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PubMed2000

Acylation of protein lysines by trichloroethylene oxide.

Stable lysine adducts were formed in proteins following reaction with trichloroethylene (TCE) oxide, the major reactive compound generated by the metabolism of TCE. The order of formation of these adducts is N(6)-formyllysine > N(6)-(dichloroacetyl)lysine >> N(6)-glyoxyllysine, with the ratio being influenced by the particular protein. Protein lysine adducts were also analyzed following the enzymatic oxidation of TCE with several different cytochrome P450 (P450) enzyme systems. The ratio of formyl/dichloroacetyl lysine adducts was influenced by the enzyme system that was used. Chloral and TCE oxide formation was more extensive with rat liver microsomes isolated from phenobarbital-treated rats than with rat microsomes in which P450 2E1 was induced by treatment with isoniazid or in human P450 2E1 systems. Glutathione (GSH) and GSH transferase had inhibitory effects on the reaction of TCE oxide with albumin, with formylation being atteunated much more than the formation of dichloroacetyllysine. GSH is likely to react with the reactive acyl chloride intermediates formed from TCE oxide hydrolysis, instead of direct reaction with TCE oxide, as judged by the lack of an effect of GSH on the rate of decomposition of TCE oxide. Studies with the model enzymes aldolase and glucose-6-phosphate dehydrogenase, both known to have sensitive lysine groups, indicate that TCE oxide has effects similar to known acylating agents that form the same adducts; concentrations of TCE oxide (or the model acylating agents) in the low-millimolar range were needed for inhibition. The characterization of TCE-derived protein adducts can be used as a basis for consideration of the exposure and risk of TCE to humans. Human P450 2E1 was less likely to oxidize TCE to form TCE oxide and protein lysine adducts than rat P450 2B1, and the difference is rationalized in terms of the influence of the protein on chloride migration in an enzyme reaction intermediate.

Acetylation↗
PubMed2001

Unexpected formation of an epoxide-derived multisubstrate adduct inhibitor on the active site of GAR transformylase.

Multisubstrate adduct inhibitors (MAI) of glycinamide ribonucleotide transformylase (GAR Tfase), which incorporate key features of the folate cofactor and the beta-GAR substrate, typically exhibit K(i)'s in the picomolar range. However, these compounds have reduced bioavailability due to the incorporation of a negatively charged phosphate moiety that prevents effective cellular uptake. Thus, a folate analogue that is capable of adduct formation with the substrate on the enzyme active site could lead to a potent GAR Tfase inhibitor that takes advantage of the cellular folate transport systems. We synthesized a dibromide folate analogue, 10-bromo-10-bromomethyl-5,8,10-trideazafolic acid, that was an intermediate designed to assemble with the substrate beta-GAR on the enzyme active site. We have now determined the crystal structure of the Escherichia coli GAR Tfase/MAI complex at 1.6 A resolution to ascertain the nature and mechanism of its time-dependent inhibition. The high-resolution crystal structure clearly revealed the existence of a covalent adduct between the substrate beta-GAR and the folate analogue (K(i) = 20 microM). However, the electron density map surprisingly indicated a C10 hydroxyl in the adduct rather than a bromide and suggested that the multisubstrate adduct is not formed directly from the dibromide but proceeds via an epoxide. Subsequently, we demonstrated the in situ conversion of the dibromide to the epoxide. Moreover, synthesis of the authentic epoxide confirmed that its inhibitory, time-dependent, and cytotoxic properties are comparable to those of the dibromide. Further, inhibition was strongest when the dibromide or epoxide is preincubated with both enzyme and substrate, indicating that inhibition occurs via the enzyme-dependent formation of the multisubstrate adduct. Thus, the crystal structure revealed the successful formation of an enzyme-assembled multisubstrate adduct and highlighted a potential application for epoxides, and perhaps aziridines, in the design of efficacious GAR Tfase inhibitors.

Binding Sites↗
PubMed2002

8-(Hydroxymethyl)-3,N(4)-etheno-C, a potential carcinogenic glycidaldehyde product, miscodes in vitro using mammalian DNA polymerases.

