Ah receptor-independent inhibition of gap junction intercellular communications in hepatoma 27 cell culture by polycyclic aromatic hydrocarbons

Gap junction intercellular communication (GJIC) is a system that regulates tissue homeostasis. It is generally accepted that GJIC down-regulation is linked to carcinogen tumor-promoting properties. The effect of carcinogenic polycyclic aromatic hydrocarbons on GJIC in cultured hepatoma 27 cells deficient in cytochrome P450 and Ah receptor has been investigated. It was found that, out of six compounds examined, only benz/a/pyrene and 3-methylcholanthrene were able to inhibit GJIC. It is concluded that in hepatoma cells there is unknown factor that interacts with some polycyclic aromatic hydrocarbons and inhibit GJIC. Application of benz/a/pyrene together with benz/e/pyrene (an agent with similar structure but without carcinogenic activity) showed that GJIC inhibition by benz/a/pyrene is at least a two-stage process.     

Effect of 7,8-benzoflavone on the incidence of skin tumors induced by polycyclic hydrocarbons]

Tumours were induced in CBA mice or (C57BL X CBA)F1 hybrids by the application of benz(a)pyrene (BP), 6-methylBP, 6-formylBP or 7,12-dimethylbenzanthracene (DMBA) to the skin. 7,8-benzoflavone (BF) used in combination with the mentioned agents decreased the carcinogenic action of methyl-substituted hydrocarbons--DMBA and 6-methylBP and had no effect on the carcinogenic action of BP and 6-formylBP. It is suggested that the inhibitory action of BF on carcinogenesis induced by methyl-substituted hydrocarbons was due to the inhibited oxidation of the methyl group.     

Effect of benz(a)pyrene and its non-carcinogenic analog 1,2-benzopyrene on the Na, K-ATPase activity of homogenates of different mouse organs and rat heart membrane preparations

The carcinogenic agent benz(a)pyrene inhibits Na, K-ATPase activity of organ homogenates where it is capable of inducing tumors. It causes no significant changes in target organ homogenates.     

Growth of Various Bacteria on Polycyclic Aromatic Hydrocarbons and N-2–Fluorenylacetamide

Several heterotrophic bacteria grew in nutrient broth medium containing high concentrations (10⁻⁵ M) of the potent carcinogenic compounds, benz[a]pyrene, 3-methylcholanthrene or N-2–fluorenylacetamide. They were capable of metabolizing the polycyclic aromatic hydrocarbons to smaller molecules and utilized these compounds as the sole source of carbon and energy. Identification of the metabolites formed from benz[a]pyrene revealed that the major metabolite (3-hydroxybenz[a]pyrene) of mammalian systems did not accumulate in any of these cultures when the standard fluorometric assay was used. Benz[a]pyrene metabolism profiles with high-pressure liquid chromatography also exhibited no accumulation of any metabolites (hydroxy, quinone and diol derivatives).     

Effect of some carcinogenic and non-carcinogenic polycyclic aromatic hydrocarbons on gap junction intercellular communication in hepatoma cell cultures

One of the systems that regulate tissue homeostasis is gap junction intercellular communication (GJIC). It is accepted that the down-regulation of GJIC is linked to the tumor-promoting properties of carcinogens. In this study, the effect of some carcinogenic and non-carcinogenic polycyclic aromatic hydrocarbons (PAH) on GJIC was investigated. It was found that in hepatoma cell culture (Hep G2) carcinogenic PAH inhibited GJIC after 24 h exposure by 75-100% depending on the PAH structure. The inhibition effect on GJIC is reversible because removing the PAH by changing of culture medium restores the GJIC. The non-carcinogenic PAH do not significantly influence GJIC. alpha-Naphthoflavone, an inhibitor of PAH metabolism, has no effect on inhibition of GJIC by carcinogenic PAH. 2,3,7,8-Tetrachloro-p-dibenzodioxin, an aryl hydrocarbon (Ah) receptor ligand, inhibits GJIC by about 50% only after 48 h exposure. To clarify the role of formation of PAH metabolites and interaction with Ah receptor on inhibition of GJIC, we determined the effect of benzo/a/pyrene on hepatoma G27 cells in which neither mRNA of CYP1A1 nor Ah receptor was determined. As in Hep G2 cells, benzo/a/pyrene, unlike non-carcinogenic benzo/e/pyrene, inhibits GJIC. We conclude that in the studied hepatoma cells carcinogenic PAH inhibit GJIC directly (that is, not via their metabolites) and this effect is not associated with Ah receptor interaction.     

