Induction of aryl hydrocarbon (benzo(a)pyrene) hydroxylase and tyrosine aminotransferase in hepatoma cells in culture.

In the Reuber (H35) hepatoma cell strain, microsomal aryl hydrocarbon (benzo[a]pyrene) hydroxylase is induced 25-fold by the polycyclic hydrocarbon benz[a]anthracene but is not induced by the steroid hormone dexamethasone. Soluble tyrosine aminotransferase is induced sixfold by dexamethasone and twofold by benz[a]anthracene. Each enzyme requires similar inducer concentrations for induction, and their induction kinetics are similar. The induction of each enzyme requires RNA and protein synthesis; in each case the transcriptional and translational steps can occur independently. The two induction systems are differentially sensitive to inhibitors of macromolecular synthesis. Simultaneous exposure to both inducers produces increases in both enzyme activities that are greater than those produced by either inducer alone. Each inducer acts at a pretranslational level to produce this synergistic effect. The results suggest that the requirements for macromolecular synthesis are similar for the induction of each enzyme, but that the turnover of enzyme-specific macromolecules may differ for each.

Variation in aryl hydrocarbon (benzo(a)pyrene) hydroxylase activity in heteroploid and predominantly diploid rat liver cells in culture.

Aryl hydrocarbon (benzo(a)pyrene) hydroxylase is present and inducible in Buffalo rat liver cells in culture. There is substantial variation in both basal and inducible hydroxylase activities among heteroploid subclones isolated from a heteroploid parent population, and among diploid subclones isolated from a diploid parent population. This variation is not related to differences in the growth characteristics of the subclones, or to differences in their chromosome number. The results indicate that substantial heterogeneity in both basal and induced hydroxylase activity develops during the growth of both heteroploid and diploid cell strains in culture. These findings indicate that diploid cell populations are not necessarily homogeneous with respect to aryl hydrocarbon hydroxylas activity. This observation may complicate the interpretation of experiments involving somatic cell hybridization or polycyclic hydrocarbon-induced transformation and/or cytotoxicity. This heterogeneity in hydroxylase activity develops rather rapidly (2-3 mo of culture), in the absence of any apparent mutational stress.

Retention of membrane proteins by the endoplasmic reticulum.

We have used a monoclonal antibody specific for a hydrocarbon-induced cytochrome P450 to localize, by electron microscopy, the epitope-specific cytochrome P450. The cytochrome was found in the rough and smooth endoplasmic reticulum (ER) and the nuclear envelope of hepatocytes. Significant quantities of cytochrome P450 were not found in Golgi stacks. We also could not find any evidence of Golgi-associated processing of the Asn-linked oligosaccharide chains of two well-characterized ER membrane glycoprotein enzymes (glucosidase II and hexose-6-phosphate dehydrogenase), or of the oligosaccharides attached to the bulk of the glycoproteins of the ER membrane. We conclude that these ER membrane proteins are efficiently retained during a process of highly selective export from this organelle.

beta-Glucuronidase catalyzed hydrolysis of benzo(a)pyrene-3-glucuronide and binding to DNA.

beta-Glucuronidase catalyzes the hydrolysis of benzo[a]pyrene-3-glucuronide to 3-hydroxybenzo[a]pyrene. During the enzymatic hydrolysis, a benzo[a]pyrene derivative is formed which binds to DNA to a far greater extent than either the 3-hydroxybenzo[a]pyrene or its glucuronide. These results suggest that conjugates of benzo(a)pyrene may be converted by beta-glucuronidase at intracellular and organ sites distal to the initial sites of oxygenation and conjugation of benzo(a)pyrene to activated intermediates that are possibly carcinogenic.

Alpha-naphthoflavone: an inhibitor of hydrocarbon cytotoxicity and microsomal hydroxylase.

Alpha-naphthoflavone inhibits the metabolism of 3,4-benzopyrene and 7,12-dimethylbenz(a)anthracene in hamster enlbryo cell cultures and protects the cells against the inhibition of cell multiplication by these carcinogens. Alphla-nalphthoflavone also inhibits the aryl hydrocarbon hydroxylase activity in homogenates of induced hamster embryo cells and in liver microsomes from rats previously treated with polycyclic aromatic hydrocarbons, but not in microsomes from control rats.

Benzpyrene hydroxylase induction by polycyclic hydrocarbons in hamster embryonic cells grown in vitro.