8-(Hydroxymethyl)-3,N(4)-etheno-C (8-HM-epsilonC) is an exocyclic adduct resulting from the reaction of dC with glycidaldehyde, a mutagen and animal carcinogen. This compound has now been synthesized and its phosphoramidite incorporated site-specifically into a defined 25-mer oligonucleotide. In this study, the mutagenic potential of this adduct in the 25-mer oligonucleotide was investigated in an in vitro primer-template extension assay using four mammalian DNA polymerases. The miscoding potentials were also compared to those of an analogous derivative, 3,N(4)-etheno C (epsilonC), in the same sequence. Both adducts primarily blocked replication by calf thymus DNA polymerase alpha at the modified base, while human polymerase beta catalyzed measurable replication synthesis through both adducts. Nucleotide insertion experiments showed that dA and dC were incorporated by pol beta opposite either adduct, which would result in a C --> T transition or C --> G transversion. Human polymerase eta, a product of the xeroderma pigmentosum variant (XP-V) gene, catalyzed the most efficient bypass of the two lesions with 25% and 32% for 8-HM-epsilonC and epsilonC bypassed after 15 min. Varying amounts of all four bases opposite the modified bases resulted with pol eta. Human polymerase kappa primarily blocked synthesis at the base prior to the adduct. However, some specific misincorporation of dT resulted, forming an epsilonC.T or 8-HM-epsilonC.T pair. From these data, we conclude that the newly synthesized glycidaldehyde-derived adduct, 8-HM-epsilonC, is a miscoding lesion. The bypass efficiency and insertion specificity of 8-HM-epsilonC and epsilonC were similar for all four polymerases tested, which could be attributed to the similar planarity and sugar conformations for these two derivatives as demonstrated by molecular modeling studies.

Aldehydes↗
PubMed2002

Etomoxir-induced oxidative stress in HepG2 cells detected by differential gene expression is confirmed biochemically.

Although they are known to be effective antidiabetic agents, little is published about the toxic effects of carnitine palmitoyltransferase-1 (CPT-1) inhibitors, such as etomoxir (ET). These compounds inhibit mitochondrial fatty acid beta-oxidation by irreversibly binding to CPT-1 and preventing entry of long chain fatty acids into the mitochondrial matrix. Treatment of HepG2 cells with 1 mM etomoxir for 6 h caused significant modulations in the expression of several redox-related and cell cycle mRNAs as measured by microarray analysis. Upregulated mRNAs included heme oxygenase 1 (HO1), 8-oxoguanine DNA glycosylase 1 (OGG1), glutathione reductase (GSR), cyclin-dependent kinase inhibitor 1A (CDKN1 [p21(waf1)]) and Mn+ superoxide dismutase precursor (SOD2); while cytochrome P450 1A1 (CYP1A1) and heat shock 70kD protein 1 (HSPA1A) were downregulated. Real time quantitative PCR (RT-PCR) confirmed the significant changes in 4 of 4 mRNAs assayed (CYP1A1, HO1, GSR, CDKN1), and identified 3 additional mRNA changes; 2 redox-related genes, gamma-glutamate-cysteine ligase modifier subunit (GCLM) and thioredoxin reductase (TXNRD1) and 1 DNA replication gene, topoisomerase IIalpha (TOP2A). Temporal changes in selected mRNA levels were examined by RT-PCR over 11 time points from 15 min to 24 h postdosing. CYP1A1 exhibited a 38-fold decrease by 4 h, which rebounded to a 39-fold increase by 20 h. GCLM and TXNRD1 exhibited 13- and 9-fold increases, respectively at 24 h. Etomoxir-induced oxidative stress and impaired mitochondrial energy metabolism were confirmed by a significant decrease in reduced glutathione (GSH), reduced/oxidized glutathione ratio (GSH/GSSG), mitochondrial membrane potential (MMP), and ATP levels, and by concurrent increase in oxidized glutathione (GSSG) and superoxide generation. This is the first report of oxidative stress caused by etomoxir.

Carcinoma, Hepatocellular↗
PubMed1997

Disposition of butadiene epoxides in Sprague-Dawley rats.

1,2-Epoxybutene (BMO) and diepoxybutane (BDE) are metabolic products of 1,3-butadiene in rodents. Both BMO and BDE are suspect in the development of tumors in rats and mice. To understand the distribution and elimination of these compounds in the absence of the rate-limiting production from butadiene, the pharmacokinetics of BMO and BDE in blood were determined in adult male Sprague-Dawley rats following intravenous administration. All animals were dually cannulated in these studies. For the BMO studies, rats were dosed with 71, 143, or 286 mumol/kg BMO (n = 3 for each dose group). For the BDE studies, rats were dosed with 523 mumol/kg BDE (n = 3). All animals tolerated the BMO and BDE doses without grossly observable adverse effects. Blood was drawn at predetermined time points and extracted in methylene chloride. BDE and BMO concentrations were quantitated by gas chromatography or gas chromatography/mass spectrometry. The BMO distribution half-lives were short and ranged from 1.4 min at the lowest dose to 1.8 min at the highest dose. Volume of distribution at steady state ranged from 0.53 +/- 0.17 to 0.59 +/- 0.31 l/kg. Systemic clearances ranged from 67 +/- 17 to 114 +/- 20 ml/min per kg. The terminal elimination half-lives were also short and ranged from 5.7 to 8.5 min among the doses. The pharmacokinetic parameters after an i.v. dose of 523 mumol/kg BDE were a distribution half-life of 2.7 min, terminal elimination T1/2 of 14 min, volume of distribution at steady state of 0.73 +/- 0.06 l/kg, and systemic clearance of 76 +/- 8 ml/min per kg. These pharmacokinetic parameters demonstrate the similarity between disposition of the two epoxides in rats, that include a rapid distribution after i.v. administration into a small extravascular body compartment as well as a rapid elimination from blood. These pharmacokinetic data provide useful blood clearance information for assessing the critical physiological and biochemical determinants underlying the disposition of butadiene epoxides.

