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We propose a novel semi-analytic design strategy for dielectric one-dimensional multilayer biosensors that is based on a relation between the angular sensitivity and the optical power flow of the Bloch surface wave guided by the multilayer. We show that our strategy can be used to optimize both the sensor’s sensitivity and figure-of-merit without the need for extensive numerical parameter sweeps.

This paper reviews recent results from electromagnetic moment measurements on isotopes in the island of inversion around N=20. The obtained moments on neutron rich Na, Mg, Al and Si isotopes allow to draw conclusions on the amount of intruder components in their ground state wave function, demonstrating a gradual transition from the normal sd-shell region into the island of inversion, starting at N=18 for Na, N=19 for Mg and N=20 for Al isotopes. A measurement of the ground state g factor of 35Si (N=21), using a polarized fragment beam at GANIL, is discussed in more detail. The magnetic moment μ(35Si, Iπ= 7/2-) = (-)1.638(4) μNis consistent with a normal ground state structure, dominated by a νf7/2neutron.

We present the design of a supporting photonic crystal structure that would allow for the excitation of the predicted transverse electric (TE) polarized excitation in a single layer of graphene. We show that it is possible to measure this excitation at room temperature, and that adding an extra layer of dielectric material on top of the structure would further facilitate the experimental observation of the graphene mode.

Abstract.: This paper reviews recent results from electromagnetic moment measurements on isotopes in the island of inversion around N=20. The obtained moments on neutron rich Na, Mg, Al and Si isotopes allow to draw conclusions on the amount of intruder components in their ground state wave function, demonstrating a gradual transition from the normal sd-shell region into the island of inversion, starting at N=18 for Na, N=19 for Mg and N=20 for Al isotopes. A measurement of the ground state g factor of 35Si (N=21), using a polarized fragment beam at GANIL, is discussed in more detail. The magnetic moment μ(35Si, Iπ= 7/2-) = (-)1.638(4) μN is consistent with a normal ground state structure, dominated by a νf7/2 neutron.

This paper reviews recent results from electromagnetic moment measurements on isotopes in the island of inversion around N=20. The obtained moments on neutron rich Na, Mg, Al and Si isotopes allow to draw conclusions on the amount of intruder components in their ground state wave function, demonstrating a gradual transition from the normal sd-shell region into the island of inversion, starting at N=18 for Na, N=19 for Mg and N=20 for Al isotopes. A measurement of the ground state g factor of 35Si (N=21), using a polarized fragment beam at GANIL, is discussed in more detail. The magnetic moment ?(35Si, I?= 7/2-) = (-)1.638(4) ?Nis consistent with a normal ground state structure, dominated by a ?f7/2neutron.

We have developed a fully automated bioreactor coupled to an on-line receptor affinity detection system. This analytical system provides detailed information on pharmacologically active metabolites of selective estrogen receptor modulators (SERMs) generated by cytochromes P450 (P450s). We demonstrated this novel concept by investigating the metabolic activation of tamoxifen and raloxifene by P450-containing pig and rat liver microsomes. The high resolution screening (HRS) system is based on the coupling of a P450-bioreactor to an HPLC-based estrogen receptor alpha (ERα) affinity assay. P450-derived metabolites of the SERMs were generated in the bioreactor, subsequently trapped on-line with solid phase extraction, and finally separated with gradient HPLC. Upon elution, the metabolites were screened on affinity for ERα with an on-line HRS assay. With this HRS system, we were able to follow, in a time-dependent manner, the formation of ERα-binding metabolites of tamoxifen and raloxifene. By analyzing the bioaffinity chromatograms with liquid chromatography-tandem mass spectrometry, structural information of the pharmacologically active metabolites was obtained as well. For tamoxifen, 15 active and 6 nonactive metabolites were observed, of which 5 were of primary, 10 of secondary, and 6 of an as yet unknown order of metabolism. Raloxifene was biotransformed in three primary and three secondary metabolites. MS/MS analysis revealed that three of the observed active metabolites of raloxifene were not described before. The present automated on-line HRS system coupled to a P450-containing bioreactor and an ERα-affinity detector proved very efficient, sensitive, and selective in metabolic profiling of SERMs.

Exposure of humans and wildlife to xenobiotics, such as halogenated biphenyls, that interfere with the endogenous estrogen balance may lead to endocrine disruption. Such compounds may either mimic or block estradiol's action by agonistic or antagonistic action, respectively. They may also affect endogenous estradiol concentrations by induction or inhibition of enzymes that metabolize estradiol. In the present study, we demonstrate that estrogenic metabolites of two brominated biphenyls, 2,2‘-dibromobiphenyl (2,2‘-DBB) and 4,4‘-dibromobiphenyl (4,4‘-DBB), are formed by rat liver microsomal cytochrome P450 (CYP) activity. Bioactivation of 2,2‘-DBB and 4,4‘-DBB yielded various mono- and dihydroxylated bromobiphenyl metabolites, which were collected by preparative HPLC and analyzed by LC/MS. Several of the metabolites bound to the estrogen receptor (ER) activated the ER and inhibited human estrogen sulfotransferase (hEST). Seven monohydroxylated metabolites were positively identified using synthetic monohydroxylated reference compounds. These synthetic monohydroxylated bromobiphenyls also bound to and activated the ER and inhibited hEST. The highest ER affinity was observed for 4-OH-2,2‘-DBB, with an EC50 of 6.6 nM. The highest ER activation was observed for 4-OH-3,4‘-DBB (EC50 of 74 nM) while 4-OH-4‘-MBB and 4-OH-2,2‘-DBB induced a supramaximal (as compared to estradiol) ER activation. The strongest hEST inhibition was found with 4-OH-3,4‘-DBB (EC50 = 40 nM). In conclusion, we show that two dibrominated biphenyls are bioactivated by CYP activity into very potent estrogenic metabolites and inhibitors of hEST. These findings are of vital importance for accurate risk assessment of exposure to environmental contaminants, such as halogenated biphenyls. Neglecting bioactivation through biotransformation will lead to underestimation of health risks of this class of xenobiotics.

