Karl L. Platt

Multi-step metabolic activation of benzene. Effect of superoxide dismutase on covalent binding to microsomal macromolecules, and identification of glutathione conjugates using high pressure liquid chromatography and field desorption mass spectrometry

Abstract Incubation of [ 14 C]benzene or [ 14 C]phenol with liver microsomes from untreated rats, in the presence of a NADPH-generating system, gave rise to irreversible binding of metabolites to microsomal macromolecules. For both substrates this binding was inhibited by more than 50% by addition of superoxide dismutase to the incubation mixtures. The decrease in binding was compensated for by accumulation of [ 14 C]hydroquinone, indicating superoxide-mediated oxidation of hydroquinone as one step in the activation of benzene to metabolites binding to microsomal macromolecules. Since our previous work had shown that binding occurred mainly with protein rather than ribonucleic acid and was virtually completely prevented by glutathione, suggesting identity of metabolite(s) responsible for binding to protein and glutathione, a conjugate was chemically prepared from p -benzoquinone and reduced glutathione (GSH) and identified by field desorption mass spectrometry (FDMS) as 2-( S -glutathionyl) hydroquinone. Microsomal incubations, containing an NADPH-generating system, with benzene, phenol, hydroquinone or p -benzoquinone in the presence of [ 3 H]glutathione or, alternatively, with [ 14 C]benzene or [ 14 C]phenol in the presence of unlabeled glutathione, were performed. All of these incubations gave rise to a peak of radioactivity eluting from the high pressure liquid chromatograph (HPLC) at a retention time identical to that of the chemically prepared 2-( S -glutathionyl) hydroquinone, whilst microsomal incubation of catechol in the presence of [ 3 H]glutathione led to a conjugate with a very different retention time which was not observed after incubation of benzene or phenol. The microsomal metabolites of p -benzoquinone, hydroquinone and phenol thus eluting from the HPLC were further identified as the 2-( S -glutathionyl) hydroquinone by field desorption mass spectrometry. The glutathione adduct formed from benzene during microsomal activation eluted from HPLC with the same retention time and its mass spectrum also contained the molecular ion (MH + ) ( m/e 416) of this conjugate as an intense peak, but the fragmentation patterns did not allow definite assignments probably due to the considerably smaller amounts of ultimate reactive metabolites formed from this pre-precursor and thus relatively larger amounts of impurities. The results indicate that rat liver microsomes activate benzene via phenol and hydroquinone to p -benzosemiquinone and/or p -benzoquinone as quantitatively important reactive metabolites.

Functional heterogeneity of UDP-glucuronyltransferase in rat tissues

Abstract Tissue distribution of UDP-glucuronyltransferase was investigated using two substrate groups which were shown to be conjugated by two different forms of this enzyme in previous studies with rat liver. These enzyme forms were found to be differentially inducible by 3-methylcholanthrene (form 1) and phenobarbital (form 2). Group 1 substrates (conjugated by form 1) include 1-naphthol, N -hydroxy-2-naphthylamine and 3-hydroxybenzo[ a ]pyrene; group 2 substrates (conjugated by form 2) comprise 4-hydroxybiphenyl, morphine and chloramphenicol. Group 1 substrates are conjugated in a number of tissues, for example, liver, kidney, small intestinal mucosa, lung, skin, testes and spleen. However, conjugation of group 2 substrates is detectable only in liver and intestine to an appreciable extent. It is concluded that enzyme(s) efficient in the conjugation of group 1 substrates is ubiquitous in the investigated organs, whilst only liver and intestine possess enzyme(s) efficient in the conjugation of group 2 substrates. In contrast to 3-hydroxybenzo[ a ]pyrene, benzo[ a ]pyrene 7,8-dihydrodiol cannot be clearly associated with only one of the 2 substrate groups. Glucuronidation of benzo[ a ]pyrene 7.8-dihydrodiol is enhanced by both phenobarbital and 3-methylcholanthrene in liver. Conjugation of the dihydrodiol is detectable in all tissues examined. However, enzyme activity towards the dihydrodiol is much lower than that towards 3-hydroxybenzo[ a ]pyrene. It is disproportionately low with skin microsomes.

Cultures with cryopreserved hepatocytes: applicability for studies of enzyme induction.