Detoxification of polycyclic aromatic hydrocarbons by fungi

Summary The polycyclic aromatic hydrocarbons (PAHs) are a group of hazardous environmental pollutants, many of which are acutely toxic, mutagenic, or carcinogenic. A diverse group of fungi, includingAspergillus ochraceus, Cunninghamella elegans, Phanerochaete chrysosporium, Saccharomyces cerevisiae, andSyncephalastrum racemosum, have the ability to oxidize PAHs. The PAHs anthracene, benz[a]anthracene, benzo[a]pyrene, fluoranthene, fluorene, naphthalene, phenanthrene, and pyrene, as well as several methyl-, nitro-, and fluoro-substituted PAHs, are metabolized by one or more of these fungi. Unsubstituted PAHs are oxidized initially to arene oxides,trans-dihydrodiols, phenols, quinones, and tetralones. Phenols andtrans-dihydrodiols may be further metabolized, and thus detoxified, by conjugation with sulfate, glucuronic acid, glucose, or xylose. Although dihydrodiol epoxides and other mutagenic and carcinogenic compounds have been detected as minor fungal metabolites of a few PAHs, most transformations performed by fungi reduce the mutagenicity and thus detoxify the PAHs.     

Formation of radical cations in a model for the metabolism of aromatic hydrocarbons

To test the hypothesis that electrophilic radical cations are the major ultimate electrophilic and carcinogenic forms of benz[a]anthracene (BA), dibenz[a,h]anthracene (DBA), and benzo[a]pyrene (BP), we have focused on a chemical model of metabolism which parallels and duplicates known or potential metabolites of some polycyclic hydrocarbons formed in cells. Studies of this model system show that radical cations are hardly formed, if at all, in the case of BA or DBA but are definitely formed in the cases of the carcinogen BP as well as the non-carcinogenic hydrocarbons, pyrene and perylene. We conclude that the carcinogenicities of BA, DBA, BP, pyrene, and perylene are independent of one-electron oxidation to radical cation intermediates.     

Potential Health Risk of Reused Creosote-Treated Old Railway Ties at Recreational Sites in Korea

Old creosote-treated railway ties reused at recreational sites in Korea are potential hazards, due to the presence of harmful substances in creosote, such as polycyclic aromatic hydrocarbons (PAHs). In such sites, PAHs in ties can be leached or emitted, and human exposure might then occur. In this study, the concentrations of 16 PAHs in soil, air, and tie surfaces in old creosote-treated railway ties reused in recreational sites were investigated, and the potential health risk of the ties was evaluated through two exposure scenarios: a recreational scenario (ingestion of and dermal contact with soil and inhalation of soil particles) and a playground scenario (ingestion after contact and dermal contact with ties). For the recreational scenario, the health risks of PAHs were safe; however, for the playground scenario, the carcinogenic risk of ingestion after contact, and dermal contact with benz(a)anthracene and benzo(a)pyrene on the tie surfaces, exceeded the acceptable risk level (10^–6). For the carcinogenic risks of ingestion after contact with ties, the probabilities of cancer development were 8 and 5 in one million people for benz(a)anthracene and benzo(a)pyrene, respectively. The carcinogenic risks for dermal contact with ties were 2.4 × 10^–6 and 1.4 × 10^–6 for benz(a)anthracene and benzo(a)pyrene, respectively.     