We report on the first measurement of the g factor of the Jπ = 9/2+ isomeric state in 65Ni. (d,p) reactions on enriched 64Ni and 62Ni have been used in order to populate and spin orient the Jπ = 9/2+ isomeric states in 65Ni and 63Ni. The hyperfine field of the Ni target, oriented by an external magnetic field, was used in order to obtain a sufficient number of spin precessions within the relatively short life time of the isomers. The g factor obtained, g(65mNi) = −0.296(3), is in good agreement with the g factors of other 9/2+ states in the region and with shell-model calculations.

A series of 3,4-methylenedioxy-N-alkylamphetamines (MDAAs) were automatically docked and subjected to molecular dynamics (MD) simulations in a cytochrome P450 2D6 (CYP2D6) protein model. The predicted substrate binding orientations, sites of oxidation, and relative reactivities were compared to the experimental data of wild-type and Phe¹²⁰Ala mutant CYP2D6. Automated docking results were not sufficient to accurately rationalize experimental binding orientations of 3,4-methylenedioxy-N-methylamphetamine (MDMA) in the two enzymes as measured with spin lattice relaxation NMR. Nevertheless, the docking results could be used as starting structures for MD simulations. Predicted binding orientations of MDMA and sites of oxidation of the MDAAs derived from MD simulations matched well with the experimental data. It appeared the experimental results were best described in MD simulations considering the nitrogen atoms of the MDAAs in neutral form. Differences in regioselectivity and stereoselectivity in the oxidative metabolism of the MDAAs by the Phe¹²⁰Ala mutant CYP2D6 were correctly predicted, and the effects of the Phe¹²⁰Ala mutation could be rationalized as well.

The popular docking programs AutoDock, FlexX, and GOLD were used to predict binding modes of ligands in crystallographic complexes including X-ray water molecules or computationally predicted water molecules. Isoenzymes of two different enzyme systems were used, namely cytochromes P450 (n = 19) and thymidine kinases (n = 19) and three different “water” scenarios: i.e., docking (i) into water-free active sites, (ii) into active sites containing crystallographic water molecules, and (iii) into active sites containing water molecules predicted by a novel approach based on the program GRID. Docking accuracies were determined in terms of the root-mean-square deviation (RMSD) accuracy and, newly defined, in terms of the ligand catalytic site prediction (CSP) accuracy. Consideration of both X-ray and predicted water molecules and the subsequent pooling and rescoring of all solutions (generated by all three docking programs) with the SCORE scoring function significantly improved the quality of prediction of the binding modes both in terms of RMSD and CSP accuracy.

The in vitro metabolism of 3,4-dihydro-6-hydroxy-2,2-dimethyl-7-methoxy-1(2H)-benzopyran (CR-6), a potent lipid peroxidation inhibitor and scavenger of nitric oxide and peroxynitrite species that is currently in phase II trials for antitumoral therapy, has been investigated in rat liver microsomes in the presence of NADP(H). Five major metabolites were identified by comparison with authentic standards, namely, the quinone 2-(3‘-hydroxy-3‘-methylbutyl-5-methoxy-1,4-benzoquinone (2a) and its ring-closed spiro form oxaspiro[4.5]-2,2-dimethyl-8-methoxy-dec-8-ene-7,10-dione (2b), the hydroquinone 2-(3‘-hydroxy-3‘-methylbutyl)-5-methoxyhydroquinone (3), the hydroxylated metabolite 3,4-dihydro-4,6-dihydroxy-2,2-dimethyl-7-methoxy-1(2H)-benzopyran (4), and the catechol 3,4-dihydro-6,7-dihydroxy-2,2-dimethyl-1(2H)-benzopyran (5). When the incubations were carried out in the presence of GSH, the HPLC peaks corresponding to the quinone metabolites 2a/b were absent and two novel products were formed showing MS fragmentation patterns consistent with the structure of GSH conjugates of quinone 2a. The time dependence on the formation of metabolites 2a,b and 3 was measured in incubations induced with phenobarbital (PB), dexamethasone, and β-naphthoflavone (βNF). For the dexamethasone-induced microsomes, the amount of hydroquinone 3 decreased from minute 10 to minute 30 while that of 2a,b increased in a complementary manner. Similar effects were observed for the incubations carried out using PB- and βNF-induced microsomes. On the other hand, CR-6 inhibited 7-ethoxyresorufin O-dealkylation activity (IC50 = 25 μM) in incubations with βNF-induced microsomes. Likewise, addition of pentoxyresorufin to the incubations of CR-6 with PB-induced microsomes showed a time-dependent inhibition (IC50= 75 μM) of the dealkylation activity. These results are in agreement with the putative generation of reactive metabolites from CR-6 that could deactivate P450 1A and P450 2B, respectively. When these incubations were carried out in the presence of 10 mM GSH, the inhibition of P450 2B could be partially prevented. Finally, preincubation of CR-6 with liver microsomes from PB-induced rats resulted in a strong increase in microsomal glutathione S-transferase (mGST) activity (up to a maximum of approximately 5-fold). When the preincubation was carried out in the presence of 10 mM GSH, the activation of mGST was blocked. Overall, these results suggest that CR-6 undergoes in vitro biotransformation indicative of the involvement of thiol-reactive metabolites.

