to dna in fish liver

Benzo[a]pyrene became bound to the hepatic DNA in juvenile English sole ($\text{Parophrys vetulus}$) force fed tritiated benzo[a]pyrene. No statistically signïficant change was observed in the level of the binding from 16 h to 2 wk after the single exposure. Specific activities of binding were similar for both DNA and protein. Moreover, a binding index was calculated to represent the number of benzo[a]pyrene molecules bound per 10⁶ nucleotides after administration of a theoretical dose of 1 mmole of hydrocarbon per kg body weight. The value for English sole liver DNA was of the same order of magnitude as the values reported for mouse skin and mammary gland in which benzo[a]pyrene is carcinogenic.     

Benzo[a]pyrene-7,8-dihydrodiol: selective binding to single stranded DNA and inactivation of 0X174 DNA infectivity.

When single-stranded ØX174 DNA is exposed to certain dihydrodiol derivatives of benzo[a]pyrene and benz[a]anthracene, inhibition of viral DNA infectivity is observed. Binding studies with labeled $\text{trans}$-7,8-dihydrodiol of benzo[a]pyrene and $\text{anti}$-benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide indicate that the diol preferentially reacts with single-stranded DNA, whereas the diolepoxide reacts equally well with both single- and double-stranded DNA, as well as with RNA. Also, the diol and diolepoxide derivatives show a marked difference in their capacity to complex with specific deoxyhomopolymers, i.e., Poly dI. These observations suggest that the diol and diolepoxide derivatives recognize different binding sites in nucleic acids, and that the diol derivative may play an important role in mutagenesis and carcinogenesis induced by polycyclic aromatic hydrocarbons.     

Kinetics of hydrolysis to tetraols and binding of benzo(a)pyrene-7,8-dihydrodiol-9,10-oxide and its tetraol derivatives to DNA. Conformation of adducts

When the major reactive metabolite of benzo(a)pyrene, $\text{trans}$ -7,8-dihydroxy - $\text{anti}$-9,10-epoxy -7,8,9,10-tetrahydrobenzo(a)pyrene ($\text{anti}$-BPDE) is incubated with DNA in aqueous solution at 25°C, both covalent binding and hydrolysis of $\text{anti}$-BPDE to its tetraols occur. Using fluorescence and absorption spectroscopy it is shown that hydrolysis of $\text{anti}$-BPDE is markedly accelerated by DNA. In the presence of 5A₂₆₀ units of DNA per ml in cacodylate buffer solution, at an initial concentration of DNA phosphate/$\text{anti}$-BPDE ratio of 100, both the extent of covalent binding to DNA ( < 7% of the total $\text{anti}$-BPDE initially present) and hydrolysis of $\text{anti}$-BPDE reach their maximum levels within less than five minutes after mixing. Absorption and electric linear dichroism spectra indicate that the tetraols bind non-covalently to DNA by an intercalation mechanism, whereas the covalent product displays the characteristics of an externally bound complex.     

Evidence for cytosolic benzo(a)pyrene carrier proteins which function in cytochrome P450 oxidation in rat liver

Solutions of cytosolic proteins from rat liver contain benzo(a)pyrene solubilizing activity capable of serving as a carrier between solid state benzo(a)pyrene and microsomal cytochrome P₄₅₀. Fractionation of benzo(a)pyrene-saturated cytosolic proteins on a Sephadex G-100 column or by sucrose density gradients produced benzo(a)pyrene peaks of about 46,000 daltons and a very high molecular weight material. The protein-bound benzo(a)pyrene obtained in both peaks was oxidized rapidly by microsomes in the presence of NADPH, indicating that the benzo(a)pyrene carrier activity is capable of presenting the substrate to the cytochrome P₄₅₀. Liver cytosolic proteins from rats receiving intraperitoneal injection of [¹⁴C] benzo(a)pyrene was chromatographed on a column of Sephadex G-75. Radioactivity eluted at the same positions of the chromatogram as did the carrier activities described above. These results indicate that these benzo(a)pyrene carrier proteins may have an $\text{in}\text{vivo}$ role in the metabolism of benzo(a)pyrene.     

2,3,7,8-Tetrachlorodibenzo-P-dioxin: potent anticarcinogenic activity in CD-1 mice.

