Metabolic activation of benzo[a]pyrene in human skin maintained in short-term organ culture

The metabolic activation of benzo[a]pyrene (BP) was examined in six samples of human skin after topical application of the hydrocarbon to the skin in short-term organ culture. The results show that all of the samples were capable of metabolizing BP to water-soluble products and to ether-soluble products that included the 4,5-, 7,8- and 9,10-dihydrodiols and a product which had chromatographic properties identical with those of authentic trans-11,12-dihydro-11,12-dihydroxybenzo[a]pyrene (BP-11,12-diol). The major BP-deoxyribonucleoside adduct detected in each skin sample appeared to be formed from the reaction of r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BP-7,8-diol 9,10-oxide) with deoxyguanosine residues in DNA.     

Metabolism and activation of benzo[a]pyrene by mouse and rat skin in short-term organ culture and in vivo

The metabolic activation of BP was examined in mouse and rat skin in vivo and in short-term organ culture. In mouse skin, larger quantities of ether- and water-soluble metabolites were formed and more BP became bound covalently to DNA and protein than in rat skin. Qualitative differences in the formation of dihydrodiol metabolites and of BP-deoxyribonucleoside adducts between mouse and rat skin were also observed. Organ culture techniques may not provide a true model of metabolic activation in vivo because it was found that the covalent binding of BP to DNA and protein was reduced in skin maintained in culture despite an accumulation of dihydrodiol and other ether-soluble metabolites. In addition, the proportions of the syn- and anti-isomers of BP-7,8-diol 9,10-oxide involved in the formation of adducts with deoxyguanosine differed between skin treated in organ culture and in vivo.     

Fate of juvenile hormone in mammalian cell culture.

The metabolism of juvenile hormone (JH) I has been examined in fetal mouse liver cells maintained in culture. Diffusion of the hormone into the cells appears to be passive. The hormone is metabolized essentially to organic-soluble metabolites (diol ester, diol acid and acid) by the action of epoxide hydrase and carboxylesterases. Conjugative reactions play a minor role, less than 3% of the hormone being excreted as conjugates (glucuronides, sulfates and mercapturic acid). About 0.8% of the cellular radioactivity is bound to macromolecules, mainly those of nuclear and mitochondrial origin. Metyrapone and SKF 525-A inhibit covalent binding of the hormone to cytoplasmic macromolecules, which suggests participation of the cytochrome P-450 system in covalent binding of the hormone.     

Effect of various substituents in the 6-position on the relative carcinogenic activity of a series of benzo[a]pyrene derivatives

To test the hypothesis that polycyclic aromatic hydrocarbons capable of being converted to a reactive ester of the mesohydroxymethyl metabolite would be carcinogenic, a series of 6-substituted derivatives of benzo[a]pyrene (B[a]P) were tested for carcinogenicity in Sprague-Dawley rats by subcutaneous injection of the compound in sesame oil on alternate days for 30 doses. At the 0.2-μmol dose level B[a]P, 6-acetoxymethyl(6-AcOCH₂)B[a]P, 6-hydroxymethyl(6-HOCH₂)B[a]P, 6-methyl(6-CH₃)B[a]P and 6-benzoyloxymethyl(6-BzOCH₂)B[a]P were nearly equipotent, 6-formyl(6-OCH)-and 6-chloromethyl(6-ClCH₂)B[a]P were less active, and 6-methoxymethyl (6-MeOCH₂)B[a]P was inactive. At lower doses the order of potency was estimated to be: 6-AcOCH₂- = 6-HOCH₂- = or > B[a]P > 6-CH₂- > 6-BzOCH₂- > 6-ClCH₂- > 6-OCH- > 6-BrCH₂B[a]P. Incubation of these compounds in the presence of cofactors or cofactors plus a microsomal preparation of rat subcutis indicated that enzymic activation was necessary for metabolism to highly polar products and for conversion of 6-AcOCH₂-, 6-BzOCH₂- and 6-OCHB[a]P to 6-HOCH₂B[a]P. The halomethyl compounds were converted to 6-HOCH₂B[a]P in the absence of enzyme by hydrolysis. 6-MeOCH₂B[a]P was unchanged in this system. These observations are consistent with the foregoing hypothesis with regard to derivatives of B[a]P and demonstrate that compounds of this series that are capable of conversion to the 6-HOCH₂-derivatives are carcinogenic.     

