A once-per-day, drug-in-food protocol for prolonged administration of antiepileptic drugs in animal models.

PURPOSE: Convenient and effective methods for administering potential antiepileptic drugs (AEDs) chronically should facilitate many experiments in animal models of chronic epilepsy with spontaneous recurrent seizures. This proof-of-principle study aimed to optimize a once-per-day, drug-in-food protocol by testing the effect of carbamazepine (CBZ) on the frequency of convulsive seizures in rats with kainate-induced epilepsy. METHODS: Adult male rats were given repeated low-dose kainate injections until convulsive status epilepticus persisted for >3 h. After the rats developed spontaneous recurrent seizures, food pellets with CBZ (30, 100, or 300 mg/kg/day) were provided once per day in three 2-week trials (n = 7-9 rats) involving 5 days of CBZ or control treatment, separated by two recovery days within a trial. The total amount of food provided and consumed per day corresponded to a normal caloric diet (60 g/kg/day). KEY FINDINGS: When provided once per day, all animals ate the CBZ-containing food irregularly but continuously throughout the 24-h day. With this daily feeding protocol, CBZ significantly reduced the frequency of spontaneous convulsive seizures in a dose-dependent manner. It is important to note that the effect of CBZ was consistent across the 5 days and throughout each day of the trials. With food administered at 9:00 a.m., and blood assayed at 5:00 p.m., higher food levels of CBZ resulted in higher plasma concentrations of CBZ. SIGNIFICANCE: This AED-in-food protocol is simple, efficient, inexpensive, reliable, and noninvasive; it allows easier long-term drug administration and is less stressful and more humane than other methods of AED administration.

Major differences among chemopreventive organoselenocompounds in the sustained elevation of cytoprotective genes.

Cytoprotective enzyme elevation through the nuclear erythroid 2 p45-related factor 2 (Nrf2)-Kelch-like ECH-associated protein 1/antioxidant response element pathway has been promulgated for cancer prevention. This study compares the redox insult and sustained cytoprotective enzyme elevation by organoselenocompounds and sulforaphane (SF) in lung cells. SF elicited a rise in reactive oxygen species (ROS) and drop in glutathione (GSH) at 2 h; nuclear accumulation of Nrf2 at 4 h; and a GSH rebound and elevation in NAD(P)H quinone oxidoreductase (NQO1), thioredoxin reductase (TR1), and glutamate-cysteine ligase (GCL) at 24 h. Selenocystine (SECY) elicited a similar 24 h response, despite lesser earlier time-point changes. 2-Cyclohexylselenazolidine-4-carboxylic acid effects were similar to SECY's but with a larger Nrf2 change and the largest 24 h increase in GSH, GCL, TR1, and NQO1 of any compound investigated. Selenomethionine elicited a similar acute rise in ROS, but lesser depletion of GSH, no 4 h increase in nuclear Nrf2, only minor 24 h elevations in TR1 and NQO1, and a GCL elevation insufficient to elevate GSH.

Uroporphyria in the Cyp1a2-/- mouse.

Cytochrome P4501A2 (Cyp1a2) is important in the development of uroporphyria in mice, a model of porphyria cutanea tarda in humans. Heretofore, mice homozygous for the Cyp1a2-/- mutation do not develop uroporphyria with treatment regimens that result in uroporphyria in wild-type mice. Here we report uroporphyria development in Cyp1a2-/- mice additionally null for both alleles of the hemochromatosis (Hfe) gene and heterozygous for deletion of the uroporphyrinogen decarboxylase (Urod) gene (genotype: Cyp1a2-/-;Hfe-/-;Urod+/-), demonstrating that upon adding porphyria-predisposing genetic manipulations, Cyp1a2 is not essential. Cyp1a2-/-;Hfe-/-;Urod+/- mice were treated with various combinations of an iron-enriched diet, parenteral iron-dextran, drinking water containing δ-aminolevulinic acid and intraperitoneal Aroclor 1254 (a polychlorinated biphenyl mixture) and analyzed for uroporphyrin accumulation. Animals fed an iron-enriched diet alone did not develop uroporphyria but uroporphyria developed with all treatments that included iron supplementation and δ-aminolevulinic acid, even with a regimen without Aroclor 1254. Hepatic porphyrin levels correlated with low UROD activity and high levels of an inhibitor of UROD but marked variability in the magnitude of the porphyric response was present in all treatment groups. Gene expression profiling revealed no major differences between genetically identical triple cross mice exhibiting high and low magnitude porphyric responses from iron-enriched diet and iron-dextran supplementation, and δ-aminolevulinic acid. Even though the variation in porphyric response did not parallel the hepatic iron concentration, the results are compatible with the presence of a Cyp1a2-independent, iron-dependent pathway for the generation of uroporphomethene, the UROD inhibitor required for the expression of uroporphyria in mice and PCT in humans.