Topical pretreatment with non-toxic doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin, a contaminant formed in the commercial synthesis of the herbicide 2,4,5-trichlorophen-oxyacetic acid, strongly inhibited the initiation of skin tumors by 7,12-dimethylbenz(a)-anthracene and benzo(a)pyrene in female CD-1 mice. 2,3,7,8-tetrachlorodibenzo-p-dioxin also produced marked induction of epidermal monooxygenase enzymes functional in the conversion of 7,12-dimethylbenz(a)anthracene to a variety of hydroxylated products. The time course of anticarcinogenic effects resulting from pretreatment with the dioxin correlated with the magnitude of induction as well as with a singnificant reduction in the quantity of 7,12-dimethylbenz(a)anthracene metabolites covalently bound $\text{in vivo}$ to epidermal DNA and RNA but not protein.     

The induction of cytochrome P-448 dependent benzo(a)pyrene hydroxylase in Saccharomycescerevisiae

When grown in high concentrations of glucose, the yeast $\text{Saccharomyces}\text{cerevisiae}$ produces a microsomal cytochrome P-450 monooxygenase system which is capable of hydroxylating benzo(a)pyrene. The addition of benzo(a)pyrene to the yeast during growth causes only a small increase in cytochrome P-448 levels but results in a dramatic improvement in the apparent kinetics of benzo(a)pyrene hydroxylation as measured by a decrease in the Michaelis constant and an increase in maximal velocity. Dimethylnitrosamine, phenobarbital and 3-methylcholanthrene also induce this enzyme to various degrees. Yeast pretreatment with β-naphthoflavone did not affect this enzyme, yet pretreatment with lanosterol resulted in a decreased affinity for benzo(a)pyrene. The addition of benzo(a)pyrene to yeast growing at low glucose concentration does not induce cytochrome P-448. The implications of these findings with regard to the presence of multiple forms of cytochromes $\text{P-448}\text{P-450}$ in yeast are briefly discussed.     

Direct fluorometric analysis of benzo(a)pyrene metabolite formation by mouse liver microsomes

We have examined the metabolites produced by in vitro incubation of benzo(a)pyrene with 3-methylcholanthrene-induced mice liver microsomes. Our objective was to observe directly a possible difference in microsomal enzyme systems of animal models having different susceptibility to chemical carcinogens. The metabolites produced by the two animal models,$\text{C57BL}\text{6J}$ and $\text{DBA}\text{2}$ mice, were analyzed by a highly sensitive, “three-dimensional” fluorescence plotting technique. The fluorescence spectra of the total ethyl acetate-soluble metabolites clearly indicate that the metabolites produced by $\text{DBA}\text{2}$ enzymes were predominantly monohydroxylated benzo(a)pyrene while those produced by the liver microsomes of $\text{C57BL}\text{6J}$ were highly enriched with the 7,8-dihydrodihydroxybenzo(a)pyrene type.     

Absorbance and fluorescence properties of protochlorophyllide in etiolated bean leaves

Primary leaves of 7-to-9 day-old etiolated bean seedlings contain a species of protochlorophyllide which is not transformed to chlorophyllide by light; this pigment species exhibits an absorption peak at 631nm $\text{in}\text{vivo}$ at ?196° and a fluorescence emission peak at 639nm $\text{in}\text{vivo}$ at room temperature. Heat-treatment of etiolated leaves converts the phototransformable protochlorophyllide holochrome to a pigment species with $\text{in}\text{vivo}$ absorption and fluorescence peaks identical to those of endogenous nontransformable protochlorophyllide. Administration of δ-amino-levulinic acid to etiolated leaves causes the synthesis of non-transformable protochlorophyllide with an absorption peak also at 631nm $\text{in}\text{vivo}$ at ?196° but with a fluorescence emission peak at 643nm $\text{in}\text{vivo}$ at room temperature. Heat-treatment of such leaves does not affect the position of these bands. The results indicate that protochlorophyllide which is derived from exogenous δ-amino-levulinic acid is in a physically different state from other forms of protochlorophyllide in the leaf.     

Fast kinetics of antagonist-acetylcholine receptor interactions: a temperature-jump relaxation study