Animals↗
PubMed1980

Evaluation of metabolic activation of 7,12-dimethylbenz(a)anthracene in vitro by aroclor 1254-induced rat liver S-9 fraction.

Short-term assays for detection of chemical carcinogens frequently rely on an Aroclor 1254-induced rat liver S-9 fraction for metabolic activation of test compounds. The ability of this in vitro system to reproduce the activation occurring in target tissue was investigated by examining the DNA adducts produced when the polycyclic aromatic hydrocarbon carcinogen, 7,12-dimethylbenz(a)anthracene (DMBA), was incubated with the S-9 fraction and calf thymus DNA. Analyses by Sephadex LH-20 column chromatography of hydrocarbon-deoxyribonucleoside adducts obtained after enzymic digestion of the [3H]DMBA-modified DNA revealed that the products of binding of DMBA to DNA in the presence of the S-9 fraction vary with the relative concentration of DMBA to S-9 fraction. Further analyses of these adducts by high-pressure liquid chromatography in the presence of the diol-epoxide-DNA adduct (isolated from mouse embryo cells exposed to [14C]DMBA) and chemically synthesized ultraviolet-absorbing markers of DMBA 5,6-oxide-deoxyribonucleoside adducts showed that, at high DMBA-S-9 ratios, DMBA 5,6-oxide-deoxyriboiucleoside adducts were prominent among the products while, at low DMBA-S-9 ratios, the products included the diol-epoxide-DNA adduct found in target tissue. However, this adduct was always accompanied by other adducts not found in intact cellular systems. Inclusion of a metabolic inhibitor (1,1,1-trichloropropylene oxide) in the Salmonella mutagenicity assay demonstrated that high levels of revertants can be obtained from rat liver S-9 fraction-activated DMBA under conditions which should prohibit formation of the diol-epoxide. These results suggest that Aroclor 1254-induced rat liver S-9 fraction does not exactly reproduce the metabolic activation of this particular carcinogen in vivo and therefore should not be assumed to do this for other carcinogens.

9,10-Dimethyl-1,2-benzanthracene↗
PubMedDate not supplied

Identification of epoxide- and quinone-derived bromobenzene adducts to protein sulfur nucleophiles.

Bromobenzene (BB) hepatotoxicity is widely attributed to the alkylation of cellular proteins by chemically reactive metabolites, particularly BB-3,4-oxide. This laboratory recently reported the first conclusive evidence that BB epoxides actually do alkylate proteins; i.e., acid hydrolysates of hepatic proteins from phenobarbital-(PB-) induced BB-treated rats contain S-(o-, S-(m-, and S-(p-bromophenyl)cysteine [Weller, P.E., and Hanzlik, R.P. (1991) Chem. Res. Toxicol. 4, 17-20]. However, these three compounds account for less than 0.5% of total protein covalent binding. Bromoquinone metabolites of BB are also suspected of alkylating proteins. To search for such adducts to protein cysteinyl or methionyl residues, we heated hepatic proteins from PB-induced BB-treated rats with a two-phase mixture of 16 N KOH and CH3I ("alkaline permethylation"). Under these conditions S-alkylated residues are cleaved via elimination and the phenoxide and thiophenoxide groups on the fragments are methylated. Product analysis by 14C HPLC and GC/MS revealed o-, m-, and p-bromothioanisoles in amounts comparable to the content of S-(bromophenyl)cysteines found by acid hydrolysis (para much greater than meta, ortho). This, too, clearly implicates protein-SH alkylation by BB-2,3- and 3,4-oxides. In addition, 2,3-dimethoxy-5-bromothioanisole and another unidentified isomer were observed. However, by far the major adduct (5-6% of total covalent binding) was 2,5-dimethoxythioanisole (i.e., a debrominated adduct). When BB-d5 was administered, the latter contained mostly 3 deuterium atoms/mol. These latter results clearly show that alkylation of protein sulfur nucleophiles in vivo by quinone metabolites is 10-15 times more extensive than their alkylation by BB epoxides. After BB-d5 was administered, the bromothioanisoles and dimethoxybromothioanisoles contained 4 and 2 deuterium atoms/mol, respectively. A weighted average calculation of deuterium retention across the six major sulfur adducts agreed well with 3H/14C retention ratios determined earlier for total liver protein covalent binding of dual-labeled [3H/14C]BB, indicating that the overall pattern of BB metabolite binding to all protein nucleophiles may closely parallel that seen here specifically for protein sulfhydryl groups. The identification of a variety of specific BB-derived adducts to protein now affords the opportunity to investigate their relative contributions to the toxicity of bromobenzene.