The use of hepatocyte cultures is well established for the study of drug-drug interactions. However, the major hindrance for the use of human hepatocyte cultures is that human hepatocytes are only occasionally available. This problem could be overcome by cryopreservation. Although cryopreserved hepatocytes have been recommended for short term applications in suspension, studies on induction of enzyme activity, requiring a more prolonged maintenance of cryopreserved hepatocytes in culture, represent a new field of research. In the present study, we established a technique that allows preparation of rat hepatocyte co-cultures, using cryopreserved hepatocytes. After incubation with phenobarbital (0.75 mM; 72 h) induction factors for the isoenzyme-dependent regio and stereoselective testosterone hydroxylations were 1.6, 2.2, 1.0, 2.1, 5.6, 2.4, 3.6, 4.5 and 0.9 for 2alpha-, 2beta-, 6alpha-, 6beta-, 7alpha-, 15beta-, 16alpha- and 16beta-hydroxytestosterone and 4-androsten-3,17 dione. Regarding induction factors of less than 2-fold, as questionable these induction factors were similar to those of cultures with freshly isolated hepatocytes and the induction pattern of the individual hydroxylation products was similar to the in vivo situation. In addition 3-methylcholanthrene (5 microM; 72 h) induced exclusively the formation of 7alpha-hydroxytestosterone (6.6-fold) in cultures with cryopreserved hepatocytes. This specificity also correlates to that obtained in rats. Although these induction factors were clearly satisfactory in cryopreserved cultures, the absolute activities of the main testosterone hydroxylation products were reduced when compared to fresh cultures. For instance, 6beta-hydroxytestosterone, the main metabolite in solvent controls was reduced to 79%, 7alpha-hydroxytestosterone, the main metabolite after induction with 3-MC, was reduced to 66% and 16beta-hydroxytestosterone, the main metabolite after induction with PB, was reduced to 52%. Similarly, EROD activity after induction with 3-methylcholanthrene in cryopreserved cultures was reduced to 62%, compared with that in fresh cultures. Although further optimization and validation is required, the data show that cytochrome P450 activities can clearly be induced in co-cultures of cryopreserved hepatocytes, in a fashion which for the investigated inducers, is similar to that in cultures from freshly isolated hepatocytes and similar to the in vivo situation.

Mutagenicity of phenanthrene and phenanthrene K-region derivatives.

Phenanthrene and 9 K-region derivatives, most of them potential metabolites of phenanthrene, were tested for mutagenicity by the reversion of histidine-dependent Salmonella typhimurium TA1535, TA1537, TA1538, TA98 and TA100 and the rec assay with Bacillus subtilis H17 and M45. The strongest mutagenic effects in the reversion assay were observed with phenanthrene 9,10-oxide, 9-hydroxyphenanthrene and N-benzyl-phenanthrene-9,10-imine. Interestingly, the mutagenic potency of the arene imine was similar to that of the corresponding arene oxide. This is the first report on the mutagenicity of arene imine. The mutagenic effects of all these phenanthrene derivatives were much weaker than that of the positive control benzo[a]pyrene 4,5-oxide. Even weaker mutagenicty was found with cis-9,10-dihydroxy-9,10-dihydrophenanthrene and with trans-9,10-dihydroxy-9-10-dihydrophenanthrene. The other derivatives were inactive in this test. However, 9-10-dihydroxyphenanthrene and 9,10-phenanthrenequinone were more toxic to the rec- B. subtilis M45 strain than to the rec+ H17 strain. This was also true for phenanthrene 9,10-oxide and 9-hydroxyphenanthrene, but not with the other test compounds that reverted (9,10-dihydroxy-9,10-dihydrophenanthrenes; N-benzyl-phenanthrene 9,10-imine; benzo[a]pyrene 4,5-oxide) or did not revert (phenanthrene, 9,10-bis-(p-chlorophenyl)-phenanthrene 9,10-oxide, 9-10-diacetoxyphenanthrene) the Salmonella tester strains. Although the K region is a main site of metabolism and although all potential K-region metabolites were mutagenic, phenanthrene did not show a mutagenic effect in the presence of mouse-liver microsomes and an NADPH-generating system under standard conditions. However, uhen epoxide hydratase was inhibited, phenanthrene was activated to a mutagen that reverted his- S. typhimurium. This shows that demonstration of the mutagenic activity of metabolites together with the knowledge that a major metabolic route proceeds via these metabolites dose not automatically imply a mutagenic hazard of the mother compound, because the metabolites in question may not accumulate in sufficient quantities and therefore the presence and relative activities of enzymes that control the mutagenically active metabolites are crucial. N-Benzyl-phenanthrene 9.10-imine was mutagenic for the episome-containing S. typhimurium TA98 and TA100 but not for the precursor strains TA1538 and TA1535. This arene imine would therefore be useful as a positive control during routine testing to monitor in the former strains the presence of the episome which is rather easily lost.