Heterocyclic polycyclic aromatic hydrocarbon carcinogenesis: 7H-dibenzo[c,g]carbazole metabolism by microsomal enzymes from mouse and rat liver

The metabolism of dibenzo[c,g]carbazole (DBC), was studied in vitro using microsomal fractions of mouse and rat liver from animals, which were treated with 3-methylcholanthrene (MC). The separation of extractable metabolites by high pressure liquid chromatography (HPLC) and thinlayer chromatography (TLC) as well as identification of most of them by nuclear magnetic resonance, mass spectrometry and comparison with synthetically obtained products are described. The microsomes of both species produced the same twelve compounds of which the following have been identified: five monohydroxylated derivatives (phenols), the product of further oxidation of one of them, and a dihydrodiol. The 5-OH-DBC (60% including its spontaneously-formed dimer) and the 3-OH-DBC (14%) are the main metabolites. Three minor metabolites cochromatographed with synthetically prepared 2-OH-DBC, 4-OH-DBC and 6-OH-DBC. The dihydrodiol detectable in small quantity (4–6%) was tentatively identified as 3,4-dihydroxy-3,4-dihydro-DBC by the sensitivity of its formation to very low concentrations of the inhibitor of microsomal epoxide hydrolase, 1,1,1-trichloropropene oxide, by its molecular ion and major fragment in mass spectrometry and by its dehydration product 3-OH-DBC. No other dihydrodiols were detected. The qualitative and quantitative effects of various modulators of metabolism (enzyme inhibitors, apparently homogeneous epoxide hydrolase, glutathione, supernatant fraction) were investigated. The results are discussed with respect to possible ultimate carcinogens.     

Binding of benzo[a]pyrene at the 1,3,6 positions to nucleic acids in vivo on mouse skin and in vitro with rat liver microsomes and nuclei

Loss of tritium from specific positions in [³H,¹⁴C] aromatic hydrocarbons can elucidate their binding site(s) to DNA and RNA and indicate the mechanism of activation. Studies of tritium loss from [6-³H,¹⁴C]benzo[a]pyrene(B[a]P), [1,3-³H,¹⁴C]B[a]P, [1,3,6-³H,¹⁴C]B[a]P, [6,7-³H,¹⁴C]B[a]P, and [7-³H,¹⁴C]B[a]P were conducted in vitro using liver nuclei and microsomes from 3-methylcholanthrene-induced Sprague-Dawley rats and in vivo on the skin of Charles River CD-1 mice. The relative loss of tritium from $\text{[}{}^3\text{H}\text{,}{}^{14}\text{C}\text{]}\text{B}\text{[a]}\text{P}$ was measured after binding to skin DNA and RNA, to nuclear DNA, and to native and denatured calf thymus and rat liver DNA's and poly(G) by microsomal activation. In skin, nuclei, and microsomes plus native DNA, virtually all B[a]P binding occurred at positions 1,3 and 6; while with microsomes plus denatured DNA or poly(G), B[a]P showed no binding at the 6 position and a small amount at the 1 and 3 positions. In vivo and with nuclei, binding at the 6 position predominated. Little loss of tritium from the 7 position was seen; this was expected because binding at this position is not thought to occur. This confirms the interpretation of loss of tritium as an indication of binding at a given position. These results demonstrate that the use of microsomes to activate B[a]P is not a valid model system for delineating the in vivo mechanism of B[a]P activation, and support previous evidence for one-electron oxidation as the mechanism of activation of hydrocarbons in binding to nucleic acids.     

The metabolism of 7,12-dimethylbenz[a]anthracene and 7-hydroxymethyl-12-methylbenz[a]anthracene by rat liver and adrenal homogenates and by rat adrenocortical cells

The metabolism of 3H-labelled 7,12-dimethylbenz[a]anthracene (DMBA) and of 7-hydroxymethyl-12-methylbenz[a]anthracene (7-OHM-12-MBA) into solvent- and water-soluble and protein-bound derivatives has been examined in rat liver and adrenal homogenates and in rat adrenocortical cells in culture. Although the overall extents of metabolism of the substrates by the two types of homogenate were similar, there was twice as much binding to protein in incubations with the 7-hydroxymethyl derivative. Rat adrenal cells in culture metabolized DMBA more extensively than 7-OHM-12-MBA and converted much more of the parent hydrocarbon into water-soluble derivatives. Both hydrocarbons were metabolized to yield dihydrodiols that were separated and identified by high performance liquid chromatography (HPLC). The 8,9-dihydrodiol was the major dihydrodiol formed from DMBA but, with 7-OHM-12-MBA as substrate, metabolism was diverted to the 10,11- and 3,4-positions in adrenal and hepatic preparations respectively. The viability of rat adrenocortical cells in culture, as measured by trypan blue exclusion, did not appear to be affected by treatment with DMBA, 7-OHM-12-MBA, the sulphate ester of 7-OHM-12-MBA or by 3,4-dihydro-3,4-dihydroxy-7-hydroxymethyl-12-methylbenz[a]anthracene.     

