Search PubMed⌕ Search

Biomedical subjects

K L Platt

Publications and source records attributed to K L Platt.

At least 73 records · Page 4Linked to original sources
PubMed1985

Host-mediated mutagenicity experiments with benzo[a]pyrene and two of its metabolites.

Benzo[a]pyrene (BP) and two of its major metabolites, the ultimate mutagen BP-4,5-oxide and the proximate mutagen trans-7,8-dihydro-7,8-dihydroxybenzo[a]pyrene (BP-7,8-diol) were investigated for mutagenicity in Salmonella typhimurium TA1538, TA98 and TA100 using an intrasanguineous host-mediated assay. BP and BP-4,5-oxide were not mutagenic under any experimental conditions. BP-7,8-diol was inactive with the strain TA1538 but was mutagenic with the strains TA98 and TA100. The effect was potentiated by pretreatment of the host mice with the cytochrome P-450 inducer 5,6-benzoflavone. We conclude: (i) one of the reasons for the observed insensitivity of the intrasanguineous host-mediated assay towards BP is that BP-4,5-oxide, which contributes to the microsome-mediated mutagenicity of BP, is inactive in the host-mediated assay; (ii) the finding that BP-7,8-diol is mutagenic in the host-mediated assay demonstrates that the lack of mutagenicity of BP is not intrinsic; (iii) the potentiated mutagenicity after treatment of the hosts with 5,6-benzoflavone suggests that cytochrome P-450 is more important in the activation of BP-7,8-diol in this system than other enzymes (e.g. prostaglandin synthase) that can also activate this compound in vitro.

Animals↗
PubMed1985

Arene imines, a new class of exceptionally potent mutagens in bacterial and mammalian cells.

K-region aziridines of polycyclic aromatic hydrocarbons reverted Salmonella typhimurium his- (TA100, TA98) and Escherichia coli trp- strains (WP2 uvrA), without requiring activation by mammalian enzymes. The number of revertants induced per nmol in S. typhimurium TA 100, the most responsive strain, variea from 6 to 10,000 for the seven monoaziridines and the two bisaziridines tested. Interestingly, the mutagenic potencies (y) of the monoaziridines were closely related (r = 0.984) with those of the corresponding epoxide analogues (x) by the equation y = 19.6 X0.97, i.e., the aziridines were about 20-fold stronger mutagens than were the epoxides. One of the aziridines, benzo(a)pyrene (BP)-4,5-imine, was investigated in several additional mutagenicity test systems: toxicity in DNA repair-deficient (rec-) and -proficient (rec+) Bacillus subtilis strains; induction of 6-thioguanine resistance in V79 Chinese hamster cells; and induction of sister chromatid exchanges in cultured human fibroblasts. In all systems, BP-4,5-imine was much more active than the epoxide analogue, BP-4,5-oxide. The difference in activity was particularly large in the two test systems with mammalian target cells in which several hundredfold higher concentrations of the epoxide had to be used in order to elicit equipotent effects. Even r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydro-BP, which is one of the most potent mutagens known for V79 cells, was less active in the mammalian cells than was BP-4,5-imine. One reason that arene imines are such potent mutagens may be that they are poorly detoxified. Addition of highly purified microsomal epoxide hydrolase, which strongly reduced the mutagenicity of BP-4,5-oxide and benz(a)anthracene-5,6-oxide in S. typhimurium, had no effect on the mutagenicity of the corresponding aziridines. Furthermore, while benz(a)anthracene-5,6-oxide was inactivated by highly purified cytosolic epoxide hydrolase, benz(a)anthracene-5,6-imine was not inactivated. It is noteworthy that the arene imines are isomeric with and structurally closely related to aromatic amines. Some aziridines derived from nonaromatic structures (ethylene imines) have been reported as metabolites of xenobiotics; others are used as chemotherapeutics. At present, however, the results are mainly of theoretical interest in that a new type of arene derivatives with exceptionally potent, probably ultimate, mutagenicity was discovered and may be exploited for the study of mechanisms of chemical carcinogenesis.