Epoxide hydratase and benzo(A)pyrene monooxygenase activities in liver, kidney and lung after treatment of rats with epoxides of widely varying structures

Abstract The effect of treatment with various epoxides on epoxide hydratase and benzo(a)pyrene monooxygenase activities in rat liver, kidney and lung was tested with the aim possibly to find a selective inducer of epoxide hydratase. In a first series of epoxides, good substrates of epoxide hydratase and relatively small molecules did not lead to an increase in epoxide hydratase activity in any organ tested. Treatment with the poor substrates dieldrin and trans -stilbene oxide(TSO), however, increased epoxide hydratase activity in the liver and with TSO also in the kidney (3-fold). In contrast to all other epoxide hydratase inducers so far discovered, TSO did not affect the benzo(a)pyrene monooxygenase activities, even after doses leading to maximal induction of epoxide hydratase ( ~ 350 per cent of controls). In an attempt to find an even more potent selective inducer of epoxide hydratase several trans -stilbene oxide derivatives were synthesized. All modifications of the TSO molecule led to compounds which were less effective inducers of liver epoxide hydratase than the parent compound and also either increased or drastically decreased the benzo(a)pyrene monooxygenase activities. With TSO, 4-methoxy-TSO, 4-chloro-TSO and 4-nitro-TSO the tests were extended to three other parameters of the monooxygenase system, the cytochrome P450 content, the NADPH-cytochrome c reductase and the aminopyrine N -demethylase activities. All these derivatives but not TSO itself affected part of the monooxygenase system. Thus, with respect to five measured monooxygenase parameters TSO was found to be a selective inducer of epoxide hydratase in rat liver. An influence of TSO on the pattern of the various cytochrome P450 forms not leading to observable changes in monooxygenase activity towards two substrates known to be preferential substrates of different cytochrome P450 forms (aminopyrine and benzo(a)pyrene) is unlikely but cannot be excluded.

Development and application of test methods for the detection of dietary constituents which protect against heterocyclic aromatic amines.

This article describes the development and use of assay models in vitro (genotoxicity assay with genetically engineered cells and human hepatoma (HepG2) cells) and in vivo (genotoxicity and short-term carcinogenicity assays with rodents) for the identification of dietary constituents which protect against the genotoxic and carcinogenic effects of heterocyclic aromatic amines (HAs). The use of genetically engineered cells expressing enzymes responsible for the bioactivation of HAs enables the detection of dietary factors that inhibit the metabolic activation of HAs. Human derived hepatoma (HepG2) cells are sensitive towards HAs and express several enzymes [glutathione S-transferase (GST), N-acetyltransferase (NAT), sulfotransferase (SULT), UDP-glucuronosyltransferase (UDPGT), and cytochrome P450 isozymes] involved in the biotransformation of HAs. Hence these cells may reflect protective effects, which are due to inhibition of activating enzymes and/or induction of detoxifying enzymes. The SCGE assay with rodent cells has the advantage that HA-induced DNA damage can be monitored in a variety of organs which are targets for tumor induction by HAs. ACF and GST-P(+) foci constitute preneoplastic lesions that may develop into tumors. Therefore, agents that prevent the formation of these lesions may be anticarcinogens. The foci yield and the sensitivity of the system could be substantially increased by using a modified diet. The predictive value of the different in vitro and in vivo assays described here for the identification of HA-protective dietary substances relevant for humans is probably better than that of conventional in vitro test methods with enzyme homogenates. Nevertheless, the new test methods are not without shortcomings and these issues are critically discussed in the present article.