Exposure to environmental estrogens has been proposed as a risk factor for disruption of reproductive development and tumorigenesis of humans and wildlife (McLachlan, J. A.; Korach, K. S.; Newbold, R. R.; Degen, G. H. Diethylstilbestrol and other estrogens in the environment. Fundam. Appl. Toxicol. 1984, 4, 686−691). In recent years, many structurally diverse environmental compounds have been identified as estrogens. A reliable computational method for determining estrogen receptor (ER) binding affinity is of great value for the prediction of estrogenic activity of such compounds and their metabolites. In the presented study, a computational model was developed for prediction of binding affinities of ligands to the ERα isoform, using MD simulations in combination with the linear interaction energy (LIE) approach. The linear interaction energy approximation was first described by Åqvist et al. (Åqvist, J.; Medina, C.; Samuelsson, J. E. A new method for predicting binding affinity in computer-aided drug design. Protein Eng. 1994, 7, 385−391) and relies on the assumption that the binding free energy (ΔG) depends linearly on changes in the van der Waals and electrostatic energy of the system. In the present study, MD simulations of ligands in the ERα ligand binding domain (LBD) (Shiau, A. K.; Barstad, D.; Loria, P. M.; Cheng, L.; Kushner, P. J.; Agard, D. A.; Greene, G. L. The structural basis of estrogen receptor/coactivator recognition and the antagonism of this interaction by tamoxifen. Cell 1998, 95, 927−937), as well as ligands free in water, were carried out using the Amber 6.0 force field (http://amber.scripps.edu/). Contrary to previous LIE methods, we took into account every possible orientation of the ligands in the LBD and weighted the contribution of each orientation to the total binding affinity according to a Boltzman distribution. The training set (n = 19) contained estradiol (E2), the synthetic estrogens diethylstilbestrol (DES) and 11β-chloroethylestradiol (E2-Cl), 16α-hydroxy-E2 (estriol, EST), the phytoestrogens genistein (GEN), 8-prenylnaringenin (8PN), and zearalenon (ZEA), four derivatives of benz[a]antracene-3,9-diol, and eight estrogenic monohydroxylated PAH metabolites. We obtained an excellent linear correlation (r² = 0.94) between experimental (competitive ER binding assay) and calculated binding energies, with Kd values ranging from 0.15 mM to 30 pM, a 5 000 000-fold difference in binding affinity. Subsequently, a test set (n = 12) was used to examine the predictive value of our model. This set consisted of the synthetic estrogen 5,11-cis-diethyl-5,6,11,12-tetrahydrochrysene-2,8-diol (THC), daidzein (DAI), equol (EQU) and apigenin (API), chlordecone (KEP), progesterone (PRG), several mono- and dihydroxylated PAH metabolites, and two brominated biphenyls. The predicted binding affinities of these estrogenic compounds were in very good agreement with the experimental values (average deviation of 0.61 ± 0.4 kcal/mol). In conclusion, our LIE model provides a very good method for prediction of absolute ligand binding affinities, as well as binding orientation of ligands.

The ligand-binding characteristics of rat and human CYP2D isoforms, i.e., rat CYP2D1−4 and human CYP2D6, were investigated by measuring IC50 values of 11 known CYP2D6 ligands using 7-methoxy-4-(aminomethyl)coumarin (MAMC) as substrate. Like CYP2D6, all rat CYP2D isozymes catalyzed the O-demethylation of MAMC with Km and Vmax values ranging between 78 and 145 μM and 0.048 and 1.122 min^-1, respectively. To rationalize observed differences in the experimentally determined IC50 values, homology models of the CYP2D isoforms were constructed. A homology model of CYP2D6 was generated on the basis of crystallized rabbit CYP2C5 and was validated on its ability to reproduce binding orientations corresponding to metabolic profiles of the substrates and to remain stable during unrestrained molecular dynamics simulations at 300 K. Twenty-two active site residues, sharing up to 59% sequence identity, were identified in the CYP2D binding pockets and included CYP2D6 residues Phe120, Glu216, and Asp301. Electrostatic potential calculations displayed large differences in the negative charge of the CYP2D active sites, which was consistent with observed differences in absolute IC50 values. MD studies on the binding mode of sparteine, quinidine, and quinine in CYP2D2 and CYP2D6 furthermore concurred well with experimentally determined IC50 values and metabolic profiles. The current study thus provides new insights into differences in the active site topology of the investigated CYP2D isoforms.

Tellurium compounds are effective antioxidants and chemoprotectors, even more active than their selenium and sulfur analogues. In addition to these properties, some selenium compounds, such as selenocysteine Se-conjugates, possess significant chemopreventive and antitumor activities, and selenol metabolites are considered as active species. In the present study, we have synthesized Te-phenyl-l-tellurocysteine and evaluated its bioactivation and cytotoxicity. The activities of this compound were compared with those of the corresponding selenium and sulfur analogues. Te-Phenyl-l-tellurocysteine is bioactivated into its corresponding tellurol, as detected by GC-MS, by cysteine conjugate β-lyase and amino acid oxidase, analogously to what has been shown previously for Se-phenyl-l-selenocysteine. The rate of β-elimination may reflect the bond strength of the corresponding C−S, C−Se, and C−Te bond. Bioactivation of Te-phenyl-l-tellurocysteine and its selenium and sulfur analogues by oxidative enzymes was evaluated by measuring NADPH-dependent activation of hepatic mGST and inhibition of EROD. Te-Phenyl-l-tellurocysteine and Se-phenyl-l-selenocysteine displayed strong and time-dependent mGST activation, while S-phenyl-l-cysteine resulted in no significant activation. Te-Phenyl-l-tellurocysteine was also a strong inhibitor of EROD activity. In addition to EROD inhibition, Te-phenyl-l-tellurocysteine was the strongest inhibitor of several human cytochrome P450 isoenzymes followed by Se-phenyl-l-selenocysteine, while S-phenyl-l-cysteine was the weakest inhibitor. Interestingly, Te-phenyl-l-tellurocysteine selectively inhibited cytochrome P450 1A1 directly, which is, for example, responsible for the activation of several procarcinogens. Preliminary cytotoxicity studies with Te-phenyl-l-tellurocysteine in freshly isolated rat hepatocytes showed a time-dependent depletion of GSH and LDH leakage comparable with the relatively nontoxic drug paracetamol, while the selenium and sulfur analogues were nontoxic toward rat hepatocytes. In conclusion, because the chemopreventive and antitumor activities of selenium compounds are thought to be mediated via their selenol metabolites and tellurium compounds might be even more active than selenium compounds, tellurocysteine Te-conjugates might be an interesting novel class of prodrugs for the formation of biologically active tellurols.

Midazolam is almost exclusively metabolized by cytochrome P450 3A (CYP3A) isoenzymes. Therefore, midazolam is used as a probe to determine CYP3A levels in humans and rats. A prerequisite for longitudinal determination of CYP3A expression levels using midazolam as a probe is that midazolam itself has no effect on the expression of CYP3A. In the present study, we analyzed the mRNA levels and enzyme activities of the major CYP isoforms in the rat liver after intraperitoneal injection of midazolam (50 mg/kg) for 3 consecutive days. CYP3A1 mRNA levels were increased 4-fold in midazolam-treated animals compared with controls, whereas the mRNA levels of CYP3A2, CYP3A9, and CYP3A18 were not altered. The increase in CYP3A1 mRNA was accompanied by a 25% increase in microsomal testosterone 6beta-hydroxylation activity. More strikingly, CYP2B1/2 mRNA levels were increased 22-fold upon midazolam treatment, leading to an 11- to 95-fold enhancement of CYP2B enzyme activity. CYP2C6 mRNA levels were 4 times higher in midazolam-treated animals. Formation of 2alpha-hydroxy-testosterone, mainly catalyzed by CYP2C11, was 2.6-fold lower in liver microsomes from midazolam-treated animals. Midazolam induced CYP2E enzyme activity 2.5-fold at the post-transcriptional level. The induction of CYP2B1/2 mRNA levels by midazolam was dose-dependent (4.5-fold increase at 10 mg/kg). Induction of CYP3A1 and CYP2B expression was also observed in isolated rat hepatocytes cultured with 100 microM midazolam. We conclude that midazolam is a phenobarbital-like CYP inducer in rats. Induction of CYP3A1 by midazolam may have implications for the longitudinal use of midazolam as a probe for analysis of CYP3A expression levels in rats.

Several selenocysteine Se-conjugates have been shown to possess potent chemopreventive activity in animal models for chemical carcinogenesis. As a mechanism of action, β-elimination reactions to form chemopreventive selenols, ammonia, and pyruvate has been proposed. The enzymes involved in these β-elimination reactions, however, have been partially elucidated. Next to cysteine conjugate β-lyases, as yet unidentified non-pyridoxal-5‘-phosphate-dependent enzymes also appear to be involved in cytosolic β-elimination reactions. In the present study, it was investigated whether amino acid oxidases contribute to the bioactivation of selenocysteine Se-conjugates. Using purified l-amino acid oxidase from Crotalus adamanteus as a model enzyme, significant β-elimination activities were indeed observed upon incubation with Se-methylselenocysteine (Km, 195 μM; kcat, 48 min^-1), Se-allylselenocysteine (Km, 608 μM; kcat, 34 min^-1), Se-phenylselenocysteine (Km, 107 μM; kcat, 57 min^-1) and Se-benzylselenocysteine (Km, 59 μM; kcat, 13 min^-1). For all selenocysteine Se-conjugates tested, the rate of pyruvate formation was comparable to that of hydrogen peroxide, one of the products of oxidative deamination. The fact that addition of catalase did not alter pyruvate formation indicated that the β-elimination reaction observed was not mediated by selenoxidation/syn-elimination due to the hydrogen peroxide formed via the oxidative deamination pathway. Using d-amino acid oxidase from porcine kidney and d-SeCys conjugates similar results were obtained. To delineate whether mammalian l-amino acid oxidases are also able to catalyze β-elimination of selenocysteine Se-conjugates, rat renal cytosol was fractionated and screened for β-elimination and oxidative deamination activities. One of the fractions isolated displayed oxidative deamination activity with several amino acids and cysteine S-conjugates. With selenocysteine Se-conjugates as substrates, however, this fraction displayed both oxidative deamination and β-elimination activities, when incubated in the presence of aminoxyacetic acid to block contribution of pyridoxal-5‘-phosphate-dependent enzymes. The potential significance of this novel bioactivation route for the chemopreventive activity of selenocysteine Se-conjugates is discussed.

Previously, it was shown that β-elimination of selenocysteine Se-conjugates by rat renal cytosol leading to pyruvate formation was not solely catalyzed by pyridoxal phosphate-dependent enzymes. It was hypothesized that selenoxidation of the selenocysteine Se-conjugates, followed by syn-elimination, may be an alternative mechanism for pyruvate formation. In this study, selenoxidation of selenocysteine Se-conjugates was studied using rat liver microsomes and recombinant human oxidative enzymes. For all six selenocysteine Se-conjugates that were tested, it was found that rat liver microsomal incubations led to the formation of pyruvate, whereas the corresponding selenoxides were not observed. Microsomal pyruvate formation from Se-benzyl-l-selenocysteine (SeBC) was NADPH-dependent, but only marginally inhibited by several P450 inhibitors. Inhibition by methimazole and by heat pretreatment and stimulation by n-octylamine indicated that flavin-containing monooxygenases are mainly responsible for pyruvate formation from the selenocysteine Se-conjugates in rat liver microsomes. In the case of S-benzyl-l-cysteine, the sulfur analogue of SeBC, pyruvate formation was not observed. For this substrate, a chemically stable sulfoxide could be observed, as previously described. By using recombinantly expressed human flavin-containing monooxygenases and P450 enzymes, it was delineated that SeBC is selenoxidized by human FMOs, but not by human P450s. The kcat/Km of selenoxidation was 3.8-fold higher for FMO-1 than for FMO-3. In conclusion, selenoxidation of selenocysteine Se-conjugates catalyzed by FMOs and subsequently syn-elimination has taken place as an alternative route for the formation of pyruvate from selenocysteine Se-conjugates. Although selenoxides are known to be easily reduced by thiol compounds, microsomal pyruvate formation from SeBC was only 75% inhibited in the presence of an excess of glutathione. This indicates that even in the presence of physiological concentrations of reducing thiol compounds, selenoxides of selenocysteine Se-conjugates may undergo syn-elimination to some extent. Whether selenoxides and/or selenenic acids that are formed are involved in the activity of chemopreventive selenocysteine Se-conjugates remains to be established.

A series of six structural analogs of 7-methoxy-4-(aminomethyl)-coumarin (MAMC), a recently developed high-throughput substrate of P450 2D6 (CYP2D6), was synthesized to investigate the influence of N-substitution on the metabolism by cytochrome P450s, as well as on P450 selectivity. The analogs were obtained by introducing alkyl substituents at the amino group of MAMC and by replacing this moiety with a pyridine group. Competition experiments using heterologously expressed CYP2D6 demonstrated that the introduction and elongation of alkyl substituents strongly decreased the IC(50) values toward dextromethorphan O-demethylation. Metabolism studies showed that the regioselectivity of metabolism was unaffected by the varying N substituents, as only O-dealkylation of the analogs and no N-dealkylation was observed. In excellent agreement with the competition experiments, metabolism studies also showed that elongation of the alkyl chain dramatically increased the affinity of the compounds toward CYP2D6, as indicated by an up to 100-fold decrease in K(m) values. The V(max) values displayed a much less pronounced decrease with an increasing N-alkyl chain, resulting in as much as a 30-fold increase in the V(max)/K(m) value. Interestingly, due to the higher fluorescent yield of the N-alkyl metabolites compared with the metabolite of MAMC, O-dealkylation of N-methyl MAMC by CYP2D6 can be measured with a more than 3-fold higher sensitivity. Studies on P450 selectivity showed that only CYP1A2 and CYP2D6 contribute to the O-dealkylation of the N-alkyl analogs in both heterologously expressed P450s and human liver microsomes. In sharp contrast to CYP2D6, N-alkylation of MAMC did not significantly affect the K(m) values of O-dealkylation by CYP1A2, but it did result in higher V(max) values. Finally, CYP1A2 also N-dealkylated the analogs.

This study was performed to evaluate whether selenocysteine Se-conjugates are substrates for human cysteine conjugate beta-lyase enzymes. By testing kidney cytosols of three different humans, we studied interindividual differences in beta-lyase enzymes in humans. A series of 22 selenocysteine Se-conjugates were tested in rat and human kidney cytosols to compare their ability to form selenol compounds by beta-elimination. All compounds appeared to be good substrates for rat and human cysteine conjugate beta-lyase enzymes. The beta-lyase activity toward the selenocysteine Se-conjugates was comparable with those of the known nephrotoxic cysteine S-conjugate S-(2-chloro-1,1,2-trifluoroethyl)-L-cysteine in rats and humans. In rat kidney cytosol, between 22- and 877-fold higher beta-elimination rates were observed compared with human kidney cytosol. Significant correlations (P <.0001) between three human kidney cytosols in beta-lyase activities were found within the tested series of 22 compounds. Specific beta-lyase activities and intrinsic clearances of beta-elimination reactions ranged up to 3-fold, indicating that there are quantitative rather than qualitative interindividual differences in beta-eliminating enzymes in humans. Furthermore, Se-alkyl selenocysteine conjugates showed a sterically dependent bioactivation to selenol compounds in humans but not in rats. The present study supports the hypothesis that selenocysteine Se-conjugates may be useful as prodrugs to target pharmacologically active selenol compounds (e.g., antitumor or chemoprotective) to the kidney in humans.

Selenocysteine Se-conjugates have recently been proposed as potential prodrugs to target pharmacologically active selenol compounds to the kidney. Although rat renal cytosol displayed a high activity of beta-elimination activity toward these substrates, the enzymes involved in this activation pathway as yet have not been identified. In the present study, the possible involvement of cysteine conjugate beta-lyase/glutamine transaminase K (beta-lyase/GTK) in cytosolic activity was investigated. To this end, the enzyme kinetics of 15 differentially substituted selenocysteine Se-conjugates and 11 cysteine S-conjugates was determined using highly purified rat renal beta-lyase/GTK. The results demonstrate that most selenocysteine Se-conjugates are beta-eliminated at a very high activity by purified beta-lyase/GTK, implicating an important role of this protein in the previously reported beta-elimination reactions in rat renal cytosol. As indicated by the rapid consumption of alpha-keto-gamma-methiolbutyric acid, purified beta-lyase/GTK also catalyzed transamination reactions, which appeared to even exceed that of beta-elimination. The corresponding sulfur analogs also showed significant transamination but were beta-eliminated at an extremely low rate. Comparison of the obtained enzyme kinetic data of purified beta-lyase/GTK with previously obtained data from rat renal cytosol showed a poor correlation. By determining the activity profiles of cytosolic fractions applied to anion exchange fast protein liquid chromatography and gel filtration chromatography, the involvement of multiple enzymes in the beta-elimination of selenocysteine Se-conjugates in rat renal cytosol was demonstrated. The identity and characteristics of these alternative selenocysteine conjugate beta-lyases, however, remain to be established.