Animals↗
PubMed1984

Dose dependent activation of rat small intestinal monooxygenase-activity towards benzo(a)pyrene and 7-ethoxycoumarin after oral pretreatment with cimetidine.

Arylhydrocarbon-hydroxylase and 7-ethoxycoumarin O-deethylase were stimulated in intestinal microsomes prepared two hours after administration of cimetidine (25 mg/kg, p.o.) to male Wistar rats. Moreover, the metabolite-pattern of benzo(a)pyrene was changed as compared to that in control (saline, 2 hr) animals. Cytochrome P-450 content was not affected. However, rats receiving higher doses of cimetidine (75 and 150 mg/kg) showed a dose dependent decrease in diolformation from benzo(a)pyrene. We were not able to achieve similar effects on monooxygenase activity by in vitro pretreatment of intestinal cells and/or microsomes. Observed interaction might implicate an effect of cimetidine on carcinogenic activity in small intestine of polycyclic aromatic hydrocarbons.

7-Alkoxycoumarin O-Dealkylase↗
PubMed1982

The mutagenicity of dibenz [a,h]anthracene activated by phenobarbital-inducible mouse-liver mono-oxygenase is potentiated by the presence of hydrophilic residues at the K-region of the molecule.

Dibenz[a,h]anthracene and synthetic K-region derivatives of the parent hydrocarbon and of benz[a]anthracene were tested for mutagenicity by the reversion of histidine-dependent Salmonella typhimurium TA98, TA100 and TA1537. The K-region metabolite 5,6-dihydroxy-5,6-dihydrodibenz[a,h]anthracene, inactive as such, was efficiently activated to mutagens for TA98 and TA100 by mouse-liver 9000 X g supernatant or microsomal fraction. Microsomes from phenobarbital- or Aroclor-1254-treated mice were efficient for this activation, while those from untreated or beta-naphthoflavone-treated mice were much less active. A study on the influence of various structural features on this efficient activation by phenobarbital-inducible mono-oxygenase of mouse-liver microsomes showed that, if the K-region were saturated, no metabolism to mutagens occurred, while substitution of the K-region by carbonyl and hydroxyl substituents led to increased mutagenic efficacy with increasing hydrophilicity (dihydro less than carbonyl less than hydroxyl). The K-region epoxide was the only derivative that did not require metabolic activation and it had a markedly different mutagenic specificity in that it was also mutagenic for TA1537.

Animals↗
PubMed1980

Rat liver cytoplasmic dihydrodiol dehydrogenase. Purification to apparent homogeneity and properties.

A method is described for the purification of an enzyme, which catalyzes the conversion of benzene dihydrodiol to catechol, from rat liver cytoplasmic fraction to apparent homogeneity. The purification involved (NH4)2SO4 fractionation, DEAE-cellulose chromatography, interfacial salting-in and gel filtration through Sephadex G-100 superfine. The end product, which was purified over 500-fold with a yield of about 14% when compared to rat liver 100,000 X g supernatant, was judged to be homogeneous by several criteria, including sodium dodecyl sulfate-polyacrylamide gel electrophoresis, analytical ultracentrifugation, gel filtration, and immunoprecipitation. Physical studies suggested that the protein was a monomer with a molecular weight of 35,000 and one NADPH binding site per molecule. Amino acid analysis showed that the enzyme had a relatively high content of acidic and neutral amino acids in agreement with its isoelectric point which was at pH 6.2 Apparent Km values for benzene dihydrodiol and NADP+ were found to be 2.2 mM and 7.7 microM, respectively. The apparent Vmax value for the conversion of benzene dihydrodiol to catechol using NADP+ as cofactor was calculated to be 6.67 mumol/mg of enzyme/min. Substrate specificity studies showed that, in addition to benzene dihydrodiol, the dehydrogenase could oxidize acenaphthenol and the 3 alpha-hydroxy group of steroids. No activity was observable with a large number of other hydroxylated steroids possessing hydroxy groups at positions 3 beta, 11 beta, 17 alpha, 17 beta, 20 alpha, 20 beta, 21, and 22 of the steroid skeleton. Furthermore, only steroids which contained a 3-keto group and no double bond at the delta 4 position were reduced. This, and the fact that a range of nonsteroidal vicinal diols did not serve as substrates, indicates a relatively narrow substrate specificity. When benzene dihydrodiol was used as substrate, NADP+ was the preferred coenzyme but NAD+ was also accepted, whereas with the hydroxylated steroids the difference between the specific activities with NAD+ and NADP+ or with NADH and NADPH was less striking. The role of the enzyme in the metabolism of carcinogenic polycyclic hydrocarbons is discussed.