The interactions of an antagonist with the membrane-bound acetylcholine receptor from $\text{Torpedo}$ have been studied using the temperature-jump relaxation method. The fluorescence emission of the antagonist, a pyrenyl-choline derivative, excited by energy transfer from the protein chromophores, enabled the observation of two relaxation processes in the lower millisecond time range. The concentration dependence of the relaxation times and associated amplitudes could be accounted for by a reaction scheme involving the binding of $\text{two}$ antagonist molecules and the subsequent isomerization of the complexes. Both binding steps appear to be diffusion-controlled and the second isomerization rate limiting. Competitive and non-competitive antagonism are discussed in relation to the proposed reaction mechanism.Recent advances in the study of the reaction mechanisms between the acetylcholine receptor (AChR)¹ and cholinergic ligands $\text{in}\text{vitro}$ have been accomplished by applying kinetic techniques (see review in (1)). In most cases extrinsic fluorescent probes have been employed in conjunction with the stopped-flow technique (2–7). Absorption spectroscopy has been used in one case (8). A slightly more direct approach utilized the intrinsic fluorescence of the membrane-bound AChR and cholinergic agonists of well characterized activity, including the natural neurotransmitter acetylcholine (9–11). From other potentially useful approaches or probes (see e.g. ref. (12)) only fragmentary and qualitative information is available. Since the introduction of dansyl-choline (13), and the subsequent demonstration of its mixed agonist-local anaesthetic action $\text{in}\text{vivo}$ (14), it became apparent that the pharmacological characterization of extrinsic fluorescent probes is an absolute prerequisite to the performance of any spectroscopic study $\text{in}\text{vitro}$. This prerequisite has been met for example in a later study (15) in which pyrene butyrylcholine was synthesized, having in mind the high molecular weight of the AChR oligomer and the need to match its rotational relaxation time with a chromophore of particularly long lifetime such as pyrene. This (15) and other (16) compounds of the series were found to be competitive $\text{antagonists}$ of the nicotinic AChR.In the present work one of these compounds is used to study the kinetics of the interaction with the membrane-bound AChR from $\text{Torpedo}\text{marmorata}$ by means of the temperature-jump relaxation technique. The simplest reaction mechanism accounting for the observed kinetics is discussed in relation to the available electrophysiological information on the action of this type of cholinergic blocking agent.     

Kinetics of repair of O6-methylguanine in DNA by O6-methylguanine-DNA methyltransferase in, vitro and in, vivo

The demethylation of O⁶-methylguanine in double stranded DNA catalyzed by rat liver O⁶-methylguanine-DNA transmethylase was found to proceed much more rapidly when the DNA substrate was methylated to a high extent. When the content of O⁶-methylguanine in DNA was equal to 1 in 2000 guanines, the reaction was 90% complete within 2 min, but when the content was 1 in 500,000 it required 27 min at 37°C. These results suggest that the repair protein either moves along the DNA substrate or else has little selectivity for binding specifically to the sites containing O⁶-methylguanine rather than to the normal DNA. The repair of O⁶-methylguanine in rat liver $\text{in}\text{,}\text{vivo}$ occurred at rates comparable to those seen $\text{in}\text{,}\text{vitro}$ with the substrates alkylated to low extents and was virtually complete within 3 hours. These results provide strong evidence that this protein is the factor responsible for O⁶-methylguanine removal $\text{in}\text{,}\text{vivo}$ and explain the wide variation in time courses reported in the literature since substrates methylated to greatly different extents have been used for such experiments.     

In vitro binding of 3H-benzo(a)pyrene to chromatin DNA

Chromatin was prepared from mouse liver and incubated in an $\text{in}$$\text{vitro}$ binding assay containing ³H-benzo(a)pyrene and a NADPH-generating system. Binding to chromatin DNA was stimulated by the presence of microsomes from 3-methylcholanthrene pretreated mice. This incubation system represents an improvement over previous studies in which purified DNA is employed as the target macromolecule in that aralkylation is being investigated under conditions which better approximate those present in the cell, i.e., the genetic material is “coated” with nuclear protein.     

A spectrofluorimetric investigation of calf thymus DNA modified by BP diolepoxide and 1-pyrenyloxirane

7β,8α-Dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BP diolepoxide, $\text{1}$) and 1-pyrenyloxirane ($\text{2}$) bind chemically to calf thymus DNA. The fluorescence efficiency of pyrenyl groups in mutagen modified DNA varies appreciably with its conformation and decreases in the order: pyrenees, modified denatured DNA and modified native DNA. A particularly interesting observation is that the fluorescence efficiency of mutagen modified DNA intensifies substantially upon denaturation. Our results suggest that the pyrenyl groups in mutagen modified DNA are intercalated between the base pairs of DNA. Since both $\text{1}$ and $\text{2}$ are powerful frame-shifting mutagens for S. typhimurium TA-98, the intercalative covalent binding of these compounds to DNA may provide a molecular basis for their mutagenic activity.     

Role of protein binding and pharmacokinetics in the lack of correlation between in vitro inhibition of PG-synthetase and in vivo antiinflammatory activity of a homologous series of nonsteroidal antiinflammatory compounds.