Blood group A glycolipid (Ax) with globo-series structure which is specific for blood group A1 erythrocytes: one of the chemical bases for A1 and A2 distinction

A new blood group A-active glycolipid fraction, termed Ax, showing a chromatographic mobility between Aa and Ab was found in blood group A1 erythrocytes but not in A2 erythrocytes. Ax was identified by its conversion to "globo H" by alpha-N-acetylgalactosaminidase and by 1H-NMR spectroscopy as GalNAc alpha l----3[Fuc alpha l----2]Gal beta l----3GalNAc beta l----3Gal alpha l----4Gal beta l----4Glc beta l----lCer. Globo-H (Fuc alpha l----2Gal beta l----3GalNac beta l----3Gal alpha l----4Gal beta l----4Glc beta l----lCer) was found in blood group A, and O but not in A1 erythrocytes. Thus, one of the A1-specific determinants must be an A determinant carried by globo-series structure.     

Fungal oxidation of 3-methylcholanthrene: Formation of proximate carcinogenic metabolites of 3-methylcholanthrene

The filamentous fungus, Cunninghamella elegans, was found to metabolize the potent carcinogen, 3-methylcholanthrene (3-MC) to 1-hydroxy-3-MC, 2-hydroxy-3-MC, 1-keto-3-MC, 2-keto-3-MC and trans-9,10-dihydrodiols of 1-hydroxy-3-MC. In addition several unidentified derivatives of 3-MC were found. The metabolites formed were separated by high pressure liquid chromatography (HPLC) and identified by comparison of retention times, absorbance, fluorescence and mass spectra with those of synthetic standards. Incubation of (±)-1-hydroxy-3-MC and (±)-2-hydroxy-3-MC with cells of C. elegans indicated that 1-hydroxy-3-MC is metabolized to form diasteromerically related trans-9,10-dihydrodiols of 1-hydroxy-3-MC. Experiments with 3-[¹⁴C]MC showed that over a 48-h period, 8.7% of the hydrocarbon was oxidized to organic solvent-soluble metabolic products. Most of the metabolites were polar products, some of which co-chromatographed with trans-9,10-dihydrodiols of 1-hydroxy-3-MC. The results show that C. elegans has the ability to oxidize 3-MC to metabolites that have been implicated as proximate carcinogenic forms of 3-MC in higher organisms.     

The DNA binding of benzo[alpha]pyrene metabolites catalysed by rat lung microsomes in vitro and in isolated perfused rat lung.

When [³H]benzo[a]pyrene is incubated in vitro together with DNA, NADPH and rat lung microsomes, covalent binding of benzo[a]pyrene (BP) metabolites to DNA occurs. These metabolite-nucleoside complexes can be resolved into several distinct peaks by elution of a Sephadex LH-20 column with a water-methanol gradient. 3-Methylcholanthrene (MC) pretreatment of animals induces the total covalent binding in vitro several-fold and increases the amounts of at least five metabolite-nucleoside complexes associated with the 7,8-diol-9,10-epoxidcs, the 7,8-oxide or quinones oxygenated further, the 4,5-oxide and phenols oxygenated further. These increases correspond well with the increases in the production of both non-K-region and K-region metabolites of BP by lung microsomes, as determined by highpressure liquid chromatography (HPLC). On the other hand, when [³H]BP is metabolized in isolated perfused rat lung, only the peak representing the 7,8-diol-9,10-epoxide bound to nucleoside(s) is readily detectable and then only in lungs from MC-treated animals. The extent of binding of BP metabolites to lung DNA is very low, about 0.0004% of the total dose applied to the perfusion medium; more than 60% of this can be accounted for by the binding of the 7,8-diol-9,10-epoxides to nucleoside(s). It is suggested that the further metabolism leading to metabolites not available to covalent binding, (e.g. conjugation) of primary BP metabolites in the intact tissue is responsible for the differences in the metabolite-nucleoside patterns observed in vivo, as compared with microsomal metabolism in vitro.     

Release of mutagenic metabolites of benzo[a]pyrene from the perfused rat liver after inhibition of glucuronidation and sulfation by salicylamide

The role of glucuronide and sulfate conjugation in presystemic inactivation of benzo[a]pyrene (BP) metabolites was investigated with rat livers perfused with BP (12 mumol). Comparisons were made between metabolite profiles and mutagenicity of medium from perfusions with and without salicylamide, a selective inhibitor of glucuronide and sulfate conjugation. After 4 h perfusion in the presence of salicylamide, certain BP metabolites (diols, quinones, phenols, and metabolites more polar than BP-9,10-diol) were significantly increased at the expense of quinones and phenols in the glucuronide fraction. Mutagenicity of medium (detected by the Ames test, using tester strains TA98 and TA100) was low in perfusion without salicylamide. Mutagenicity detected with tester strain TA98 was significantly increased in perfusions with salicylamide. Involvement of glucuronidation in BP inactivation was also observed at the subcellular level; when cofactors of glucuronidation were added to liver homogenates along with the NADPH regenerating system in the Ames test, BP mutagenicity was markedly decreased. Both the activation of BP to mutagenic metabolites and the inactivation of BP metabolites by glucuronidation was much more pronounced with liver homogenates from 3-methylcholanthrene-treated rats than with those from phenobarbital-treated animals or untreated controls. The results suggest an important role for glucuronidation and sulfation in the inactivation and elimination of polycyclic aromatic hydrocarbons.     

The transformation of chlorophenols by lactoperoxidase

The lactoperoxidase-catalyzed transformations of penta-, 2,3,4,6,-tetra-, 2,4,6,-tri, 2,4,-di- and 4-monochlorophenol were followed spectrophotometrically. Apparent stoichiometries of chlorophenol: H₂O₂ ranged from 1:1 for the tri- and tetrachlorophenol at pH 7 to 5:2 for pentachlorophenol at pH 4. The initial velocity (ν₀) was only slightly influenced by changes in [H₂O₂] ? 5 μM. ν₀ responded to [chlorophenol] according to the empirical expression ν₀ = [lactoperoxidase]·(k₁[chlorphenol] + k₂[chlorophenol]²). The constant k₁ was found to be 5.8 · 10⁵, 1.8 · 10⁶, 1.9 · 10⁶ M^?1 · s^?1 for the protonated forms of penta-, tetra- and trichlorophenol, respectively, at pH 7. With the di- and monochlorophenol the solution soon became opaque, and the reaction ceased. The results show that more than one reaction occurs. Some comparisons were also made with horseradish peroxidase A and C. Cetyltrimethylammonium bromide prevented opaqueness, but was shown to be a substrate for lactoperoxidase. Assuming an average concentration of 0.1 μM for H₂O₂ and pentachlorophenol in man, the metabolic rate becomes 30 ng/h per g peroxidase-containing tissue, possibly with deposition of the products.     

Metabolism and nucleic acid binding of 7-fluoro-2-acetamidofluorene in rats: oxidative defluorination and apparent dissociation from hepatocarcinogenesis of 8-(N-arylamide) guanine adducts on DNA

The significance in hepatocarcinogenesis of various arylamine/amide adducts with nucleic acid was investigated by the use of comparison studies on several different parameters. Female Fischer and Sprague-Dawley rats are comparably sensitive to hepatocarcinogenesis by 2-acetamidofluorene (AAF), while male rats are more sensitive. 7-Fluoro-AAF is more carcinogenic in Sprague-Dawley rats than is AAF, but is strikingly so toward the liver of the female rat. Based on these observations, binding of both compounds to liver nucleic acids was determined for male and female Fischer rats at 1 and 3 days after a single injection of carcinogen, and in female Sprague-Dawley rats from 1 to 28 days after a single injection. As shown by others, no 8-(N-2-fluorenylacetamido)guanine adduct could be found in RNA or DNA of female Sprague-Dawley rats treated with AAF (nor was the corresponding 7-fluoro derivative detectable). These adducts were present, however, in comparable amounts in both male and female Fischer rats. The binding of 7-fluoro-AAF derivatives was higher than that of AAF derivatives in female Sprague-Dawley rats. Feeding of either AAF or 7-fluoro-AAF to Sprague-Dawley rats for 4 weeks before a single injection of [3H]7-fluoro-AAF resulted in reduction of the 8-(N-2-(7-fluoro)fluorenylacetamido)guanine adduct in males to undetectable levels in DNA and to 10% of control level in RNA. Non-acetylated adducts were increased in males, but decreased in females by AAF prefeeding; 7-fluoro-AAF prefeeding resulted in little change in adduct formation in females and in a major increase in non-acetylated adducts in males. AAF adducts disappeared from DNA more rapidly than did 7-fluoro-AAF adducts. Assay of the urinary metabolites from the animals in the prefeeding experiment showed that all compounds fed (including the non-hepatocarcinogens 4-acetamidobiphenyl and 2-acetamidophenanthrene) increased the proportion of N-hydroxy-7-fluoro-AAF among the metabolites. Defluorination of 7-fluoro-AAF to 7-hydroxy-AAF was also demonstrated and the ratio of 7-hydroxy-AAF to 5-hydroxy-7-fluoro-AAF was comparable to that observed for 7-hydroxy-AAF/5-hydroxy-AAF and AAF itself, suggesting that fluoro substitution does not increase activity by preventing detoxication.     