In this study, a selective substrate for cytochrome P450 2D6 was designed using a small molecule model developed by M. J. De Groot et al. [(1997) Chem. Res. Toxicol. 10, 41−48]. The substrate, 7-methoxy-4-(aminomethyl)coumarin (MAMC), and its putative O-demethylated metabolite 7-hydroxy-4-(aminomethyl)coumarin (HAMC) were synthesized, and their respective fluorescence properties were characterized. The selectivity of MAMC for P450 2D6 was characterized using microsomes containing single human P450 isoenzymes and human liver microsomes. Formation of the metabolic product HAMC was easily assessed in real time with fluorescence spectroscopy, since MAMC and HAMC excitation and emission wavelengths differed significantly. HPLC analysis confirmed that HAMC was the single metabolic product of MAMC and that HAMC formation accounts for the total increase in fluorescence. It was found that, in microsomes from yeast or lymphoblastoid cells selectively expressing P450 isoenzymes, MAMC was selective for P450 2D6 at a concentration of 25 μM with only P450 1A2 contributing significantly to the formation of HAMC. P450s 2A6, 2B6, 2C8, 2C9, 2C19, 2E1, 3A4, and 3A5 were shown not to metabolize MAMC at a concentration of 25 μM. Km and vmax values of MAMC for P450 2D6 were found to be 26.2 ± 2.8 μM and 2.9 ± 0.07 min^-1, respectively. For P450 1A2, MAMC was found to have a Km value of 29.7 ± 6.2 μM and a vmax of 0.57 ± 0.07 min^-1. Formation of HAMC in human liver microsomes could be completely inhibited by quinidine, at a concentration of 0.5 μM selective for P450 2D6, and furafylline, at a concentration of 30 μM selective for P450 1A2. In conclusion, O-demethylation of 7-methoxy-4-(aminomethyl)coumarin is a rapid and easily determined parameter for P450 2D6 activity and, due to the fluorescent properties of the metabolite formed, may be a valuable new tool for high-throughput screening purposes.

The recent development of several promising new thiourea-containing drugs has renewed interest in the thiourea functionality as a potential toxicophore. Most adverse reactions of thiourea-containing compounds are attributed to the thionocarbonyl moiety. Oxidation of these thionocarbonyl compounds by flavin-containing monooxygenases (FMO) and cytochrome P450 isoenzymes (P450) to reactive sulfenic, sulfinic, or sulfonic acids leads to alkylation of essential macromolecules. To more rationally design thiourea-containing drugs, structure−toxicity relationships (STRs) must be derived. Since for the development of STRs a large number of thiourea-containing compounds must be investigated, it is important to develop rapid in vitro assays for alkylating potential. In this study, the utility of activation of microsomal glutathione S-transferase (mGST) and inactivation of P450 1A1 as markers of the alkylating potential of metabolites of thiourea-containing compounds was investigated. It was found that metabolites of thiourea-containing compounds inactivate P450 1A1 in a time-dependent manner, as evidenced by a decrease in 7-ethoxyresorufin O-dealkylation (EROD) activity. An extent of inactivation of P450 1A1 by 100 μM N-phenylthiourea (PTU) of 64% was found after 10 min. This inactivation was dependent on the presence of NADPH and the presence of the thionosulfur, since the carbonyl analogue of PTU was not found to inactivate P450 1A1, and was partially prevented by heat treatment of the microsomes which is known to selectively inactivate FMO enzymes. Inactivation of P450 1A1 could be reversed by treatment with dithiothreitol, indicating the formation of disulfide bonds. However, thiourea-containing compounds also inhibited the EROD activity of P450 1A1 in a competitive manner. This property complicates the usefulness of the EROD activity of P450 1A1 as a marker for the alkylating potential of thiourea-containing compounds. It was found that metabolites of thiourea-containing compounds could transiently activate the mGST. A maximal level of activation by 100 μM PTU of 162 ± 16% was found after 10 min. Activation of mGST by 100 μM PTU was dependent on the presence of NADPH and the presence of the thionosulfur, since the carbonyl analogue of PTU was not found to activate mGST. Activation was completely prevented by heat treatment of the microsomes, indicating involvement of FMO in the bioactivation process. Finally, a series of structurally diverse thiourea-containing compounds were tested for their ability to activate mGST. It appeared that their potency in alkylating mGST was inversely related to their Vmax/Km value for the FMO enzyme. From this study, it is concluded that, whereas activation of mGST in rat liver microsomes may be a useful system with which to investigate the relationship between structure and alkylating potential of thiourea-containing compounds in vitro, inactivation of P450 1A1 is not.

The metabolic clearance (CL) and half-life of racemic hexobarbitone and sleeping time were studied in rats following intra-arterial (i.a.), intraperitoneal (i.p.) and oral (p.o.) administration, at dose levels of 25 and 100 mg kg−1of its sodium salt. CLp.o.was higher than CLi.a.at both 25 and 100 mg kg−1. CLi.a.and CLi.p.values were much lower, but CLi.p.was higher than CLi.a.at 25 mg kg−1and lower than CLi.a.at 100 mg kg−1. There was no distinct dependency of the half-lives on route of administration, but a slight increase upon increasing the dose was observed. Hexobarbitone blood concentrations at which the rats awoke were significantly higher after 100 mg kg−1i.p. than after 100 mg kg−1i.a., although there was only a small difference in sleeping time. It is postulated that the rate of uptake of the barbiturates into the portal system after i.p. administration is so high that transient saturation of hepatic first-pass metabolism occurs. Therefore neither CLi.p.nor sleeping times can be used as an accurate reflection of drug-metabolizing enzyme activity in the rat; instead CLp.o.should be used.

The electron impact and chemical ionization (ammonia and methane) mass spectral behaviour of the cis- and trans-(2-hydroxycyclohexyl)-mercapturic acid methyl esters, metabolites of cyclohexeneoxide, was examined. Only with chemical ionization was it possible to differentiate between these two configurational isomers, by observing the stereoselective loss of H2O from the protonated molecular ions. Using this stereoselective process, a method was developed for the assessment of the relative amounts of stereoisomeric mercapturic acids, excreted as metabolites in the urine of rats treated with cyclohexeneoxide.

TOXICOLOGY: A PRIMER ON TOXICOLOGY PRINCIPLES AND APPLICATIONS, edited by M. A. Kamrin. Pages I-XIII and 1-145. ISBN 0-873711335, Lewis Publishers, Chelsea, Michigan (1988), $21.85ENVIRONMENTAL TOXICOLOGY: ORGANIC POLLUTANTS by J. K. Fawell and S. Hunt. Pages 1-440. ISBN 0-7458-0194-3, Ellis Harwood Ltd., Publishers, Chichester, UK (1988).BASIC GAS CHROMATOGRAPHY—MASS SPECTROMETRY, PRINCIPLES AND TECHNIQUES by F. W. Karasek and R. R. Clement, Elsevier Science Publishers (1988).GC/MS, A KNOWLEDGE BASE, by F. A. Settle and M. A. Pleva, Elsevier Science Publishers (1988).

We have studied the effects of oxygen radical scavengers on the inactivation of ss ΦX174 DNA by the semi-quinone free radical of the antitumor agent etoposide (VP 16-213), which was generated from the ortho-quinone of etoposide at pH ≥ 7.4. A semi-quinone free radical of etoposide is thought to play a role in the inactivation of ss ΦDX174 DNA by its precursors 3',4'-ortho-quinone and 3',4'-ortho-dihydroxy-derivative. The possible role of oxygen radicals formed secondary to semi-quinone formation in the inactivation of DNA by the semi-quinone free radical was investigated using the hydroxyl radical scavengers t-butanol and DMSO. the spin trap DMPO, the enzymes catalase and superoxide dismutase, the iron chelator EDTA and potassium superoxide. Hydroxyl radicals seem not important in the process of inactivation of DNA by the semi-quinone free radical, since t-butanol, DMSO, catalase and EDTA had no inhibitory effect on DNA inactivation. The spin trapping agent DMPO strongly inhibited DNA inactivation and semi-quinone formation from the ortho-quinone of etoposide at pH ≥ 7.4 with the concomitant formation of a DMPO-OH adduct. This adduct probably did not arise from OH· trapping but from trapping of O2-. DMSO increased both the semi-quinone formation from and the DNA inactivation by the ortho-quinone of etoposide at pH ≥ 7.4. Potassium superoxide also stimulated ΦDX174 DNA inactivation by the ortho-quinone at pH ≤ 7. From the present study, it is also concluded that superoxide anion radicals probably play an important role in the formation of the semi-quinone free radical from the orthoquinone of etoposide, thus indirectly influencing DNA inactivation.

Summary Voluntary bystanders, simulating a situation of non-occupational exposure to Z- and E-1,3-dichloropropene (Z- and E-DCP), were exposed during field application of this nematocide in the Dutch flower-bulb culture. Environmental monitoring revealed that mean respiratory exposure concentrations of Z- and E-DCP varied from non-detectable levels to 1.12 mg/m3 8-h time-weighted average (TWA) for Z-DCP and to 0.91 mg/m3 8-h TWA for E-DCP. Biological monitoring was executed by determining urinary mercapturic acid metabolites of Z- and E-DCP according to a method recently validated in occupationally exposed applicators. A linear relationship between respiratory exposure to Z-and E-DCP and the urinary excretion of both mercapturic acids was observed in bystanders. Dermal uptake did not contribute significantly to the internal dose of Z- or E-DCP. The urinary mercapturic acid of Z-DCP was a more sensitive parameter for the detection of exposure than was respiratory air monitoring. In future studies it would be worthwhile to determine the extent of exposure of real bystanders to DCP on the basis of urinary mercapturic acid excretion.

In the present study structure−activity relationships (SAR's) are described for the experimentally determined kinetic parameters (Km, kcat, and kcat/Km) of the GST 4-4-catalyzed reaction between GSH and 10 2-substituted 1-chloro-4-nitrobenzenes. Steric, lipophilic, and electronic parameters were correlated with the kinetic parameters. Moreover, charge distributions and several energy values were calculated for the substrates and the corresponding Meisenheimer intermediates with MeS^- as a model nucleophile for the thiolate anion of GSH and used in the regression analyses. The correlations obtained were compared with the corresponding SAR's for the base-catalyzed GSH conjugation reaction at pH 9.2. A high correlation coefficient was found between the kinetic parameter ks for the base-catalyzed reaction and the Hammett substituent constant (σp). Much lower correlation coefficients were obtained with kcat and σp and with kcat/Km and σp. Moreover, the reaction constant ρ was significantly higher for the base-catalyzed than for the enzyme-catalyzed reaction. Also, high correlations were found between the kinetic parameters and the charges on the p-nitro substituent in the substrates. When ks was plotted against these charges, a linear relationship was found in which the slope was larger than the slope of a corresponding plot with kcat/Km. The Hammett σp can be divided into an inductive (F) and a resonance (R) component. With multiple regression between the kinetic parameters and F and R, higher correlation coefficients were obtained than with σp alone. Our observations suggest that the transition states for the base-catalyzed and the GST 4-4-catalyzed GSH conjugation reaction are different. Moreover, single classical physicochemical and computer-calculated molecular parameters and combinations of them can be an alternative approach for examining SAR's for spontaneous and GST-catalyzed GSH conjugation reactions.