Alkylation↗
PubMed1997

Comparison of the disposition of butadiene epoxides in Sprague-Dawley rats and B6C3F1 mice following a single and repeated exposures to 1,3-butadiene via inhalation.

1,3-Butadiene (BD), a compound used extensively in the rubber industry, is a potent carcinogen in mice and a weak carcinogen in rats in chronic carcinogenicity bioassays. While many chemicals are known to alter their own metabolism after repeated exposures, the effect of exposure prior to BD on its in vivo metabolism has not been reported. The purpose of the present research was to examine the effect of repeated exposure to BD on tissue concentrations of two mutagenic BD metabolites, butadiene monoepoxide (BDO) and butadiene diepoxide (BDO2). Concentrations of BD epoxides were compared in several tissues of rats and mice following a single exposure or ten repeated exposures to a target concentration of 62.5 ppm BD. Female Sprague-Dawley rats and female B6C3F1 mice were exposed to BD for 6 h or 6 h x 10 days. BDO and BDO2 were quantified in blood and several other tissues following preparation by cryogenic vacuum distillation and analysis by multidimensional gas chromatography-mass spectrometry. Blood and lung BDO concentrations did not differ significantly (P < or = 0.05) between the two exposure regimens in either species. Following multiple exposures to BD, BDO levels were 5- and 1.6-fold higher (P < or = 0.05) in mammary tissue and 2- and 1.4-fold higher in fat tissue of rats and mice, respectively, as compared with single exposures. BDO2 levels also increased in rat fat tissue following multiple exposures to BD. However, in mice, levels of this metabolite decreased by 15% in fat, by 28% in mammary tissue and by 34% in lung tissue following repeated exposures to BD. The finding that the mutagenic epoxide BDO, which is the precursor to the highly mutagenic BDO2, accumulates in rodent fat may be important in assessing the potential risk to humans from inhalation of BD.

Adipose Tissue↗
PubMed1987

Acute and 2-week inhalation toxicity studies on aerosols of selected ethylene oxide/propylene oxide polymers in rats.

The ethylene oxide/propylene oxide (EO/PO) polymers evaluated in this study have previously been shown to have a low order of toxicity and/or irritancy by ocular, dermal, or oral routes of administration. These studies evaluated the acute inhalation toxicity of respirable aerosols of three EO/PO compounds (U-660, U-2000, and U-5100) that differ in chain length, molecular weight, and viscosity. The respective 4-hr LC50 values (95% confidence limits) for U-660, U-2000, and U-5100 in Wistar albino rats were 4670 (4090-5320), 330 (227-480), and 106 (45-245) mg/m3. Occasionally, slight increases in respiration rate and slight hyperactivity were observed during the postexposure period. All deaths were delayed for 2-5 days postexposure. Body weight gains were transiently depressed in rats exposed to U-2000 and U-5100. Discolored lungs and livers occurred in animals which died during the 14-day postexposure period. Subsequently, a repeated-exposure study was conducted on U-5100 in F-344 rats exposed for 6 hr/day, 5 days/week, for 9 exposures at mean concentrations of 0, 5, 26, and 50 mg/m3. Portions of the control and 50 mg/m3 groups were maintained for an additional 2-week recovery period. Exposure-related effects included transient urogenital wetness in 50 mg/m3 group females; decreased body weight gain (7-29%) in all U-5100 groups except the 5 mg/m3 group females; increases in absolute (17-52%) and relative lung weights in all U-5100 groups; macroscopic red foci in the lungs; and microscopic findings of congestion and hemorrhage of pulmonary alveolar capillaries and necrosis of alveolar epithelial cells. Lung weights remained elevated after the 2-week recovery period, but the severity of the microscopic lesions was noticeably less, indicating partial reversibility of the lesions. In conclusion, EO/PO polymers have a higher order of toxicity by inhalation in comparison to other routes of administration, vary considerably in their acute lethal toxicity as a function of chain length/molecular weight, and induce pulmonary hemorrhage, and possibly edema, following repeated aerosol exposures at concentrations as low as 5 mg/m3.

Aerosols↗
PubMed1999

Production in vitro by the cytochrome P450 CYP94A1 of major C18 cutin monomers and potential messengers in plant-pathogen interactions: enantioselectivity studies.

The major C(18) cutin monomers are 18-hydroxy-9,10-epoxystearic and 9,10,18-trihydroxystearic acids. These compounds are also known messengers in plant-pathogen interactions. We have previously shown that their common precursor 9,10-epoxystearic acid was formed by the epoxidation of oleic acid in Vicia sativa microsomes (Pinot, Salaün, Bosch, Lesot, Mioskowski and Durst (1992) Biochem. Biophys. Res. Commun. 184, 183-193). Here we determine the chirality of the epoxide produced as (9R,10S) and (9S,10R) in the ratio 90:10 respectively. We further show that microsomes from yeast expressing the cytochrome P450 CYP94A1 are capable of hydroxylating the methyl terminus of 9,10-epoxystearic and 9,10-dihydroxystearic acids in the presence of NADPH to form the corresponding 18-hydroxy derivatives. The reactions were not catalysed by microsomes from yeast transformed with a void plasmid or in absence of NADPH. After incubation of a synthetic racemic mixture of 9,10-epoxystearic acid with microsomes of yeast expressing CYP94A1, the chirality of the residual epoxide was shifted to 66:34 in favour of the (9S,10R) enantiomer. Both enantiomers were incubated separately and V(max)/K(m) values of 16 and 3.42 ml/min per nmol of P450 for (9R, 10S) and (9S,10R) respectively were determined, demonstrating that CYP94A1 is enantioselective for the (9R,10S) enantiomer, which is preferentially formed in V. sativa microsomes. Compared with the epoxide, the diol 9,10-dihydroxystearic acid was a much poorer substrate for the omega-hydroxylase, with a measured V(max)/K(m) of 0.33 ml/min per nmol of P450. Our results indicate that the activity of CYP94A1 is strongly influenced by the stereochemistry of the 9, 10-epoxide and the nature of substituents on carbons 9 and 10, with V(max)/K(m) values for epoxide>>oleic acid>diol.

Chromatography, High Pressure Liquid↗
PubMed2004

Adducts of N-terminal valines in hemoglobin with isoprene diepoxide, a metabolite of isoprene.

Isoprene (2-methylbuta-1,3-diene) is a multi-site carcinogen in rodents. To evaluate the role of the diepoxide metabolite (1,2:3,4-diepoxy-2-methylbutane) in carcinogenesis, measurements of in vivo doses of the diepoxide are needed. The in vivo dose may be inferred from levels of reaction products with hemoglobin (Hb adducts). This report presents in vitro studies of the adduct formation by the diepoxide of isoprene with valinamide and oligopeptides as model compounds of N-terminal valines in hemoglobin (Hb). In the reaction with valinamide it was shown that isoprene diepoxide forms as the main product a ring-closed adduct, which is a pyrrolidine derivative [N,N-(2,3-dihydroxy-2-methyl-1,4-butadiyl)valinamide, MPyr-Val]. The analysis was performed by gas chromatography/mass spectrometry (GC/MS) (EI and PICI) after acetylation. The ring-closed adduct was also identified by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) as the main product in the reaction between isoprene diepoxide and standard hepta- or (2H8)octapeptides, corresponding to the N-terminal peptides of the alpha-chains in mouse and rat Hb. These peptides, alkylated with isoprene diepoxide, to be used as internal standards and calibration standards for quantification of MPyr-adduct levels in vitro and in vivo, were analyzed with respect to the degree of MPyr-alkylation by two independent methods, amino acid analysis and HPLC-UV; similar results were obtained using these methods. A method for measurement of Hb adducts as modified peptides, used earlier to measure a similar adduct to N-terminal valines in Hb from the diepoxide of 1,3-butadiene, has in the present work been tested for application to isoprene diepoxide. The method is based on tryptic degradation of globin and LC/ESI-MS analysis of N-terminal Pyr-heptapeptides of the Hb alpha-chain enriched by HPLC. MPyr-adduct levels in isoprene diepoxide alkylated hemolysate from mouse erythrocytes incubated with different concentrations of isoprene diepoxide (2 and 10 mM) for 1 h were quantified. The adduct level was about 50 nmol/g alpha-chain Hb per mM x h. From the adduct levels the rate constant of isoprene diepoxide for reaction with N-terminal valine was calculated to be about 1.6 times faster than for diepoxybutane.

Alkylation↗
PubMed2003

Dosimetry by means of DNA and hemoglobin adducts in propylene oxide-exposed rats.

The main purpose of the study was to establish the relation between exposure dose of propylene oxide (PO) and dose in various tissues of male F344 rats exposed to the compound by inhalation. The animals were exposed to 0, 5, 25, 50, 300, or 500 ppm PO in the air for 3 days (6 h/day) or 4 weeks (6 h/day, 5 days/week). Blood, nasal respiratory epithelium, lung, and liver were collected. 2-Hydroxypropylvaline (HPVal) in hemoglobin was quantified using the N-alkyl Edman method and gas chromatography/tandem mass spectrometry. 7-(2-Hydroxypropyl)guanine (7-HPG) in DNA was quantified using (32)P postlabeling. The levels of 7-HPG in DNA of nasal respiratory epithelium and lung increased linearly with concentration as measured both after 3 days and 4 weeks of exposure. Similarly, 7-HPG in liver DNA and HPVal in hemoglobin showed a linear increase with PO concentration in the 3-day exposure group, whereas a deviation from linearity was observed above 300 ppm in the 4-week exposure group. The new results confirm previous observations of a dose difference between tissues with the highest dose present in the nasal respiratory epithelium. The measured adduct levels were used for calculation of adduct increments and corresponding tissue doses per unit of external exposure dose. For this purpose, the buildup of adducts was modeled considering the different kinetics of formation and elimination of adducts with DNA and hemoglobin, respectively, and also considering the increasing body weight of the animals. The half-life of 7-HPG in vivo, as well as tissue doses, could be solved from DNA adduct data at the 3rd and 26th days. Within the range of concentrations where the dose-response curves for adduct formation are linear, the relationship between exposure dose and resulting tissue doses could be based equally well on adduct data from the short-term exposure as on adduct data from the prolonged exposure.

Animals↗
PubMed2006

A novel methionine aminopeptidase-2 inhibitor, PPI-2458, inhibits non-Hodgkin's lymphoma cell proliferation in vitro and in vivo.

PURPOSE: Fumagillin and related compounds have potent antiproliferative activity through inhibition of methionine aminopeptidase-2 (MetAP-2). It has recently been reported that MetAP-2 is highly expressed in germinal center B cells and germinal center-derived non-Hodgkin's lymphomas (NHL), suggesting an important role for MetAP-2 in proliferating B cells. Therefore, we determined the importance of MetAP-2 in normal and transformed germinal center B cells by evaluating the effects of MetAP-2 inhibition on the form and function of germinal centers and germinal center-derived NHL cells. EXPERIMENTAL DESIGN: To examine the activity of PPI-2458 on germinal center morphology, spleen sections from cynomolgus monkeys treated with oral PPI-2458 were analyzed. Antiproliferative activity of PPI-2458 was assessed on germinal center-derived NHL lines in culture. A MetAP-2 pharmacodynamic assay was used to determine cellular MetAP-2 inhibition following PPI-2458 treatment. Finally, inhibition of MetAP-2 and proliferation by PPI-2458 was examined in the human SR NHL line in culture and in implanted xenografts. RESULTS: Oral PPI-2458 caused a reduction in germinal center size and number in lymphoid tissues from treated animals. PPI-2458 potently inhibited growth (GI(50) = 0.2-1.9 nmol/L) of several NHL lines in a manner that correlated with MetAP-2 inhibition. Moreover, orally administered PPI-2458 significantly inhibited SR tumor growth, which correlated with inhibition of tumor MetAP-2 (>85% at 100 mg/kg) in mice. CONCLUSIONS: These results show the potent antiproliferative activity of PPI-2458 on NHL lines in vitro and oral antitumor activity in vivo and suggest the therapeutic potential of PPI-2458 as a novel agent for treatment of NHL should be evaluated in the clinical setting.

Aminopeptidases↗
PubMed2002

Theoretical study of the mechanism of alkane hydroxylation and ethylene epoxidation reactions catalyzed by diiron bis-oxo complexes. The effect of substrate molecules.

The hybrid density functional method B3LYP was used to study the mechanism of the hydrocarbon (methane, ethane, methyl fluoride, and ethylene) oxidation reaction catalyzed by the complexes cis-(H(2)O)(NH(2))Fe(mu-O)(2)(eta(2)-HCOO)(2)Fe(NH(2))(H(2)O), I, and cis-(HCOO)(Imd)Fe(mu-O)(2)(eta(2)-HCOO)(2)Fe(Imd)(HCOO) (Imd = Imidazole), I_m, the "small" and "medium" model of compound Q of the methane monooxygenase (MMO). The improvement of the model from "small" to "medium" did not change the qualitative conclusions but significantly changed the calculated energetics. As in the case of methane oxidation reported by the authors previously, the reaction of all the substrates studied here is shown to start by coordination of the substrate molecule to the bridging oxygen atom, O(1) of I, an Fe(IV)-Fe(IV) complex, followed by the H-atom abstraction at the transition state III leading to the bound hydroxy alkyl intermediate IV of Fe(III)-Fe(IV) core. IV undergoes a very exothermic coupling of alkyl and hydroxy groups to give the alcohol complex VI of Fe(III)-Fe(III) core, from which alcohol dissociates. The H(b)-atom abstraction (or C-H bond activation) barrier at transition state III is found to be a few kcal/mol lower for C(2)H(6) and CH(3)F than for CH(4). The calculated trend in the H(b)-abstraction barrier, CH(4) (21.8 kcal/mol) > CH(3)F (18.8 kcal/mol) > or = C(2)H(6) (18.5 kcal/mol), is consistent with the C-H(b) bond strength in these substrates. Thus, the weaker the C-H(b) bond, the lower is the H(b)-abstraction barrier. It was shown that the replacement of a H-atom in a methane molecule with a more electronegative group tends to make the H(b)-abstraction transition state less "reactant-like". In contrast, the replacement of the H-atom in CH(4) with a less electronegative group makes the H(b)-abstraction transition state more "reactant-like". The epoxidation of ethylene by complex I is found to proceed without barrier and is a highly exothermic process. Thus, in the reaction of ethylene with complex I the only product is expected to be ethylene oxide, which is consistent with the experiment.

