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.     

Metabolism of (−)-trans-(3R,4R)-dihydroxy-3,4-dihydrochrysene to diol epoxides by liver microsomes

Metabolism of biosynthetic (?)-trans-(3R,4R)-dihydroxy-3,4-dihydrochrysene by liver microsomes from control, phenobarbital-treated and 3-methylcholanthrene-treated rats was investigated. Although previous studies of the metabolism of related benzo[a]pyrene and benzo[e]pyrene dihydrodiols which also prefer the diaxial conformation had indicated that diol epoxides were minor metabolites, the diastereomeric chrysene 3,4-diol-1,2-epoxides-$\text{1}$ and $\text{?2}$ were major metabolites (66–90%). All three types of microsomes metabolized the chrysene 3,4-dihydrodiol at low but essentially similar rates (0.5–0.7 nmol substrate/nmol cytochrome P-450/min).     

High mutagenicity and toxicity of a diol epoxide derived from benzo[a]pyrene

(±)-7β,8α-Dihydroxy-9β,10β-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BP 7,8-diol-9,10-epoxide) is a suspected metabolite of benzo[a]pyrene that is highly mutagenic and toxic in several strains of $\text{Salmonella}\text{typhimurium}$ and in cultured Chinese hamster V79 cells. BP 7,8-diol-9,10-epoxide was approximately 5, 10 and 40 times more mutagenic than benzo[a]pyrene 4,5-oxide (BP 4,5-oxide) in strains TA 98 and TA 100 of $\text{S.}\text{typhimurium}$ and in V79 cells, respectively. Both compounds were equally mutagenic to strain TA 1538 and non-mutagenic to strain TA 1535 of $\text{S.}\text{typhimurium}$. The diol epoxide was toxic to the four bacterial strains at 0.5–2.0 nmole/plate, whereas BP 4,5-oxide was nontoxic at these concentrations. In V79 cells, the diol epoxide was about 60-fold more cytotoxic than BP 4,5-oxide.     

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.     

In vitro reaction of radioactive 7beta, 8alpha, --dihydroxy--9alpha, 10alpha-epoxy--7,8,9,10--tetrahydrobenzo (A) pyrene and 7beta,8alpha--dihydroxy--9beta, 10beta--epoxy--7,8,9,10--tetrahydrobenzo (A) pyrene with DNA.

The $\text{in vitro}$ reaction of bacteriophage T7-DNA with the radioactive diastereomeric benzo(a)pyrene-diol-epoxides, (±) [${}^3\text{H}{}_9$, ${}^3\text{H}{}_{10}$]-7β,8α-dihydroxy-9α,10β-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene, and (±) [${}^3\text{H}{}_9$, ${}^3\text{H}{}_{10}$]-7β,8α-dihydroxy-9β,19β-epoxy-7,8,9,10-tetrahydrobenzo(1)pyrene, was investigated. Chromatographic analysis of digests of the DNA allowed the distinction of characteristic deoxynucleoside adduct peaks for the two benzo(a)pyrene-diol-epoxides. Our results, together with data from the literature, allow the identification of these adducts as mostly N₂-(10-7β,8α,9α-trihydroxy-7,8,9,10-tetrahydrobenzo(a)pyreney1)deoxyguanosine and N₂-(10-7β,8α,9β-trihydroxy-7,8,9,10-tetrahydrobenzo(a)pyreney1)deoxyguanosine, respectively. DNA-benzo(a)pyrene adducts with the same chromatographic properties were formed in mouse embryo fibroblasts upon treatment with benzo(a)pyrene.     

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.     

Liver microsomal DT diaphorase: noninvolvement in hydroxylation of benzo(a)pyrene.