Regiospecific oxidation of polycyclic aromatic dihydrodiols by rat liver dihydrodiol dehydrogenase

Rat liver dihydrodiol dehydrogenase (DDH, E.C. 1.3.1.20) has recently been shown to oxidize the highly carcinogenic benz[a]anthracene-3,4- dihydrodiol in an NADP(+)-dependent reaction to its corresponding catechol. The present study is a systematic investigation of the substrate specificity of the purified enzyme towards synthetic trans-dihydrodiol metabolites of phenanthrene, benz[a]anthracene, chrysene, dibenz[a, h]anthracene and benzo[a]pyrene. DDH exhibited a remarkable regiospecificity of enzymatic catalysis with regard to the site of the dihydrodiol moiety of the parent hydrocarbon. M-region- and, with lower efficiency, bay-region dihydrodiols were found to be good substrates of the enzyme with maximal velocities between 20-80 nmol/min per mg enzyme and Km values in the micromolar range. K-region dihydrodiols were not accepted as substrates. Dihydrodiols situated at the terminal ring of an anthracene-type structure such as benz[a]anthracene-8,9-dihydrodiol as well as the corresponding dihydrodiol epoxides were also not oxidized by DDH at measurable rates. The results provide evidence for a detoxifying role of DDH in the metabolism of the chemical carcinogens benz[a]anthracene, chrysene and dibenz[a, h]anthracene.     

Mutagenicity of non-K-region diols and diol-epoxides of benz(a)anthracene and benzo(a)pyrene in S. typhimurium TA 100

When incubated with a 9,000 x g rat-liver supernatant, benzo(a)pyrene 7,8-diol and benz(a)anthracene 8,9-diol were more active than the parent hydrocarbons in inducing his⁺ revertant colonies of S. typhimurium TA 100. Benzo(a) pyrene 9,10-diol was less active than benzo(a)pyrene; the K-region diols, benz(a)anthracene 5,6-diol and benzo(a)pyrene 4,5-diol, were inactive. None of the diols was active when the cofactors for the microsomal mono-oxygenase were omitted. The diol-epoxides benzo(a)pyrene 7,8-diol 9,10-oxide, benz(a)anthracene 8,9-diol 10,11-oxide and 7-methylbenz(a)anthracene 8,9-diol 10,11-oxide and the K-region epoxides, benzo(a)pyrene 4,5-oxide and benz(a)anthracene 5,6-oxide, were mutagenic without further metabolism.     

Mutagenic and carcinogenic actions of some polycyclic aromatic hydrocarbons]

The carcinogenic and mutagenic actions of benz(a)pyrene (BP) and its derivatives 6-methyl-, 6-formyl-, 6-chloro-, 6-hydroxy-, 6-acetoxy-, 6-methoxy- and 4(5)-methoxy-BP were studied in mice and bacteria Salmonella typhimurium TA-98 and TA-100, respectively. The potent carcinogenic agents BP, 6-methyl-, 6-formyl-BP proved also mutagenic in bacteria of both strains. Weak carcinogenic compounds (6-chloro-, 6-methoxy-, 4(5)-methoxy-BP) and noncarcinogenic ones (6-acetoxy-, 6-hydroxy-BP) either turned out nonmutagenic or mutagenic in one of two bacterial strains tested. The differences in carcinogenic and mutagenic actions of the substances under study are not relative to the rate of their oxidation in the enzymatic system.     

Profiles of polycyclic aromatic hydrocarbon metabolites after treatment with various inducers of microsomal rat liver monoxygenases (author's transl)

The microsomal oxidation of 12 frequently occurring environmental polycyclic aromatic hydrocarbons after incubation with rat-liver microsomes has been studied and their metabolites characterized by means of gas-liquid chromatography/mass spectrometry. The method enables the detection and characterisation of phenols, diols, triols, and tetrols as trimethylsilyl ethers beside the original hydrocarbons. Moreover, the induction properties of some carcinogenic and non-carcinogenic hydrocarbons (benz[a]anthracene, pyrene, chrysene, benzo[a]-pyrene, benzo[e]pyrene, benzo[b]fluoranthene, benzo[j]fluoranthene, benzo[k]fluoranthene) have been studied. Except pyrene and benzo[e]pyrene, all compounds investigated significant but different induction factors. The relevance of the induction for an estimation of the biological effect of environmental polycyclic aromatic hydrocarbons is discussed.     

Rate of microbial transformation of polycyclic aromatic hydrocarbons: a chromatographic quantification procedure.

A chromatographic procedure has been developed for isolating and quantifying microbial transformation products of 14C-labeled polycyclic aromatic hydrocarbons. Transformation rates of naphthalene, anthracene, benz(a)anthracene, and benz(a)pyrene by a mixed bacterial population have been measured. With this procedure, extremely slow or incomplete transformations may be quantified that would not be detectable by previously used techniques.     

New approach to synthesis of peptide immunomimetic of benzo[a]pyrene

A novel approach to discovery of the peptide with an internal immunological image of carcinogen is suggested in this work. The hybridomas producing monoclonal antibodies (mAb) B2 against benzo[a]pyrene and benz[a]antracene were prepared. Polyclonal Ab against benzo[a]pyrene (Bp) and benz[a]antracene (Ba), antracene, chrysene, and pyrene were also prepared. We identified the related peptide-presenting phage specificity binding to mAT B2 and polyclonal Ab against Bp from 12 large random peptide libraries using phage display method. ICR mice were immunized with specific binding of positive phage clone for studying its immunogenicity. The Bp antibodies were found in the blood serum. All positive phage clones carried the sequence. Thus, the unique peptide with an internal immunological image of Bp may be the new candidate for anticarcinogenic vaccine.     

Diethylstilbestrol potentiates and testosterone antagonizes the action of 3-methylcholanthrene on benzo(a) pyrene metabolism in hep G2 cells

We have used the human hepatoma cell line, Hep G2, to examine the ability of hormones and xenobiotics to modulate the hepatic induction of benzo(a)pyrene hydroxylase and epoxide hydrolase. Hep G2 cells were cultured in Eagle's Minimum Essential Medium supplemented with 10% fetal calf serum. 3-Methylcholanthrene, diethylstilbestrol, testosterone propionate, and combinations of 3-meth-ylcholanthrene, and each of the hormones were added directly to the culture media. We subsequently studied the metabolism of benzo(a)pyrene using cell lysates of the Hep G2 cells. Metabolites were quantitated by high-performance liquid chromatography (HPLC) using fluorodetection. Exposure to 3-methyl-cholanthrene alone resulted in an eightfold increase in total benzo(a)pyrene metabolites with a change of the predominant metabolite from the 3-hydroxy-benzo(a)pyrene to the carcinogenic pathway of the benzo(a)pyrene-7,8-diol. Diethylstilbestrol and testosterone propionate resulted in small, but significant, decreases in metabolism of benzo(a)pyrene. When exposed in combination with 3-methyl-cholanthrene, testosterone propionate antagonized and diethylstilbestrol potentiated the metabolism of benzo(a)pyrene. 3-Methylcholanthrene, diethylstilbestrol, and combinations of 3-methylcholanthrene and diethylstilbestrol or testosterone propionate resulted in increased epoxide hydrolase activity as compared to controls. These results, carried out in a human hepatoma cell line, lend support to a concern for potentiated toxicity and carcinogenicity following exposure to complex chemical mixtures.     

Diethylstilbestrol potentiates and testosterone antagonizes the action of 3-methylcholanthrene on benzo(a)pyrene metabolism in Hep G2 cells

We have used the human hepatoma cell line, Hep G2, to examine the ability of hormones and xenobiotics to modulate the hepatic induction of benzo(a)pyrene hydroxylase and epoxide hydrolase. Hep G2 cells were cultured in Eagle's Minimum Essential Medium supplemented with 10% fetal calf serum. 3-Methylcholanthrene, diethylstilbestrol, testosterone propionate, and combinations of 3-methylcholanthrene, and each of the hormones were added directly to the culture media. We subsequently studied the metabolism of benzo(a)pyrene using cell lysates of the Hep G2 cells. Metabolites were quantitated by high-performance liquid chromatography (HPLC) using fluorodetection. Exposure to 3-methylcholanthrene alone resulted in an eightfold increase in total benzo(a)pyrene metabolites with a change of the predominant metabolite from the 3-hydroxybenzo(a)pyrene to the carcinogenic pathway of the benzo(a)pyrene-7,8-diol. Diethylstilbestrol and testosterone propionate resulted in small, but significant, decreases in metabolism of benzo(a)pyrene. When exposed in combination with 3-methylcholanthrene, testosterone propionate antagonized and diethylstilbestrol potentiated the metabolism of benzo(a)pyrene. 3-Methylcholanthrene, diethylstilbestrol, and combinations of 3-methylcholanthrene and diethylstilbestrol or testosterone propionate resulted in increased epoxide hydrolase activity as compared to controls. These results, carried out in a human hepatoma cell line, lend support to a concern for potentiated toxicity and carcinogenicity following exposure to complex chemical mixtures.     

Induction of cytochrome P-450 forms in liver microsomes of rats in the early neonatal period after administration of phenobarbital and 3-methylcholanthrene

The activity of cytochrome P-450 dependent monooxygenase system from rat liver microsomes after induction by phenobarbital and 3-methylcholantrene in early neonatal period (3-16 days after birth) was studied. It was found that the total amount of cytochrome P-450 increases after injection of these inducers in neonatal rats of all age groups. In parallel, in the case of 3-methylcholantrene induction the benz(a)pyrene hydroxylase and 7-ethoxyresorufin deethylase activities increase; phenobarbital induction causes a rise in the benzphetamine-N-demethylase and benz(a)pyrene hydroxylase activities. Immunochemical analysis involving the use of antibodies specifically directed against cytochrome P-450 of adult rats revealed that the level of cytochrome P-450 in the case of 3-methylcholantrene induction increases from 5 to 50%, whereas that of cytochrome P-450 upon phenobarbital induction increases from 5 to 40% in liver microsomes of 3- and 16-day-old rats. The mode of inhibition of various substrates metabolism by antibodies in neonatal rat microsomes suggests that the 3-methylcholantrene-induced cytochrome P-448, like in adult rats, participates in the hydroxylation of benz(a)pyrene and O-deethylation of 7-etoxyresorufin. The participation of phenobarbital-induced cytochrome P-450 in the metabolism of benzphetamine and aldrin in neonatal rats is much lower than in the adult ones. The metabolism of benz(a)pyrene in phenobarbital-induced neonatal rat microsomes in all age groups is not inhibited by antibodies. The age-dependent differences in inhibition of metabolism and the increase in the benz(a)pyrene hydroxylase activity in phenobarbital-induced rats suggest that the spectrum of inducible forms of cytochrome P-450 in neonatal rats differ from that in adult animals.     

Benz[j]aceanthrylene: a novel polycyclic aromatic hydrocarbon with bacterial mutagenic activity

Initial studies on the mutagenicity and metabolism of a novel cyclopenta-PAH, benz[j]aceanthrylene, are reported in the Salmonella bacterial system. The spectrum of activity of benz[j]aceanthrylene over the 5 Ames tester strains is similar to that of benzo[a]pyrene, and the dose-response curves for strain TA98 are comparable. Like other biologically active PAH, benz[j]aceanthrylene is a frame-shift mutagen requiring metabolic activation. An interesting feature of the S9 dependence of activity is the low concentration (congruent to 10-fold smaller than for benzo[a]pyrene) at which optimal activity is observed. The 1,2-dihydro-1,2-diol (product of metabolism of the cyclopenta-ring) appears to be the predominant metabolite, and implicates the 1,2-oxide as the ultimate mutagenic species.