Alkanes↗
PubMedDate not supplied

Triptolide: a potential male contraceptive.

The antifertility effect of triptolide and other related compounds, isolated from Tripterygium wilfordii, has been demonstrated in male rats. The exact sites and mechanism of action of triptolide remain unknown. Our objectives were to determine whether triptolide at selected dose levels that induce infertility has any detrimental effects on the testes and to determine the sites and the possible mechanisms of its action. Groups of six adult male Sprague-Dawley rats were given oral administration of either vehicle (control group) or triptolide (50 or 100 microg/kg body weight) daily for 35 or 70 days. Body weight gain was normal in all treated groups. All six rats treated with a high dosage of triptolide were infertile during the second (63-70 days) mating trial. A lower dose (50 microg) of triptolide gave intermediate fertility values. Plasma levels of luteinizing hormone, follicle-stimulating hormone, testosterone, and intratesticular testosterone were not significantly different between control and triptolide-treated groups. Cauda epididymal sperm content was decreased by 68% and the motility, which averaged 58.2% in the control rat, was reduced to almost zero. No effects of triptolide were observed on testis and accessory organs weight, volumes of tubular lumen and the total Leydig cells, tubule diameter, and the number of Sertoli cells, spermatogonia, preleptotene (PL), and pachytene (P) spermatocytes. There were, however, modest but significant decreases in tubule volume and the number of round spermatids at stages VII-VIII. No changes in the germ cell apoptotic index measured at stages VII-VIII and XIV-I were noted between controls and rats rendered infertile with a high dose of triptolide. Thus, triptolide, at a dose level that induces complete infertility in the adult rats, has minimal adverse effects on the testes and acts primarily on the epididymal sperm making triptolide an attractive lead as a post-testicular male contraceptive.

Administration, Oral↗
PubMed1985

Stereoselectivity of microsomal epoxide hydrolase toward diol epoxides and tetrahydroepoxides derived from benz[a]anthracene.

We have examined the selectivity of rat liver microsomal epoxide hydrolase (EC 3.3.2.3) toward all of the possible positional isomers of benzo-ring diol epoxides and tetrahydroepoxides of benz[a]anthracene, as well as the 1,2-diol 3,4-epoxides of triphenylene. This set includes compounds with no bay region in the vicinity of the benzo-ring, a bay-region diol group, a bay-region epoxide group, and (for the triphenylene derivatives) both a bay-region diol and a bay-region epoxide. In all cases where both the tetrahydroepoxides and the corresponding diol epoxides were examined, there is a large retarding effect of hydroxyl substitution on the rate of the enzyme-catalyzed hydration. When the tetrahydroepoxides are fair or poor substrates (epoxide group in the 1,2-, 8,9-, or 10,11-position), the additional retardation introduced by adjacent hydroxyl groups causes the enzyme-catalyzed hydrolysis of the corresponding diol epoxides to be insignificantly slow or nonexistent. In contrast, a benz[a]anthracene derivative with an epoxide group in the 3,4-position, (-)-tetrahydrobenz[a]anthracene (3R,4S)-epoxide, has been identified as the best substrate known for epoxide hydrolase, with a Vmax at 37 degrees C and pH 8.4 of 6800 nmol/min/mg of protein, and the two diastereomeric (+/-)-benz[a]anthracene 1,2-diol 3,4-epoxides, unlike all the other diol epoxides examined to date, are moderately good substrates for epoxide hydrolase. This novel observation is accounted for by the fact that the very high reactivity of the tetrahydrobenz[a]anthracene 3,4-epoxide system towards epoxide hydrolase is large enough to overcome a kinetically unfavorable effect of hydroxyl substitution. The enantioselectivity and positional selectivity of the enzyme have been determined for the tetrahydro-1,2- and -3,4-epoxides of benz[a]anthracene as well as the 1,2-diol 3,4-epoxides. When the epoxide is located in the 3,4-position, the benzylic carbon is the preferred site of attack, whereas for the enantiomers of the bay-region tetrahydro-1,2-epoxides, the chemically less reactive non-benzylic carbon is preferred. The regio- and enantioselectivity of epoxide hydrolase are discussed in terms of a possible model for the hydrophobic binding site of this enzyme.