The antiinflammatory activity of a homologous series of α-alkyl substituted [4-(1-oxo-2-iso-indolinyl)-phenyl]-acetic acid has been assayed by some $\text{in}$$\text{vitro}$ and $\text{in}$$\text{vivo}$ tests.These compounds were shown to be particularly active in inhibiting prostaglandin biosynthesis from bovine seminal vesicles, and their potency was seen to increase as the size of the substituents in the side chain increased.The antiinflammatory activity $\text{in}$$\text{vivo}$ is not correlated with $\text{in}$$\text{vitro}$ inhibition of PG-synthetase. Discussion of the data takes into account the plasma protein binding and pharmacokinetics of these compounds.     

Nuclear 115cadmium: uptake and disappearance correlated with cadmium-binding protein synthesis.

The intracellular distribution of ¹¹⁵cadmium was determined following a pulsed exposure to the metal. The uptake and disappearance of label from rat liver nuclei was correlated with the appearance of a cytoplasmic Cd-binding protein. By coupling $\text{in}\text{vivo}$ - $\text{in}\text{vitro}$ experiments it was shown that unspecifically bound cadmium is free to enter the nucleus while specifically bound cadmium remains in the cytoplasm.     

Formation of the 11,12-diol as a metabolite of benzo(a)pyrene by rat skin in vivo

The dihydrodiols present as metabolites in rat skin after topical application of ³H-labelled benzo(a)pyrene included a significant amount of radioactivity that cochromatographed with synthetic $\text{trans}$-11,12-dihydro-11,12-dihydroxybenzo(a)pyrene. Treatment of the radioactive metabolite with hot mineral acid gave a product that had chromatographic properties identical to those of the phenol similarly formed from the synthetic dihydrodiol and acetylation of the metabolite yielded a product that cochromatographed with the diacetate of the synthetic dihydrodiol. These observations show that the 11,12-dihydrodiol is formed as a metabolite of BP in rat skin $\text{in vivo}$. The metabolite was not detected in mouse skin.     

The non-covalent binding of benzo[a]pyrene and its hydroxylated metabolites to intracellular proteins and lipid bilayers

The non-covalent interactions of benzo[a]pyrene (BP) and several of its hydroxylated metabolites with ligandin, aminoazodye-binding protein A (Z-protein, fatty acid binding protein) and lecithin bilayers have been studied by equilibrium dialysis, an adsorption technique and fluorescence spectroscopy. Binding affinities expressed as $\text{v}\text{/c}$ (where $\text{v}$ = moles of BP or BP metabolite bound per mole of protein or lipid and c = unbound concentration), were measured at concentrations sufficiently low that there was no self-association of the unbound compounds as judged by their fluorescence characteristics. 3-Hydroxybenzo[a]pyrene (BP-3-phenol), 4,5-dihydro-4,5-dihydroxybenzo[a]pyrene (BP-4,5-dihydrodiol) and 7,8-dihydro-7,8-dihydroxybenzo[a]pyrene (BP-7,8-dihydrodiol) bind more strongly ($\text{v}\text{/c = 10}{}^5\text{?5 · 10}{}^5\text{l}\text{·}\text{mol}{}^{\mathrm{?1}}$) to all three binders than does BP itself ($\text{v}\text{/c = 10}{}^4\text{?7 · 10}{}^4\text{l}\text{·}\text{mol}{}^{\mathrm{?1}}$). 9,10-Dihydro-9,10-dihydroxybenzo[a]pyrene (BP-9,10-dihydrodiol) binds to ligandin with an affinity similar to those of the other BP metabolites studied here, but binds much less strongly to both protein A and lecithin ($\text{v}\text{/c = 10}{}^4$ and 3 · 10⁴ l · mol^?1, respectively). The low affinity of BP-9,10-dihydrodiol for lecithin would account for earlier findings that on incubation of BP with isolated rat hepatocytes, this metabolite egressed from the cells to the extracellular medium much more readily than either BP-4,5-dihydrodiol or BP-7,8-dihydrodiol.Calculations based on these results suggest that within hepatocytes BP and its metabolites, including BP-9,10-dihydrodiol, will be found almost exclusively associated (>98%) with lipid membranes.     

Unscheduled DNA synthesis in isolated hepatic nuclei after treatment of rats with methylnitrosourea in vivo

Administration of the carcinogenic methylating agent, methylnitrosourea, to rats caused a significant increase in endogenous DNA synthesis assayed subsequently in isolated hepatic nuclei $\text{in}\text{vitro}$. DNA synthesis was related directly to the dose of carcinogen and inversely to the interval between treatment and isolation of nuclei. This synthesis appears to represent the continuation $\text{in}\text{vitro}$ of unscheduled, reparative DNA synthesis initiated in damaged cells $\text{in}\text{vivo}$.