Amino Acids↗
PubMed1979

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.

Drug Evaluation, Preclinical↗
PubMedDate not supplied

Metabolizing systems in short-term in vitro tests for carcinogenicity.

Most mutagens require metabolic activation or can be metabolically inactivated. Lipophilic xenobiotics are typically metabolized by introduction and/or modification of a functional group and subsequent conjugation with a hydrophilic endogenous compound to allow excretion. Functional groups are most often introduced by the various cytochromes P-450. The risk that a reactive intermediate is generated is relatively high at this stage. However, many reactive intermediates may be efficiently inactivated by other enzymes, which modulate functional groups or introduce hydrophilic moieties. In other apparently rare cases, activation may also occur during these metabolic steps. Since bacteria and most cultured mammalian cells used as targets in short-term in vitro carcinogenicity tests are largely deficient in the cytochromes P-450, exogenous metabolizing systems (purified enzymes, crude subcellular preparations, intact cells or (in host-mediated tests) whole animals) are routinely used in such tests. Purified enzymes and crude subcellular preparations supplemented with individual or several cofactors are useful in elucidating the enzymatic control of mutagenic metabolites. As crude subcellular preparations are easy to prepare and to store, they are the metabolizing system most frequently used (supplemented with NADPH) in short-term tests. They favour the enzymes preferentially involved in activation. In contrast, intact cells (e.g. freshly isolated hepatocytes) and host animals additionally elicit the various enzyme activities preferentially involved in inactivation, so the results strongly depend on the kind of metabolizing system used. The choice of system plays a pivotal role in tests in bacteria, which are deficient not only in the 'preferentially activating enzymes' but also in the 'preferentially inactivating xenobiotic-metabolizing enzymes'. Mammalian cells in culture retain substantial activity of the latter enzymes. In our experience, results from in vitro mutagenicity tests in which adequate levels of activity of 'inactivating enzymes' are provided, either in the added metabolizing system or in the target cells, correlate better with carcinogenicity than tests in which the 'activating enzymes' are largely favoured.

Alkenes↗
PubMedDate not supplied

Bisdihydrodiols, rather than dihydrodiol oxides, are the principal microsomal metabolites of tumorigenic trans-3,4-dihydroxy-3,4-dihydrodibenz[a,h]anthracene.

Several studies on metabolism and biological activity of tumorigenic dibenz[a,h]anthracene (DBA) and its derivatives have led to the conclusion that the M-region dihydrodiol, trans-3,4-dihydroxy-3,4-dihydro-DBA (DBA-3,4-dihydrodiol), is the precursor of the ultimate mutagenic and tumorigenic metabolite of DBA with the presumed structure of a bay-region dihydrodiol oxide. Incubations of DBA-3,4-dihydrodiol (50 microM) with the microsomal hepatic fraction of Sprague-Dawley rats pretreated with Aroclor 1254 yielded more than 13 metabolites upon separation by HPLC. anti-3,4-Dihydroxy-1,2-epoxy-1,2,3,4-tetrahydro-DBA [0.27 nmol/(nmol of P450.15 min)] could be identified for the first time by UV spectroscopy, by cochromatography with the synthetic reference compound, and by its nonenzymatic hydrolysis to r-1,t-2,t-3,c-4-tetrahydroxy-1,2,3,4-tetrahydro-DBA, while firm evidence for the presence of the diastereomeric syn-dihydrodiol oxide was not obtained. Major microsomal metabolites of the M-region dihydrodiol were however three bisdihydrodiols: trans,trans-3,4:8,9-tetrahydroxy-3,4,8,9-tetrahydro-DBA [0.32 nmol/(nmol of P450.15 min)], trans,trans-3,4:10,11-tetrahydroxy-3,4,-10,11-tetrahydro-DBA [DBA-3,4:10,11-bisdihydrodiol; 1.44 nmol/(nmol of P450.15 min)], and trans,trans-3,4:12,13-tetrahydroxy-3,4,12,13-tetrahydro-DBA [0.70 nmol/(nmol of P450.15 min)], whose structures were verified by UV and mass spectrometry as well as cochromatography with synthetic reference compounds and by the observation that they were not formed when epoxide hydrolase was inhibited (1,1,1-trichloro-2-propene oxide, 1 mM).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMedDate not supplied