Animals↗
PubMed2000

Micronuclei and gene mutations in transgenic big Blue((R)) mouse and rat fibroblasts after exposure to the epoxide metabolites of 1, 3-butadiene.

1,3-Butadiene (BD) is a commodity compound and by-product in the manufacture of synthetic rubber that elicits a differential carcinogenic response in rodents after chronic exposure. Mice are up to approximately 1000-fold more sensitive to the tumorigenicity of inhaled BD than rats, thereby confounding human risk assessment analyses. Rodent transgenic in vivo and in vitro models have been recently utilized for generating genetic toxicology data in support of risk assessment studies. However, studies have not been extended to investigate multiple endpoints of genetic damage using in vitro transgenic models. The goal of this study was to evaluate possible differences in the production of genetic damage in transgenic Big Blue((R)) mouse (BBM1) and rat (BBR1) fibroblasts exposed to three predominant epoxide metabolites of BD. Analyses of cytotoxicity, micronucleus (MN) formation, cII mutant frequency (MF) and apoptosis were assessed after in vitro exposure of BBM1 and BBR1 cells exposed to various concentrations of butadiene monoepoxide (BMO), diepoxybutane (DEB) and butadiene diolepoxide (BDE). Both BMO and DEB reduced cell survival in BBM1 and BBR1 cells. However, BDE decreased cell survival only in BBM1 cells at the concentrations evaluated. Concentration-dependent increases in the formation of MN was observed in both BBM1 and BBR1 cells, with DEB being the most potent followed by BDE and then BMO. The dose-response for mutations induced at the cII locus was essentially equal after DEB exposure of BBM1 and BBR1 fibroblasts. In contrast, the cII MF was significantly increased only in BBM1 cells after exposure to either BMO or BDE. These data demonstrate a differential genetic response for gene mutations but not for MN formation in transgenic BBM1 and BBR1 fibroblasts and suggest a rodent species-specific difference in the persistence of DNA damage that results in gene mutations. In addition, apoptosis was observed in BBR1 cells but not in BBM1 cells when treated with any of the three BD epoxide metabolites. This response may partially explain the differential response to mutations induced by BMO and BDE. These data offer insight into specific differences in mouse and rat cells with respect to their response to BD epoxide metabolites. Such data may help to explain the different tumorigenicity results observed in rodent BD carcinogenicity studies.

Analysis of Variance↗
PubMed1995

Gender differences in the metabolism of 1,3-butadiene in Sprague-Dawley rats following a low level inhalation exposure.

1,3-Butadiene (BD), a compound used extensively in the rubber industry, is carcinogenic in the male and female Sprague-Dawley rats after chronic exposures to 1000 and 8000 p.p.m. In terms of incidence of tumors the majority were in mammary tissue, thus the incidence of tumors in female rats exceeded that in males in chronic carcinogenicity studies. In the present study the production and disposition of butadiene monoepoxide (BDO) and butadiene diepoxide (BDO2), mutagenic BD metabolites, were examined in male and female Sprague-Dawley rats following a low level inhalation exposure to BD. The rats were exposed to a target concentration of 62.5 p.p.m. BD by nasal inhalation for 6 h. Immediately after exposure blood, bone marrow, lung and fat samples were removed from all the animals and mammary tissue was removed from females. The samples were prepared by cryogenic-vacuum line distillation and analyzed for the epoxides using multidimensional gas chromatography-mass spectrometry. Levels of BDO in the blood were 25.9 +/- 2.9 and 29.4 +/- 2 pmol/g in male and females respectively. The levels of this metabolite were also similar in males and females in the other tissues examined. The greatest amounts of BDO were in fat (175 +/- 21 and 203 +/- 13 pmol/g in males and females respectively). Levels of BDO2 were approximately 5-fold greater in the blood of female rats compared with male rats. In the other tissues examined BDO2 was also consistently greater in tissues from females. In fat BDO2 was present at a concentration of 7.7 +/- 1.3 and 1.1 +/- 0.1 pmol/g tissue in females and males respectively. Mammary tissue from female rats contained 10.5 +/- 2.4 pmol/g BDO2, a level slightly lower than that observed in blood. The ratios of the two epoxides differed markedly between males and females in all tissues examined. Differences were most pronounced in lung and fat tissues, where BDO/BDO2 ratios were 9 and 0.6 (lung) and 159 and 26 (fat) for males and females respectively. This study is the first to describe a gender difference in the metabolism of BD. The greater production of the highly mutagenic BDO2 in females may play a role in the increased incidence of mammary tumors after chronic exposure to BD.

Administration, Inhalation↗