Inhibition of mutagenesis of 2-amino-3-methylimidazo(4,5-f)quinoline (IQ) by coumarins and furanocoumarins, chromanones and furanochromanones

Abstract A total of 51 natural and synthetic simple coumarins, furanocoumarins, chromanones, furanochromanones and some structurally related compounds were tested for their antimutagenic potencies with respect to IQ in Salmonella typhimurium TA 98. Antimutagenic potencies were quantified by the inhibitory dose for 50% reduction of mutagenic activity (ID 50 ) and by the remaining mutagenic activity at the highest dose tested. Antimutagenic activities of the parent compounds were weak (ID 50 : 500–750 nmol/plate) but increased in the coumarin series with introduction of hydroxy, methoxy and (or) methyl groups at carbons 4, 5, 6 and 7 (ID 50 : 70–400 nmol/plate). However, the antimutagenicity of compounds with hydroxy or methoxy substituents at C-5, C-6 and C-7 all together was low. Moreover, a hydroxy or methoxy function at C-8 greatly reduced antimutagenic potency. This was in part also true for substituents at C-3. Coumarin glycosides and glucuronides of antimutagenic aglycones were, however, inactive. Introduction of a carboxyl function rendered the respective coumarin inactive. Surprisingly, some synthetic coumarins with a bromo or iodo substituent at C-8 or a benzyloxy function at C-5 were found to be very potent antimutagens (ID 50 : 9.3–14.5 nmol/plate), whereas analogues possessing a bromo or formyl function at C-5 were less effective (ID 50 : 176 and 395 nmol/plate). Furanocoumarins and furanochromanones were very potent antimutagens (ID 50 : 5.1–26.5 nmol/plate). In enzyme kinetic experiments with Salmonella the inhibition mechanisms of xanthotoxin and visnagin were concentration dependent, being non-competitive at low concentrations. Reduction of the activity of 7-ethoxy- and 7-methoxyresorufin- O -dealkylases with IC 50 values of 1.2–11.7 μM indicated strong inhibition of cytochrome P -450 1A1 and 1A2 dependent monooxygenases by some of the furanocoumarines and -chromanones. The mutagenic activity of N -hydroxy-IQ in Salmonella, however, was not reduced by any of these compounds. In various experiments designed for modulation of the mutagenic response inhibition of activation of IQ to N-OH-IQ was found to be the only relevant mechanism of antimutagenesis of psoralen, angelicin and khellin.

Identification of Dibenzo[a,l]pyrene-DNA Adducts Formed in Cells in Culture and in Mouse Skin

Abstract The DNA adducts formed from the potent carcinogen dibenzo[a,l]pyrene (DB[a,l]P) in cultures of Sencar mouse embryo cells and a mouse keratinocyte cell line and in Sencar mouse epidermis in vivo were analyzed by 35S−phosphorothioate postlabeling, immobilized boronate chromatography and reverse-phase HPLC. The results demonstrate that the major DNA adducts result from the activation of DB[a,l]P to DB[a,l]P-11,12-diol-13,14-epoxide. Adducts from both the syn− and anti−isomers of this diol epoxide are formed in cells in culture, but in mouse epidermis only anti−isomer adducts are detected.

Studies on pathways of ring opening of benzene in a Fenton system

Ring-opened products of benzene metabolism have been postulated to play a role in hematotoxicity and leukemogenesis. The reaction of benzene in the Fenton system was reexamined to determine the presence of compounds which might serve as intermediates in the formation of trans, trans-muconaldehyde (MUC), a microsomal hematotoxic metabolite of benzene. Benzene dihydrodiol (DHD) was found in this system based on coelution with authentic standard, ultraviolet (UV) absorption characteristics, and molecular weight. Incubation of DHD in the Fenton system resulted in the formation of phenol (PH), catechol (CAT), and products which reacted with thiobarbituric acid to form chromogens absorbing at 495 nm and 532 nm, consistent with products containing an alpha, beta-unsaturated aldehyde group. However, muconaldehyde was not detected in the Fenton system incubated with DHD, indicating that MUC is not formed via ring opening of DHD. When benzene was incubated in the Fenton system, MUC, cis,trans-muconaldehyde, PH, hydroquinone (HQ), and CAT were identified. Identification of cis,trans-muconaldehyde, an isomer which can quickly rearrange to MUC, suggests that cis,cis-muconaldehyde is originally formed from benzene and converted to cis,trans- and then trans,trans-muconaldehyde.

Synthesis of non-k-region ortho-quinones of polycyclic aromatic hydrocarbons from cyclic ketones

Non-K-region o-quinones of polycyclic aromatic hydrocarbons are prepared in four steps from cyclic ketones via dehydrogenation of tetrahydrodiols with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone.