Ethoxyquin feeding to rats increases liver microsome-catalyzed formation of benzo(a)pyrene diol epoxide — DNA adduct

The ability of rat liver microsomes to catalyze the formation of benzo(a)pyrene 7,8-diol-9,10-epoxide — DNA nucleoside adduct was increased threefold by feeding 0.5% ethoxyquin to the animals. Microsomal epoxide hydratase activity was enhanced i parallel by a factor of 3 while aryl hydrocarbon hydroxylase activity was not induced. Liver microsomes from rat pretreated with 3-methylcholanthrene produced an increased proportion of diol epoxide — DNA adduct when ethoxyquin had been fed to the animals. The main chromatographic peak formed by microsomes from 3-methylcholanthrene treated rats which contains DNA adducts of secondary benzo(a)pyrene phenol metabolites is reduced when the animals had received ethoxyquin.     

Effects of harman and norharman on the mutagenicity and binding to DNA of benzo[a]pyrene metabolites in vitro and on aryl hydrocarbon hydroxylase induction in cell culture

Harman and norharman, two β-carboline derivatives known to exist in certain foods and to be formed during pyrolysis of tobacco and meat, were tested for mutagenic activity in the presence of benzo[a]pyrene, mouse liver enzymes, and Salmonella typhimurium TA98 in vitro. Both harman and norharman inhibit benzo[a]pyrene mutagenicity, benzo[a]pyrene metabolism (as measured by aryl hydrocarbon hydroxylase activity), and the binding of all benzo[a]pyrene metabolites to DNA in vitro. Moreover, harman and norharman are quite toxic to cultures of hepatoma-derived H-4-II-E and Hepa-1 established cell lines and therefore were found to be very weak inducers of aryl hydrocarbon hydroxylase activity.     

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.     

Binding of polycyclic hydrocarbons to nuclear components in vitro.

Rat liver nuclei were incubated with microsomes, a NADPH-generating system, microsomes and ³H-benzo (a) pyrene. Binding of polycyclic hydrocarbon was noted to nuclear DNA, nuclear proteins and microsomal proteins. When nuclei or microsomes from 3-methylcholanthrene treated animals were used, binding to nuclear DNA and microsomal protein was increased. These data confirm t the presence of a nuclear aryl hydrocarbon hydroxylase, extend previous studies on macromolecular acceptors to include nuclear proteins and demonstrate reduced binding to nuclear proteins and DNA when microsomes are included in the incubation system with nuclei.     

Radioactive assay for aryl hydrocarbon hydroxylase. Improved method and biological importance

By addition of two volumes of a 1M aqueous KOH/dimethylsulfoxide ($\text{15}\text{85}$; v/v) mixture to the enzymatic incubation medium, it is possible to selectively extract the unmetabolized benzo(a)pyrene in hexane. Therefore, the radio-activity remaining in the water phase corresponds to all the in vitro synthesized metabolites. This isotopic method is very sensitive (2 × 10^?11 moles) and is almost insensitive to the room lighting. The aryl hydrocarbon hydroxylase activities found with this method are 2,3 and 10 times higher in the liver, lung and kidney respectively compared to those obtained with the fluorimetric method.     

Comparison of the metabolism of benzo[alpha]pyrene and binding to DNA caused by rat liver nuclei and microsomes.

Administration of 3-methylcholanthrene (3MC) to rats greatly enhanced the aryl hydrocarbon hydroxylase (AHH) activity of liver nuclei. However, the binding in vitro [3H]benzo[alpha]pyrene (BP) to DNA within the nuclei which occurred at the same time as hydroxylation of BP was much less enhanced. Thin layer chromatography of the metabolites of BP produced by these nuclei revealed the same metabolites in similar relative amounts as were produced by rat liver microsomes prepared from rats which had received 3MC. The binding to DNA was further analysed by hydrolysis of the DNA and fractionation on a Sephadex column. This analysis revealed that the binding to DAN in nuclei was very similar in nature to that which occurred when calf-thymus DNA was added to microsomes metabolising BP.     

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.     

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.     

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).     

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     

Effects of asbestos on membrane transport and metabolism of benzo(a)pyrene

The effect of asbestos on benzo(a)pyrene uptake by microsomal membranes and lipid micelles has been investigated. Asbestos mediates a rapid transport of the carcinogen into the membrane and also impairs benzo(a)pyrene metabolism in rabbit and rat liver microsomes by markedly inhibiting aryl hydrocarbon hydroxylase.     

Aryl hydrocarbon hydroxylase induction in human lymphocyte cultures by 2,3,7,8-tetrachlorodibenzo-p-dioxin

Aryl hydrocarbon (benzo[a]pyrene) hydroxylase activity is induced in cultured human lymphocytes by 2,3,7,8-tetrachlorodibenzo-$\text{p}$-dioxin (TCDD) at a concentration in the growth medium 40 to 60 times less than the concentration of 3-methylcholanthrene (MC) necessary for maximal hydroxylase induction. In cultured lymphocytes from 19 individuals, the extent of hydroxylase induction by TCDD or MC ranged between 1.7- and 2.9-fold. Those individuals having (presumably under genetic control) lower basal and MC-inducible hydroxylase activities in their lymphocytes also have lower TCDD-inducible hydroxylase activity. Because of the day-to-day experimental variability, the variations within each assay, and for several other reasons discussed, we suggest that the observed variance of expression of hydroxylase induction more closely fits a unimodal, polygenic ($\text{i.e.}$ 2 or more genes) pattern rather than the trimodal (single gene) form of inheritance proposed recently by Kellermann and coworkers.     