The major role of glutathione in the metabolism and excretion of N,N-dimethyl-4-aminoazobenzene in the rat

In the normal rat given a single dose of one mg N,N-dimethyl-4-aminoazobenzene (DAB) via the hepatic portal vein the following biliary metabolites reached their maximal rates of excretion in the sequence: 4'-sulphonyloxy-DAB, N-(glutathione-S-methylene)-4-aminoazobenzene (GSCH2AB), 4'-sulphonyloxy-N-methyl-4-aminoazobenzene (4'-sulphonyloxy-MAB) 4'-sulphonyloxy-GSCH2AB and MAB-4'-beta-glucuronide. The unusual and relatively unstable N-methylene glutathione conjugates were major metabolites accounting for up to 70% of the whole. It was shown that all the 4-aminoazobenzene (AB) and perhaps all of the 4'-sulphonyloxy-AB, which may be observed in bile, are artefacts due to decomposition of GSCH2AB and 4'-sulphonyloxy-GSCH2AB respectively and that biliary excretion of N-methyl oxidised products of MAB and 4'-hydroxy-MAB is dependent on their conversion to the GSH conjugates, GSCH2AB and 4'-hydroxy-GSCH2AB respectively. Sulphotransferase inhibition by pentachlorophenol caused a reduction in the excretion of all sulphate conjugates, but biliary excretion as a whole was not reduced significantly due to a compensatory increase in the excretion of MAB-4'-beta-glucuronide and the appearance of 4'-OH-GSCH2AB. Glutathione (GSH) depletion by diethylmaleate caused a reduction in biliary metabolites of DAB by lowering the levels of GSH conjugates. This was because the amount of N-methyl oxidation of MAB and 4'-hydroxy-MAB were proportional to the amount of GSH present. The fall in N-methyl oxidation was not compensated for by an increase in 4'-hydroxylation and was accompanied by a delay in the appearance of 4'-hydroxylated metabolites. The administration of potential precursors of 4'-sulphonyloxy-GSCH2AB establishes the sequence of reactions resulting in its formation to be 4'-hydroxylation, N-methyl oxidation, GSH conjugation and O-sulphation.     

Identification of N-methylprotoporphyrin IX in livers of untreated mice and mice treated with 3, 5-diethoxycarbonyl- 1, 4-dihydrocollidine: source of the methyl group

Administration of the porphyrogenic agent, 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) to mice, leads to the accumulation of N-methylprotoporphyrin IX in liver. This porphyrin is a potent inhibitor of ferrochelatase activity and accounts for the porphyria produced after DDC administration. The N-methylprotoporphyrin IX extracted from DDC-treated mice is primarily of one isomeric form, as shown by nuclear magnetic resonance spectroscopy. The methyl group of N-methylprotoporphyrin IX isolated from DDC-treated mice is derived mostly from the 4-methyl group of DDC. The transfer of this methyl group and its subsequent covalent attachment to protoporphyrin IX may be mediated by a form of hepatic microsomal cytochrome P-450. N-Methylprotoporphyrin IX is also found in livers of untreated mice at levels that are low but significant.     

Inhibition of phorbol ester-induced polyamine accumulation in mouse epidermis by anti-inflammatory steroid

Application of the tumor-promoting phorbol diester 12-$\text{O}$-tetradecanoylphorbol-13-acetate to mouse epidermis causes a large increase in the activity of ornithine decarboxylase and in polyamine accumulation. Concurrent application of fluocinolone acetonide, an anti-inflammatory steroid that dramatically inhibits tumor promotion, resulted in a dose-dependent decrease in the 12-$\text{O}$-tetradecanoylphorbol-13-acetate-stimulated ornithine decarboxylase activity and the subsequent rise in spermidine levels. Spermine and putrescine levels were not greatly affected by fluocinoline acetonide treatment except that maximal putrescine values occurred later in time. Doses of the glucocorticoid as low as 0.1 μg inhibited the 12-$\text{O}$-tetradecanoylphorbol-13-acetate-induced ornithine decarboxylase activity by as much as 50% and the rise in spermidine accumulation by 30% after coincident treatment of female Sencar mice.