A highly purified reconstituted system isolated from the microsomes of 3-methylcholanthrene-treated rats consisting of cytochrome P-448, NADPH-cytochrome $\text{c}$ reductase and synthetic dilauroyl phosphatidylcholine had no DT diaphorase activity, but hydroxylated benzo[a]pyrene at a faster rate than microsomes from 3-methylcholanthrene-treated rats. DT diaphorase purified from liver microsomes of 3-methylcholanthrene-treated rats when added to this reconstituted system did not stimulate or inhibit benzo[a]pyrene hydroxylation, nor could it replace or NADPH-cytochrome $\text{c}$ reductase in supporting the reaction. We therefore conclude that microsomal DT diaphorase is not involved in microsomal hydroxylation of benzo[a]pyrene to its phenolic products despite the observation that both DT diaphorase activity and the hydroxylation of benzo[a]pyrene are induced by 3-methylcholanthrene and 2,3,7,8-tetrachlorodibenzo-$\text{p}$-dioxin     

Cellular binding of benzo(a)pyrene to DNA characterized by low temperature fluorescence

A new approach has been developed to detect ultra low concentrations of benzo(a)pyrene products bound to nucleic acids $\text{in}\text{vivo}$. The binding to DNA of hamster embryo cell cultures was characterized by low temperature fluorescence spectroscopy. The method can detect less than one polycyclic hydrocarbon residue per 50,000 nucleotides. The fluorescence spectra indicate that the benzo(a)pyrene derivative bound to DNA has a pyrene-like chromophore and resembles that obtained when DNA is reacted $\text{in}\text{vitro}$ with the 7,8-diol-9,10-oxide of benzo(a)pyrene. This confirms that metabolism of the 7,8,9,10 ring on benzo(a)pyrene precedes reaction with DNA. The method should be useful for detecting and characterizing the $\text{in}\text{vivo}$ binding of other fluorescent carcinogens to nucleic acids.     

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.     

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.     

Presence of cytochrome P-450- and cytochrome P-448-dependent monooxygenase functions in hepatoma cell lines

Cell lines derived from Reuber H-4-II-E hepatoma cells and their hybrids that differ in the expression of liver-specific functions are shown to contain different forms of monooxygenases. According to 1) the specificity toward the substrates benzo(a)pyrene, aldrin and chenodexycholic acid, 2) the kinetics of the epoxidation of aldrin, 3) the response to inducers, such as benz(a)anthracene and dexamethasone, and 4) the $\text{in}$$\text{vitro}$ modifier 7,8-benzoflavone, the monooxygenases predominating in differentiated cell lines belong to the cytochrome P-450-dependent enzyme(s), those in the less differentiated lines belong to the cytochrome P-448-dependent form(s).     

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.     

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.     

Glutathione S-transferase activities in rat and mouse sperm and human semen

Glutathione $\text{S}$-transferase activity was found in sperm of the rat and $\text{DBA}\text{2J}$ and C57 $\text{BL}\text{6J}$ mice. In rat sperm activities with benzo(a)pyrene 4,5-oxide, styrene 7,8-oxide, and 1-chloro-2,4-dinitrobenzene were 0.88, 1.07, and 26.1 nmoles/min/mg protein, respectively. Δ⁵-3-Ketosteroid isomerase activity of rat sperm was 4.9 nmoles/min/mg protein. These specific glutathione $\text{S}$-transferase and Δ⁵-3-ketosteroid isomerase activities in sperm represent 0.4–4.1% of rat liver cytosol values. Human semen also contained significant glutathione $\text{S}$-transferase activity. It is postulated that these enzymes could function in the metabolism and detoxification of certain electrophilic xenobiotics, if present in sperm.     