Glutathione S-transferases (GSTs) are an important class of phase II (de)toxifying enzymes, catalyzing the conjugation of glutathione (GSH) to electrophilic species. Recently, a number of cytosolic GSTs was crystallized. In the present study, molecular modeling techniques have been used to derive a three-dimensional homology model for rat GST 4-4 based upon the crystal structure of rat GST 3-3, both members of the mu class. GST 3-3 and GST 4-4 isoenzymes share a sequence homology of 88%. GST 4-4 distinguishes itself from GST 3-3 in being much more efficient and stereoselective in the nucleophilic addition of GSH to epoxides and α,β-unsaturated ketones. GST 3-3, however, is much more efficient in catalyzing nucleophilic aromatic substitution reactions. In this study, several known substrates of GST 4-4 were selected and their GSH conjugates docked into the active site of GST 4-4. GSH conjugates of phenanthrene 9(S),10(R)-oxide and 4,5-diazaphenanthrene 9(S),10(R)-oxide were docked into the active site of both GST 3-3 and GST 4-4. From these homology modeling and docking data, the difference in stereoselectivity between GST 3-3 and GST 4-4 for the R- and S-configured carbons of the oxirane moiety could be rationalized. The data acquired from a recently derived small molecule model for GST 4-4 substrates were compared with the results of the present protein homology model of GST 4-4. The energy optimized positions of the conjugates in the protein model agreed very well with the original relative positions of the substrates within the substrate model, confirming the usefulness of small molecule models in the absence of structural protein data. The protein homology model, together with the substrate model, will be useful to further rationalize the substrate selectivity of GST 4-4, and to identify new potential GST 4-4 substrates.

A stereospecific synthesis of N1-(2H3)-labelled R(-)-hexobarbital is described. A sensitive and rapid selected ion monitoring assay procedure for pseudoracemic hexobarbital, consisting of equal amounts of S(+)-hexobarbital (1a) and (2H3)-R(-)-hexobarbital (1b) in 100 µl blood samples of rats was developed. Both the parent enantiomers in blood and three major metabolites excreted in urine were quantified. An application of the method in rats is described, and the results are compared to previously obtained data of separately administered enantiomers.

The electron impact mass spectral behaviour of 14 synthetically prepared mercapturic acid methyl esters was examined. The most important and characteristic fragmentation reactions are discussed and special emphasis is made on the occurrence and the mechanism of two fragmentation pathways, which were recognized as ‘retro-Michael’ reactions. In principle, each reaction may result in the formation of two typical ions. In addition, three characteristic ‘key-ions’ originating from the N-acetyl(-l-)cysteine methyl ester part of the mercapturic acids were recognized in all mercapturic acid methyl esters investigated; these ‘key-ions’ can most probably be used as a tool for the qualitative as well as the quantitative analysis of mercapturic acids by gas chromatogra–phy mass spectrometry in biological fluids.

The cytochrome P-450-mediated activation of phenacetin (PHEN) to reactive intermediates by two hypothetical mechanisms has been studied by use of SV 6-31G ab initio energy and spin distribution calculations. In our calculations, the cytochrome P-450 enzyme system has been substituted by a singlet oxygen atom in order to reduce the computational efforts and to fulfill the requirements as to spin conservation. Both mechanisms are based on the currently increasingly accepted view that radical intermediates, formed via sequential one-electron steps, play a crucial role in the metabolic activation of substrates by cytochrome P-450. The first pathway is proposed to involve an initial abstraction of an electron and a proton from the alpha-methylene carbon atom in the ethoxy side chain and can explain the O-deethylation products paracetamol and acetaldehyde. In the second pathway, an initial abstraction of an electron and a proton from the nitrogen atom in the acetylamino side chain is proposed. The calculated spin densities of the formed nitrogen radical indicate that the unpaired electron is primarily localized at the nitrogen atom and to a smaller extent at the ortho- and paracarbon atoms relative to the acetylamino group. Radical recombination reactions between a hydroxyl radical and the spin delocalization-radicalized reactive centers of the nitrogen radical can explain the formation of the metabolites N-hydroxy-PHEN, 2-hydroxy-PHEN, and the arylating metabolite N-acetyl-p-benzoquinone imine (NAPQI), which forms a 3-(S-glutathionyl)paracetamol conjugate in the presence of glutathione. NAPQI is proposed to be formed via intermediate formation of a hemiketal. Proposals are made for the decomposition of this hemiketal into NAPQI that are consistent with currently available experimental data on 14C- and 18O-labeled PHEN.

The therapeutic effect of ebselen has been linked to its peroxidase activity. In the present study, the peroxidase activity of ebselen toward H2O2 with the endogenous thiols GSH and dihydrolipoate [L(SH)2] as cofactors was determined. When GSH was used, peroxide removal was described by a ter uni ping pong mechanism with Dalziel coefficients for GSH and H2O2 of 0.165 +/- 0.011 and 0.081 +/- 0.005 mM min, respectively. When L(SH)2 was used, peroxidase activity was independent of the concentration of L(SH)2 in the concentration range studied (5 microM to 2 mM) and peroxide removal was only dependent on the concentration of H2O2 and ebselen, with the second-order rate constant being 12.3 +/- 0.8 mM-1 min-1. To elucidate the difference between GSH and L(SH)2, the molecular mechanism of the peroxidase activity of ebselen was investigated, using UV spectrophotometry, high pressure liquid chromatography, 77Se NMR, and mass spectrometry. GSH was found to react quickly with ebselen to give a selenenyl sulfide, an adduct of GSH to ebselen. Subsequently, the GSH-selenenyl sulfide is converted into the diselenide of ebselen. Finally the diselenide reacts with a peroxide and ebselen is regenerated. The formation by GSH of the diselenide from the GSH-selenenyl sulfide of ebselen is slow and linearly dependent on the concentration of free thiol; however, no net consumption of GSH was observed. Furthermore, it is likely that a selenol is an intermediate in diselenide formation. After reaction between ebselen and L(SH)2 the diselenide of ebselen was immediately detected. The fast formation of the diselenide with L(SH)2 versus the slow formation of the diselenide with GSH accounts for our observation that L(SH)2 is a better cofactor than GSH in the peroxidase activity of ebselen. Our results suggest that the interaction between ebselen and L(SH)2 might be of major importance in the mechanism by which ebselen exerts its therapeutic effect.