Treatment of hamster embryonic cells with 1,2-benzanthracene for 4 to 48 hours induced a three- to tenfold increase in the activity of benzpyrene hydroxylase. That the increase in enzyme activity was completely prevented by puromycin suggested an induction of enzyme synthesis.

Dimethylbenzanthracene tumorigenesis and aryl hydrocarbon hydroxylase in mouse skin: inhibition by 7,8-benzoflavone.

Mouse skin contains aryl hydrocarbon hydroxylase which is highly inducible. The enzyme system is inhibited when 7,8-benzoflavone is added to homogenates of skin epidermis. 7,8-Benzoflavone also inhibits mouse skin tumorigenesis caused by repeated treatment with 9,10-dimethylbenzanthracene or by a single treatment with this chemical followed by weekly treatment with croton oil. These findings suggest that this enzyme system may be responsible for the activation of 9,10-dimethylbenzanthracene to its carcinogenic form.

Polyinosinic-polycytidylic acid inhibits chemically induced tumorigenesis in mouse skin.

Polyinosinic-polycytidylic acid administered intraperitoneally inhibits (i) the formation of skin tumors induced by a single topical application of 9,10-dimethylbenzanthracene followed by weekly applications of croton oil and (ii) the formation of skin tumors induced by a single large application of 9,10-dimethylbenzanthracene.

Benzo(a)pyrene metabolites: efficient and rapid separation by high-pressure liquid chromatography.

High-pressure liquid chromatography can separate eight metabolites of benzo[a] pyrene formed by rat liver microsomes. This method offers major advantages over previous techniques used for the separation of oxygenated polycyclic aromatic hydrocarbons.

Aryl hydrocarbon hydroxylase is inhibited by antibody to rat liver cytochrome P-450.

Antibody to the major purified cytochrome P-450 induced by 3-methylcholanthrene in rat liver strongly inhibits aryl hydrocarbon hydroxylase activity of uninduced and benz[a]anthracene-induced human monocytes and lymphocytes. Antibody to the cytochrome P-450 induced by phenobarbital has relatively little or no effect on the aryl hydrocarbon hydroxylase activity of the same human cells.

Benzo[a]pyrene diol epoxides: mechanism of enzymatic formation and optically active intermediates.

Studies of the mechanism of benzo[a]pyrene metabolism to reactive diol epoxides and of their disposition indicate that the metabolic intermediates of the activation pathways, 7,8-epoxide and trans-7,8-diol, as well as the two stereoisomeric diol epoxides are all optically active. Benzo[a]pyrene is converted to optically active 9,10-epoxides of (-)trans-7,8-diol by three enzymatic steps: (i) stereospecific oxygenation at the 7,8 double bond of benzo[a]pyrene by the mixed-function oxidases to essentially a single enantiomer of 7,8-epoxide, (ii) hydration of the 7,8-epoxide by epoxide hydratase to an optically pure (-)trans-7,8-diol, and (iii) stereoselective oxygenation by the mixed-function oxidases at the 9,10 double bond of the (-) trans-7,8-diol to optically active r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene and optically active r-7,t-8-dihydroxy-c-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene in a ratio of approximately 10 to 1.

Rapid and marked inhibition of rat-liver RNA polymerase by aflatoxin B-1.

From 15 minutes to 2 hours after the administration of aflatoxin B(1) invivo there is a 35-to 70-percent inhibition of DNA-directed RNA synthesis. The inhibition was reversed 12 and 24 hours later.

Monoclonal antibody-directed radioimmunoassay detects cytochrome P-450 in human placenta and lymphocytes.

A multiplicity of cytochromes P-450 is responsible for the detoxification and activation of xenobiotics such as drugs and carcinogens. Individual differences in sensitivity to these agents may reside in the cytochrome P-450 phenotype. A monoclonal antibody-directed radioimmunoassay was developed that detects epitope-specific cytochromes P-450 in human placentas and peripheral lymphocytes. Placentas from women who smoked cigarettes contained greater amounts of cytochrome P-450 with the monoclonal antibody-specific epitope than placentas from nonsmokers. The amount of this cytochrome P-450 in human peripheral lymphocytes increased after treatment of the mitogenized lymphocytes with the cytochrome P-450 inducer benz[a]anthracene.

Pretreatment with black tea polyphenols modulates xenobiotic-metabolizing enzymes in an experimental oral carcinogenesis model.

The objective of this study was to evaluate the chemopreventive potential of the black tea polyphenols Polyphenon-B and BTF-35 during the preinitiation phase of 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinogenesis. Hamsters were divided into six groups. Animals in groups 2 and 3 received diet containing Polyphenon-B and BTF-35, respectively, 4 weeks before carcinogen administration when they were 6 weeks of age and continued until the final exposure to carcinogen. At 10 weeks of age, animals in groups 1, 2, and 3 were painted with 0.5% DMBA three times a week for 14 weeks. Animals in groups 4 and 5 were given Polyphenon-B and BTF-35 alone, respectively, as in groups 2 and 3. Animals in group 6 served as control. All the animals were sacrificed after an experimental period of 18 weeks. Phase I and phase II xenobiotic-metabolizing enzymes and 8-hydroxy-deoxyguanosine (8-OH-dG) in the buccal pouch and liver were used as biomarkers of chemoprevention. Hamsters painted with DMBA showed increased expression of 8-OH-dG and enhanced activities of phase I (CYP450; total as well as CYP1A1, 1A2, and 2B isoforms and cytochrome b5) and phase II (GST and quinone reductase) xenobiotic-metabolizing enzymes with increased immunohistochemical expression of CYP1A1, and CYP1B1 isoforms in the buccal pouch. This was accompanied by increased phase I and decreased phase II enzyme activities in the liver. Administration of Polyphenon-B and BTF-35 significantly decreased tumor incidence, oxidative DNA damage, phase I enzyme activities as well as expression of CYP1A1 and CYP1B1 isoforms, while enhancing phase II enzyme activities in the buccal pouch and liver. Our results provide a mechanistic basis for the chemopreventive potential of black tea polyphenols. Furthermore, the greater efficacy of BTF-35 in chemoprevention of HBP carcinomas via inhibition of oxidative DNA damage and modulation of xenobiotic-metabolizing enzymes may have a major impact in human oral cancer prevention.

In vitro antioxidative potential of lactoferrin and black tea polyphenols and protective effects in vivo on carcinogen activation, DNA damage, proliferation, invasion, and angiogenesis during experimental oral carcinogenesis.

The present study was designed to evaluate the in vitro antioxidant potential of bovine lactoferrin (bLF) and black tea polyphenols [Polyphenon-B (P-B)] as well as in vivo inhibitory effects on the development of 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinomas. Antioxidant activity was screened using a panel of assays including 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2'-azinobis-(3-ethyl-benzothiazoline-6-sulfonic acid) (ABTS), hydroxyl radical anion (OH*), superoxide anion (O2*-), and nitric oxide (NO) radical scavenging assays as well as assay for reducing power. The chemopreventive potential of bLF and P-B was assessed in the HBP model based on the modulatory effects on DMBA-induced oxidative DNA damage as well as the expression of proteins associated with carcinogen activation (CYP1A1, CYP1B1), cell proliferation [cyclin D1, proliferating cell nuclear antigen (PCNA), glutathione S-transferase pi (GST-P)], angiogenesis [vascular endothelial growth factor (VEGF), VEGF receptor 1 (VEGFR1)], and invasion and metastasis [matrix metalloproteinase-9 (MMP-9) and tissue inhibitors of MMP-2 (TIMP-2)]. Both bLF and P-B showed high radical scavenging activity and reductive potential. Although administration of bLF and P-B alone suppressed DMBA-induced HBP tumors, combined administration of bLF and P-B was more effective in inhibiting HBP carcinogenesis by inhibiting oxidative DNA damage, carcinogen activation, cell proliferation, invasion, and angiogenesis. Our study suggests that the antioxidative property of bLF and P-B may be responsible for chemoprevention of HBP carcinogenesis by modulating multiple molecular targets.

Aryl hydrocarbon (benzo[a]pyrene) hydroxylase in guinea pig lymphoid tissue.

Aryl hydrocarbon hydroxylase (AHH) activity was detected in a variety of guinea pig lymphoid tissues and was increased by intradermal or ip administration of 3-methylcholanthrene AHH levels were generally greater in spleen than in lymph node, bone marrow, or thymus. Similar AHH activities were observed in lymphoid tissues from Sewall-Wright inbred strain 2 and strain 13 guinea pigs. Greater AHH activities were associated with peritoneal and alveolar macrophages than with lymph nodes lymphocytes in the absence of mitogens.

Nonenzymatic reduction of benzo(a)pyrene diol-epoxides to trihydroxypentahydrobenzo(a)pyrenes by reduced nicotinamide adenine dinucleotide phosphate.