Microsomal benzo(a)pyrene hydroxylase in Aspergillus ochraceus TS: Assay and characterization of the enzyme system

Microsomal preparations of $\text{Aspergillus ochraceus}$ TS oxidised benzo(a)pyrene very efficiently in the presence of NADPH and O₂ and exhibits a pH optimum of 8.0–8.2. The hydroxylation is also effected in presence of NaI04. Hydroxylation was inhibited by metyrapone, SKF-525A, PCMB, imidazole, carbon monoxide and flavone but not by cyanide, azide and antimycin A indicating thereby the involvement of cytochrome P-450 in this reaction. Inhibition by cytochrome C is consistant with the participation of NADPH-cytochrome C reductase in this hydroxylation. Reduced microsomes and its solubilized preparation, when treated with carbon monoxide, showed absorption maxima at 453 and 449 respectively. Different classical inducers of cytochrome P-450 induce the benzo(a)pyrene hydroxylase activity to varying degree and as such suggests the existence of multiple forms of cytochrome P-450 in this fungus.     

Morphological transformation of C3H/10T1/2 cells subcultured at low cell densities

Morphological transformation in $\text{C3H/10T}\text{1}\text{2}$ cells treated with varied concentrations of benzo (α) pyrene (BP) was measured following subculture at low cell densities. Subconfluent cultures exposed to BP were allowed to grow to confluence, trypsinized, and reseeded at cell densities ranging from 5 to 2,300 surviving cells/cm². These secondary cultures were incubated for 8 to 9 weeks, stained, and examined for evidence of morphological transformation. BP-treated cells reseeded in virtual isolation in microwells (approx. 5 surviving cells/cm²) transformed at frequencies up to 14.5%. At these low initial cell densities, transformation frequency did not demonstrate a significant dependence on BP concentration. However, BP-treated cells reseeded at higher densities (11 to 2,300 surviving cells/cm²) showed both density-dependent transformation frequencies and BP-concentration dependence of transformation. As reported previously (Haber et al., Cancer Res. 37 1644, 1977), the subculturing of treated cells did not affect the BP-concentration dependence of focus formation in the $\text{C3H/10T}\text{1}\text{2}$ transformation assay. Cell density-dependent suppression of morphological transformation has now been observed over a wide range of BP concentration. We suggest that this phenomenon is associated with colony interactions and consider various possible mechanisms of BP involvement.     

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.     

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.     

Metabolism of benzo(a)pyrene to reactive intermediate(s) via prostaglandin biosynthesis.

We have examined the oxidation of benzo (a) pyrene (BP) to electrophilic metabolites during the formation of prostaglandins (PG) and thromboxanes (TX) from arachidonic acid (AA) by guinea pig lung microsomal protein. In the presence of NADPH or AA, electrophilic metabolites of [¹⁴C]-BP were generated which were non-extractable from microsomal protein and thus assumed to be covalently bound. The total amount of BP metabolized in the presence of NADPH was 2–2 $\text{1}\text{2}$ times the amount of BP metabolized in the presence of AA. Only 4–5% of BP metabolized by the NADPH mediated mixed-function oxidase system was covalently bound, whereas 12–15% of the BP metabolized in the presence of AA was covalently bound to tissue protein and DNA. Quinones were the major metabolites produced by the AA dependent system, while dihydrodiols were the major metabolites formed by the NADPH dependent system. 7, 12-Dimethyl-benzanthracene, and 7,8-BP-dihydrodiol, but not 3 hydroxy-BP were also oxidized by PG synthetase to reactive metabolites.     

Metabolism of cyclopenta(cd)pyrene at the K-region by microsomes and a reconstituted cytochrome P-450 system from rat liver

Liver microsomes and reconstituted cytochrome P-450 systems purified from phenobarbital or 3-methylcholanthrene pre-treated rats metabolize cyclopenta(cd)pyrene at its K-region to $\text{trans}$-9,10-dihydroxy-9,10-dihydrocyclopenta(cd)pyrene. The rate of formation of the K-region product is from 5% to 25% that of $\text{trans}$-3,4-dihydroxy-3,4-dihydro-cyclopenta(cd)pyrene. The preference of microsomes and purified cytochromes P-450 for oxygenating cyclopenta(cd)pyrene at the ethylenic C(3)–C(4) position is explainable in part by the fact that the C(4) position has the greatest electron density in the highest occupied molecular orbital.