Genetic expression of aryl hydrocarbon hydroxylase activity in the mouse: Distinction between the “responsive” homozygote and heterozygote at the Ah locus

β-Napththoflavone administration induces certain monooxygenase activities, such as aryl hydrocarbon (benzo[a]pyrene) hydroxylase, and cytochrome P₁-450 formation in the “responsive” C57BL/6 and C3H/He inbred mouse strains, whereas these changes are absent or relatively small in the so-called nonresponsive $\text{DBA}\text{2}$ inbred strain. Dose-response curves—with the use of large numbers of animals of the same age and sex and with either β-naphthoflavone or the much more potent 2,3,7,8-tetrachlorodibenzo-p-dioxin as inducer—reveal a small, but statistically significant, difference in the hydroxylase induction between the $\text{C}\text{57}\text{BL}\text{6}\text{J}$ homozygote and the ($\text{C}\text{57}\text{BL}\text{6}\text{J}$)($\text{DBA}\text{2}\text{J}$)F₁ heterozygote in liver, kidney, bowel, and lung. The (C3H/HeJ)($\text{DBA}\text{2}\text{J}$)F₁ heterozygote displays additive inheritance in each of these same tissues.     

Involvement of cytochrome b5 in the hepatic microsomal metabolism of benzo(a)pyrene

Ethanol consumption decreased the specific content of microsomal cytochrome b5 in both chow-and liquid diet-fed hamsters while cytochrome P450 levels were unchanged in chow-fed animals and increased in liquid diet-fed animals. Microsomes from animals receiving ethanol in their drinking water exhibited decreased rates of microsomal aryl hydrocarbon hydroxylase activity and postmitochondrial supernatant mediated mutagenicity of benzo(a)pyrene. In contrast, microsomes from hamsters receiving ethanol in liquid diets showed no changes in either of these two activities. When the observed rates of 7,8 and 9,10 diol formation per nmole P450 for chow-fed animals are plotted vs. the b5/P450 ratio a positive correlation was observed suggesting that cytochrome b5 participates directly in the microsomal metabolism of benzo(a)pyrene.     

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.     

Aryl hydrocarbon hydroxylase: Degradation of product due to contaminant in acetone

A contaminant present in reagent grade acetone causes degradation of fluorescent phenolic metabolites of benzo(a)pyrene as measured in the aryl hydrocarbon hydroxylase assay. Although the contaminant was not identified, its properties suggest that it is a relatively volatile organic material, possibly an oxidizing agent. The acetone may be readily purified by distillation.     

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.     

Induction of microsomal aryl hydrocarbon (3,4-benzo(α)pyrene) hydroxylase and cytochrome P-450k in rat kidney cortex: I. Characteristics of the hydroxylase system

Both the rat kidney cortex aryl hydrocarbon hydroxylase activity and cytochrome P-450_K are induced by benzo(α)pyrene treatment. Following a single injection of benzo(α)pyrene, maximal hydroxylase activity and cytochrome P-450_K content occur at 24 hr, returning to control levels within 72–96 hr. Induction of both the enzyme activity and hemoprotein is inhibited by cycloheximide. The enzyme system is localized in the microsomal fraction, has an absolute requirement for NADPH and molecular oxygen, and a pH optimum at 7.4; the induced activity is linear with microsomal protein concentration up to 0.8 mg and with time up to 20 min. Both the hydroxylase activity and cytochrome P-450_K follow the same pattern of inactivation with increasing temperature. The apparent K_m for the induced hydroxylase was 7.7 μm and V was increased fourfold above control value. In the presence of laurate, a substrate for the kidney microsomal cytochrome P-450_K-dependent monooxygenase system, the amount of inhibition of hydroxylase activity corresponded to the level of activity present in untreated kidney cortex microsomes. α-Naphthoflavone (10^?5m), a type I inducer (36) produced a greater inhibitory effect on the induced hydroxylase activity than on the control (55% vs 20%). The presence of cytochrome c or carbon monoxide markedly decreased hydroxylase activity. This evidence in addition to aforementioned characteristics of the enzyme suggests a cytochrome P-450_K-dependent aryl hydroxylase activity which differs from that present in the control rat.     

Metabolism of monohydroxybenzo(a)pyrenes by rat liver microsomes and mammalian cells in culture.

The 3-, 6-, and 9-monohydroxybenzo(a)pyrenes are metabolized by microsomes from rat liver in vitro. The metabolism of 3-hydroxybenzo(a)pyrene requires the presence of NADPH and is inhibited by carbon monoxide, suggesting that the reaction is mediated by a microsomal mixed-function oxygenase. The metabolic activity can be induced by in vivo treatment with 3-methylcholanthrene. 7,8-Benzoflavone strongly inhibits the induced activity but has little effect on the constitutive enzyme. The inducibility and inhibition characteristics, as well as the metabolic rate of the conversion of 3-hydroxybenzo(a)pyrene, closely resemble those of the oxidative metabolism of benzo(a)pyrene. The microsomal NADPH-dependent metabolism of [3H]3-hydroxybenzo(a)pyrene leads to the formation of a number of products of which a major fraction cochromatographs with the 3,6-quinone of benzo(a)pyrene. In mammalian cell cultures 3-hydroxybenzo(a)pyrene is converted by a mechanism different from that in hepatic microsomes. The disappearance of the phenol in cultures of hamster embryo cells is independent of the action of inducers or inhibitors of the aryl hydrocarbon hydroxylases and also occurs in the mouse L-cell line, A9, which lacks detectable aryl hydrocarbon hydroxylase activity. In A9 cells, [3H]3-hydroxybenzo(a)pyrene is largely converted to water soluble derivatives.