Characterization of highly polar DNA adducts derived from dibenz[A,H]anthracene (DBA), 3,4-dihydroxy-3,4-dihydro-DBA, and 3,4,10,11-tetrahydroxy-3,4,10,11-tetrahydro-DBA.

Two highly polar DNA adducts were found after metabolic activation of 3,4,10,11-tetrahydroxy-3,4,10,11-tetrahydrodibenz[a,h]anthracene (DBA-3,4,10,11-bisdiol) by liver microsomes isolated from male Sprague-Dawley rats pretreated with Aroclor 1254 in presence of calf thymus DNA. These DNA adducts could be assigned to the metabolites of dibenz[a,h]anthracene (DBA), of 3R,4R,10R,11R-tetrahydroxy-3,4,10,11-tetrahydro-DBA and of 3R,4R,10S,11S-tetrahydroxy-3,4,10,11-tetrahydro-DBA. DNA adducts derived from metabolites of 3S,4S,10S,11S-tetrahydroxy-3,4,10,11-tetrahydro-DBA were not found. These highly polar adducts also could be detected by reversed phase HPLC after incubation of dibenz[a,h]anthracene, 3R,4R-dihydroxy-3,4-dihydro-DBA ((-)-DBA-3,4-diol) and 3S,4S-dihydroxy-3,4-dihydro-DBA ((+)-DBA-3,4-diol) with DNA in presence of the activating system. After incubation of 14C labelled DBA DNA adducts derived from DBA-3,4,10,11-bisdiol were found in a fraction of 38% and bay region 3,4-dihydroxy-1,2-epoxy-1,2,3,4-tetrahydro-DBA-DNA adducts at a level of 25%. DBA-3,4,10,11-bisdiol exhibited a higher DNA binding yield (38 +/- 12 pmol/mg DNA) than (-)-DBA-3,4-diol (23 +/- 6 pmol/mg DNA), the most mutagenic 3,4-diol enantiomer. For (+)-DBA-3,4-diol the highly polar DNA adducts derived from DBA-3,4,10,11-bisdiol were by far the most predominant adducts in vitro.

Animals↗
PubMedDate not supplied

Applications of stable V79-derived cell lines expressing rat cytochromes P4501A1, 1A2, and 2B1.

1. Chinese hamster V79-derived cell lines, stably expressing cytochromes P4501A1, 1A2, and 2B1 activities, were constructed by genetic engineering in continuation of our work to establish a battery of V79 derived cell lines designed to study the metabolism of xenobiotics. 2. Cell lines XEM1 and XEM2, expressing cytochrome P4501A1, were capable of the O-dealkylation of 7-ethoxycoumarin and the hydroxylation of benzo[a]pyrene. 3. Cell lines XEMd.MZ and XEMd.NH, expressing P4501A2, were shown to hydroxylate 17 beta-estradiol and 2-aminofluorene. 4. Cell line SD1, expressing cytochrome P4502B1, was able to hydroxylate testosterone stereo- and regio-specifically at the 16 alpha and 16 beta positions. 5. Cell lines were validated in mutagenicity, cytotoxicity, and metabolism studies employing benzo[a]pyrene, trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene, cyclophosphamide, ifosfamide, and picene. 6. Construction of metabolically-competent V79-derived cell lines be recombinant DNA technology will be a fundamental improvement for the evaluation of the cytotoxic, genotoxic and pharmacological properties of a chemical.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