Induction of cytotoxicity and mutagenesis is facilitated by fatty acid hydroperoxidase activity in Chinese hamster lung fibroblasts (V79 cells)

The metabolic activation of benzo[a]pyrene and 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene was studied in V79 Chinese hamster fibroblasts after supplementations with arachidonic acid or treatments with linoleic acid hydroperoxide. The extent of metabolic activation was estimated using cytotoxicity and mutagenesis as endpoints. Pretreatment of cells with arachidonic acid for 24 h resulted in significant elevations in the content of this fatty acid in cell phospholipids and increased prostaglandin synthesis. Arachidonic acid and linoleic acid hydroperoxide facilitated 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene cytotoxicity and mutagenesis, and to a lesser extent increased the cytotoxicity and mutagenicity of benzo[a]pyrene. No other compounds tested were mutagenic under these conditions, however, linoleic acid hydroperoxide markedly increased their cytotoxicity. Arachidonic acid-facilitated toxicity and mutagenesis was inhibited by indomethacin, whereas no inhibition was seen when linoleic acid hydroperoxide was used. Nordihyroquairaretic acid abolished the cytotoxicity and mutagenesis facilitated by arachidonic acid and linoleic acid hydroperoxide. Our findings demonstrate that induction of cytotoxicity and mutagenesis following treatment of V79 cells with carcinogens may be limited by low levels of arachidonic acid in these cells. A peroxidatic mechanism is proposed, with limited substrate specificity, for the metabolic activation of chemicals in V79 cells.     

Prostaglandin synthetase dependent activation of 7,8-dihydro-7,8-dihydroxy-geno (a) pyrene to mutagenic derivativies.

The combination of arachidonic acid and a Tween 20 solubilized enzyme preparation from sheep seminal vesicles converts 7,8-dihydro-7,8-dihydroxy-benzo(a)pyrene to derivatives strongly mutagenic to $\text{Salmonella typhimurium}$ tester strain TA 98. Under similar conditions no activation of benzo(a)pyrene, 4,5-dihydro-4,5-dihydroxy-benzo(a)pyrene, or 9,10-dihydro-9,10-dihydroxy-benzo(a)pyrene occurs. The activation of 7,8-dihydro-7,8-dihydroxy-benzo(a)-pyrene is markedly reduced by the omission of arachidonic acid and is inhibited by the prostaglandin synthetase inhibitor indomethacin. 100 μM butylated hydroxyanisole is also an effective inhibitor. This is the first report of the metabolic activation of 7,8-dihydro-7,8-dihydroxy-benzo(a)pyrene by an enzyme system distinct from the mixed-function oxidases.     

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.     

Prostaglandins and their precursors can modify genetic damage-induced by gamma-radiation and benzo(a)pyrene

Experiments were performed to study the effect of various prostaglandins (PGs) and their precursors, gamma-linolenic acid (GLA) and arachidonic acid (AA) on gamma-radiation and benzo (a) pyrene (BP)-induced genetic damage to the bone marrow cells of mice, using the sensitive micronucleus (MN) test. Thromboxane B2 prostaglandin E1 and GLA completely prevented BP-induced and reduced to a great degree radiation-induced genetic damage, where as PGE2, PGF2 alpha and AA were without any effect. Since GLA and AA are widely distributed in the cell membranes, and as PGs can be formed virtually in response to any type of stimulus, it is likely that GLA and PGE1 may function as endogenous anti-mutagenic chemicals.     

Prostaglandins and their precursors can modify genetic damage-induced by gamma-radiation and benzo (a) pyrene

Experiments were performed to study the effect of various prostaglandins (PGs) and their precursors, gamma-linolenic acid (GLA) and arachidonic acid (AA) on gamma-radiation and benzo (a) pyrene (BP) -induced genetic damage to the bone marrow cells of mice, using the sensitive micronucleus (MN) test. Thromboxane B2 prostaglandin E1 and GLA completely prevented BP-induced and reduced to a great degree radiation-induced genetic damage. where as PGE2, PGF2alpha and AA were without any effect. Since GLA and AA are widely distributed in the cell membranes, and as PGs can be formed virtually in response to any type of stimulus., it is likely that GLA and PGE1 may function as endogenous anti-mutagenic chemicals.     

Effects of phorbol-12,13-diesters on prostaglandin production and phospholipase activity in canine kidney (MDCK) cells

Two tumor promoting phorbol-12,13-diesters, 12-0-tetradecanoyl-phorbol-13-acetate and phorbol-12,13-didecanoate, at concentrations of 10^?9 to 10^?10 M, stimulated prostaglandin production by dog kidney (MDCK) cells cultured in serum-supplemented medium. The non-tumor producing phorbol diester, 4α-phorbol-12,13-didecanoate, at a concentration of 10^?7 M, had no effect. The two biologically active phorbol diesters, but not the non-tumor promoting analog, stimulated deacylation of the cellular phospholipids of MDCK cells radioactively labelled with [³H]arachidonic acid. Most of the arachidonic acid was converted into prostaglandins.     

Inhibition of binding of benzo(a)pyrene to DNA by 7,8-benzoflavone in organ cultures of human bronchus

Levels of binding of exogeneously added benzo(a)pyrene to DNA in organ culture were examined in nine specimens of normal human bronchus obtained by bronchoscopy of tumor patients. The specimens were divided into two portions and incubated with [3H]benzo(a)pyrene in the absence or presence of 2 microM 7,8-benzoflavone for 24 h. 7,8-benzoflavone inhibited [3H]benzo(a)pyrene-DNA binding from 24 to 60%. Generally, the levels of binding of [3H]benzo(a)pyrene to DNA in the presence of 7,8-benzoflavone were relatively low and closely bracketed the mean value for the nine specimens. This appears to indicate that there are at least two components to [3H]benzo(a)pyrene-DNA binding catalyzed by the human bronchus. One component is quite variable in activity and is sensitive to inhibition by 7,8-benzoflavone, and may be an environmentally induced activity. The second component is lower in activity, and may be a constitutive portion of the mixed-function oxidase.     

UDP-glucuronosyl transferase and the conjugation of benzo(a)pyrene metabolites to DNA.

Addition of UDP-glucuronic acid to microsomal incubations containing benzo(a)pyrene caused a dose-dependent conjugation of principally quinone and phenol metabolites. Total benzo(a)pyrene oxidation was also stimulated with a maximum increase at 2 nM UDPGA. In the presence of calf thymus DNA, UDPGA caused a 2.7-fold increase in benzo(a)pyrene diol-oxide modification of DNA, as analyzed by Sephadex LH-20 chromatography. Maximum DNA modification by diol-oxides occurred at a UDPGA concentration which gave the highest level of free benzo(a)pyrene 7,8-dihydrodiol; likewise, the amount of DNA adduct derived from benzo(a)pyrene phenols declined in parallel with levels of free phenol metabolites. The UDPGA-induced increase in benzo(a)pyrene oxidation and concomitant increase in diol-oxide modification of DNA is consistent with removal of product inhibition by glucuronide conjugation of an inhibitory benzo(a)pyrene metabolite.     

The effect of norharman on the metabolism of benzo[alpha]pyrene by rat-liver microsomes in vitro in relation to its enhancement of the mutagenicity of benzo[alpha]pyrene.

The effect of norharman on the metabolism of benzo[alpha]pyrene by rat-liver microsomes was studied. Separation of the metabolites into hydrophilic and hydrophobic fractions showed that norharman inhibited the conversion of hydrophobic metabolites to hydrophilic ones. Analysis of the hydrophobic metabolites by high-pressure liquid chromatography showed that norharman also inhibited the disappearance of benzo[alpha]pyrene itself. However, large amounts of hydrophobic metabolites, such as phenol, quinones and diols, were formed in the presence of norharman, and formation of the strong mutagen 7,8-dihydroxybenzo[alpha]pyrene was increased 10-fold by norharman. The increase in formation of this compound may be one of the chief reasons why norharman enhances the mutagenicity of benzo[alpha]pyrene on Salmonella typhimurium.     

Differences in the repair of DNA strand breaks induced by 9-hydroxy-benzo(a)pyrene and trans-7,8-dihydro-7,8-dihydroxy-benzo(a)pyrene in cultured human fibroblasts

Two benzo(a)pyrene metabolites were found to induce DNA strand breaks in cultured human fibroblasts. DNA strand breaks induced by the non- or weakly carcinogenic 9-hydroxy-benzo(a)pyrene were repaired within two hours, while those induced by the strongly carcinogenic trans-7,8-dihydro-7,8-dihydroxy-benzo(a)pyrene were repaired at a much slower rate.     

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.     

Epidermal growth factor stimulates prostaglandin biosynthesis by canine kidney (MDCK) cells.

Serum and/or arachidonic acid stimulated prostaglandin production by dog kidney (MDCK) cells. Epidermal growth factor (EGF) at concentrations of 10^?9 to 10^?10 M stimulated the biosynthesis of prostaglandins by MDCK cells but not that by human fibroblasts (D-550), mouse fibroblasts (3T3), transformed mouse fibroblasts (MC5-5), and rabbit aorta endothelial cells (CLO). EGF also stimulated the release of radioactivity from MDCK cells radioactively labelled with [³H]arachidonic acid.     

Effect of purified epoxide hydrolase on metabolic activa-tion and binding of benzo(a)pyrene to exogenous DNA. Shift of the activation pathway

The effect of purified epoxide hydrolase (E.C. 3.3.2.3) on the binding of benzo(a)pyrene metabolites 9-hydroxybenzo(a)pyrene and 7,8-dihydro-7,8-dihydroxybenzo(a)pyrene to DNA catalyzed by cytochrome P 448 from liver microsomes of methylcholanthrene pretreated rats has been investigated. The total binding and the major binding species derived from 9-hydroxybenzo(a)pyrene were strongly inhibited by the presence of purified epoxide hydrolase and the species derived from 7,8-dihydro-7,8-dihydroxybenzo(a)pyrene was slightly increased. By modifying the balance between cytochrome P 448 and epoxide hydrolase it is possible to shift quantitatively the binding of these two main reactive intermediates to DNA.     

Prostaglandin synthesis by isolated cells of rat uterus: production rates, and effects of indomethacin and histamine

Luminal epithelial and residual cells (mainly of the endometrial stromal tissue) of proestrous rat uteri have been isolated and cultured in defined medium. The prostaglandins produced during a short-term incubation (2 h) in the presence of 10 microM arachidonic acid (to optimize PG production) were determined by direct assay of the culture medium. For the epithelial cells, PGF2 alpha was produced in greatest amounts, followed by 6-keto PGF1 alpha and PGE, while low levels were synthesized by the residual cells. The synthesis of PGF2 alpha by the epithelial cells was inhibited by incorporating indomethacin into the medium and an IC50 value of 2.3 microM was obtained. Incubations performed with histamine in the absence of exogenous arachidonic acid indicated that the pathways for the production of individual prostaglandins were followed to different relative extents, with the production of 6-keto PGF1 alpha being enhanced for both groups of cells when compared to incubations with arachidonic acid.     

Evidence of protein kinase C involvement in phorbol diester-stimulated arachidonic acid release and prostaglandin synthesis

Many stimulators of prostaglandin production are thought to activate the Ca2+- and phospholipid-dependent protein kinase first described by Nishizuka and his colleagues (Takai, Y., Kishimoto, A., Iwasa, Y., Kawahara, Y., Mori, T., and Nishizuka, Y. (1979) J. Biol. Chem. 254, 3692-3695. In this paper we report evidence that the activation of protein kinase C caused by 12-O-tetradecanoylphorbol-13-acetate (TPA) is involved in the increased prostaglandin production induced by 12-O-tetradecanoylphorbol-13-acetate in Madin-Darby canine kidney (MDCK) cells. We have shown that TPA activates protein kinase C in MDCK cells with similar dose response curve as observed for TPA induction of arachidonic acid release in MDCK cells. Activation of protein kinase C was associated with increased phosphorylation of proteins of 40,000 and 48,000 daltons. We used two compounds (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OMe) and 1-(5-isoquinolinesulfonyl)piperazine) known to inhibit protein kinase C by different mechanisms to further examine if activation of protein kinase C was involved in the increased synthesis of prostaglandins in TPA-treated MDCK cells. We found that both compounds inhibited protein kinase C partially purified from MDCK cells and that ET-18-OMe inhibited the phosphorylation of proteins by protein kinase C in the intact cells. Addition of either compound during or after TPA treatment decreased both release of arachidonic acid from phospholipids and prostaglandin synthesis. Release of [3H]arachidonic acid from phosphatidylethanolamine in TPA-treated cells was blocked by ET-18-OMe or 1-(5-isoquinolinesulfonyl)piperazine addition. However, arachidonic acid release stimulated by A23187 is not blocked by Et-18-OMe. When assayed in vitro, treatment of cells with Et-18-OMe did not prevent the enhanced conversion of arachidonic acid into prostaglandins induced by pretreatment of cells with TPA. Our results suggest that the stimulation of phospholipase A2 activity by TPA occurs via activation of protein kinase C by TPA.     

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.     

Magnesium ions affect the quantitative but not the qualitative microsome mediated binding of benzo[alpha]-pyrene to DNA

Five distinct hydrocarbon-deoxyribonucleoside adducts are separated by high pressure liquid chromatography after reaction of benzo[alpha]pyrene with calf thymus DNA in the presence of liver microsomes from 3-methylcholanthrene treated rats. The two major adducts co-chromatography with deoxyribonucleoside adducts obtained after hydrolysis of calf thymus DNA previously reacted with liver microsomal metabolically activated 9-hydroxy-benzo[alpha]pyrene or trans-7,8-dihydro-7,8-dihydroxybenzo[alpha]pyrene. High magnesium ion concentrations in the microsomal incubations cause a significant decrease in the covalent binding of the hydrocarbon to DNA but do not affect the qualitative distribution of the individual benzo[alpha]pyrene-deoxyribonucleoside adducts.     

Postreplication repair of DNA in human fibroblasts after UV irradiation or treatment with metabolites of benzo(a)pyrene

We have examined the ability of normal fibroblasts and of excision-deficient xeroderma pigmentosum (XP) and XP variant fibroblasts to perform postreplication DNA repair after increasing doses of either ultraviolet (UV) irradiation or mutagenic benzo(a)pyrene derivatives. XP cells defective in the excision of both UV-induced pyrimidine dimers and guanine adducts induced by treatment with the 7,8-diol-9,10-epoxides of benzo(a)pyrene were partially defective in their ability to synthesize high molecular weight DNA after the induction of both classes of DNA lesions. This defect was more marked in XP variant cells, despite their ability to remove by excision repair both pyrimidine dimers and the diol epoxide-induced lesions to the same degree as observed in normal cells. The benzo(a)pyrene 9,10-oxide had no effect in any of the 3 cell lines. The response of the excision and postreplication DNA repair mechanisms operating in human fibroblasts treated with benzo(a)pyrene 7,8-diol-9,10-epoxides, therefore, appears to resemble closely that seen after the induction of pyrimidine dimers by UV irradiation.     

The conversion of benzo(alpha)pyrene 4,5-oxide into 4-hydroxybenzo(alpha)pyrene in the presence of polyriboguanylic acid.

Incubation of benzo[alpha] pyrene 4,5-oxide with poly(G) in neutral aqueous ethanol resulted in the formation of covalent adducts and in the production of free 4-hydroxybenzo[alpha]pyrene. This phenol, which was identified by its UV spectral properties and by its chromatographic characteristics, was also formed but at a much slower rate when the epoxide was incubated with DNA or with GMP. Phenol formation was not detected when benzo[alpha]-pyrene 4,5-oxide was incubated for prolonged periods in the presence of poly(A), poly(C) or poly(U) or in the absence of nucleic acid. Formation of 4-hydroxybenzo[alpha] pyrene from the epoxide in the presence of poly(G) was not accompanied by detectable base modifications or by breakage of phosphodiester linkages.     

Effect of various chemicals on the metabolism of benzo(a)pyrene by cultured rat colon

The effect of various co- and anti-carcinogens of colon carcinogenesis on the metabolism of benzo(a)pyrene (BP) in cultured rat colon is reported. Rat colon enzymatically converted BP into metabolites which bind to cellular macromolecules i.e., DNA and protein. Activity of aryl hydrocarbon hydroxylase (AHH) activity and binding levels of BP to macromolecules were higher in the descending colon when compared to other segments. The major metabolites of BP, extractable with ethylacetate, were quinones, tetrols, 7,8-diol and a peak containing 9,10-dihydroxy-9,10-dihydrobenzo(a)pyrene and 7,8,9-trihydroxy-7,8-dihydrobenzo(a)pyrene. The binding levels of BP to DNA and protein in the explant was lowered by co-incubation with 7,8-benzoflavone (7,8-BF) (3.6 and 18.0 μM), a known inhibitor of AHH, and with disulfiram (100 μM), an anti-oxidant. The absence of vitamin A in the media also resulted in a lower level of BP binding to DNA and protein and in lower activity of AHH. Pretreatment with known inducers of AHH such as phenobarbital (PB) or benz(a)anthracene (BA), did not have any significant effect on the binding levels of BP to DNA or on the AHH activity. of the bile acids investigated only taurodeoxycholic acid significantly increased the binding level of BP to DNA.