The diol-epoxide r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo(a)pyrene is a potent mutagen and possibly the ultimate carcinogenic form of benzo(a)pyrene. A (7/8,9)-trihydroxy-7,8,9,10,10-pentahydrobenzo(a)pyrene is formed from the diol-epoxide r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydroxybenzo(a)pyrene by reduction with reduced nicotinamide adenine dinucleotide phosphate. Its formation is linear with reduced nicotinamide adenine dinucleotide phosphate concentration and does not require the presence of enzyme. A (7,9/8)-trihydroxy-7,8,9,10,10-pentahydrobenzo(a)pyrene is similarly formed from the diol-epoxide r-7,t-8-dihydroxy-c-9,10-oxy-7,8,9,10-tetrahydrobenzo(a)pyrene by reduction with reduced nicotinamide adenine dinucleotide phosphate. The structures of the trihydroxypentahydrobenzo(a)pyrenes were established by their ultraviolet absorption and mass spectra and their reaction with potassium triacetylosmate.

Mutagenic activation of 2-amino-3-methylimidazo[4,5-f]quinoline by complementary DNA-expressed human liver P-450.

Eight forms of human liver microsomal P-450 were individually expressed in human hepatoma Hep G2 cells with a vaccinia virus cDNA expression system. Using the Ames test, each expressed P-450 was examined for its ability to activate to mutagenic products the compounds, 2-amino-3-methylimidazo[4,5-f]quinoline, 2-amino-3,4-dimethylimidazo[4,5-f]quinoline, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, and 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline, respectively. Three forms of human P-450 significantly activated 2-amino-3-methylimidazo[4,5-f]quinoline when the latter was at high substrate concentrations, but only a single form, P-450IA2, showed very high activation of all promutagens at lower substrate concentrations. Human IA2 had extraordinarily high affinity towards four promutagens tested and is likely the predominant P-450 enzyme responsible for their mutagenic activation in human liver.

High-pressure liquid chromatographic separation of 10 benzo(a)pyrene phenols and the identification of 1-phenol and 7-phenol as new metabolites.

The separation of ten isomeric benzo(a)pyrene phenols has been accomplished by the use of high-pressure liquid chromatography utilizing a newly developed recycling technique and new column and solvent systems. Using this new system and comparing the metabolites obtained with authentic standards, we have isolated 1-hydroxybenzo(a)pyrene and 7-hydroxybenzo(a)pyrene and identified them as metabolites formed by rat liver microsomes. In previously reported chromatography systems, the new metabolites migrated with another metabolite, 3-hydroxybenzo(a)pyrene.

Quantification of CYP2B7, CYP4B1, and CYPOR messenger RNAs in normal human lung and lung tumors.

Cytochrome P450 (CYP) enzymes expressed in human lung can metabolize a variety of xenobiotics, drugs, and endogenous compounds. Metabolism of these substrates may lead to their detoxification or activation and may affect the homeostasis of the lung, its susceptibility to disease, response to therapy, and clinical prognosis. We analyzed the expression of CYP2B7, CYP4B1, and NADPH-cytochrome P450 oxidoreductase (OR) mRNAs in normal lung controls, normal lung from lung cancer patients, and lung tumors using the sensitive technique of RNase protection. The mRNAs of CYP2B7, CYP4B1, and OR were detected in all the normal and a majority of neoplastic tissues. The three mRNAs were quantified and found at an average ratio of 0.89, 4.03, and 0.88% relative to actin mRNA in normal lung, respectively. There was no correlation between the levels of expression of the three mRNAs and the histological diagnosis of tumors. The amounts of each of the three mRNAs varied considerably between patients, but analysis of frequency distribution of the levels of CYP2B7 and CYP4B1 mRNAs did not present evidence for genetic polymorphism as a possible source of the observed interindividual variability. Levels of expression of the two P450 mRNAs were reduced (2.3- and 2.4-fold) in the neoplasms compared to normal lung. The level of OR mRNA expression was uniform with no significant differences between normal and neoplastic tissues, and its interindividual variability was the lowest amongst the three mRNAs studied. All mRNAs had increased interindividual variability in neoplastic tissues. Analysis of the patients' smoking histories and the level of CYP2B7, CYP4B1, and OR mRNAs revealed no evidence for their induction by compounds present in cigarette smoke. This study identifies and characterizes lung and lung tumor mRNAs encoding enzymes that may participate in the metabolism of xenobiotics in humans.