Nanosilver particle effects on drug metabolism in vitro.

Nanosilver particles are present in consumer and health care products. Their effects on human microsomal cytochrome P450 (P450) activities and induction in luciferase reporter-engineered Caco-2 (MDR1.C) and HepG2 (DPX2 and 1A2DRE) cells have been investigated. The LD(50) values were ∼ 4 µg silver/ml for HepG2 and 5 µg/ml for Caco-2 cells. At silver concentrations that showed no decreased cell viability (<1 µg silver/ml), the pregnane X receptor (PXR)-driven 4.5-fold induction response of MDR1.C cells to 50 µM omeprazole was unaffected. In DPX2 cells, the PXR-driven 5.5- and 6.5-fold induction responses to omeprazole and 10 µM rifampicin were attenuated to 4- and 3.5-fold, respectively. Nanosilver particles alone showed no induction. In 1A2DRE cells, the aryl hydrocarbon receptor-driven 5.5-fold induction response to omeprazole was attenuated to 4-fold. In 1A2DRE cells, nanosilver alone elicited slight induction at 1 µg/ml. The inhibition of human P450-selective activities by nanosilver particles in vitro was proportional to the silver/microsomal protein ratio. At a fixed (0.5 mg/ml) protein concentration, P450-selective activities differed in sensitivity (IC(50) value). Coumarin 7-hydroxylation and 7-ethoxy-4-trifluoromethylcoumarin O-deethylation exhibited the highest IC(50) values (33.5 and 31.9 µM, respectively) and S-mephenytoin 4-hydroxylation exhibited the lowest (6.4 µM). Other IC(50) values were, in ascending order, 8.0 to 9.3 µM (testosterone 6ß-hydroxylation, 7-benzyloxyquinoline debenzylation, and diclofenac 4-hydroxylation), 16.0 µM (chlorzoxazone 6-hydroxylation), 21.2 µM [7-methoxy-4-(aminomethyl)-coumarin O-demethylation], and 24.4 µM (7-methoxyresorufin O-demethylation). An investigation of 70 µM nanosilver particles showed that microsomal NADPH cytochrome c reductase activities were inhibited <12%. From our in vitro observations, we extrapolated that nanosilver particles reaching the liver may be a potential source of drug-drug interactions.

2-Diethylaminoethyl-2,2-diphenylvalerate-HCl (SKF525A) revisited: comparative cytochrome P450 inhibition in human liver microsomes by SKF525A, its metabolites, and SKF-acid and SKF-alcohol.

When incubated with human liver microsomes, 2-diethylaminoethyl-2,2-diphenylvalerate-HCl (SKF525A) undergoes cytochrome P450 (P450)-dependent oxidative N-deethylation to the secondary amine metabolite 2-ethylaminoethyl-2,2-diphenylvalerate (SKF8742). P450-selective inhibitors indicated CYP3As catalyzed this reaction, and the deethylation rate correlated best with the CYP3A activity across a range of human liver microsomes. SKF525A and its metabolite and primary amine analog all inhibited CYP2B6-, CYP2C9-, CYP2C19-, CYP2D6-, and CYP3A-selective reactions to varying degrees but had little effect on CYP1A2, CYP2A6, and CYP2E1 reactions. Only the inhibition of CYP3A showed major enhancement when the inhibitors were preincubated with NADPH-fortified microsomes, and the extent of metabolic intermediate (MI) complex formation approximated typical CYP3A content. Two "lost with time" SKF525A derivatives devoid of the ethylamine moiety, 2,2-diphenylpropylethanol (SKF-Alcohol) and 2,2-diphenylpropylacetic acid (SKF-Acid) did not form an MI complex and were identified as selective inhibitors of CYP2C9. Although without detectable metabolism, their CYP2C9 inhibition fitted best with a competitive mechanism. Thus, not all the human P450s are inhibited by SKF525A and related compounds, and the mechanisms contributing to those that are inhibited vary with the isoform. P450 MI-complex formation only seems to play a role with CYP3As.

The anticonvulsant profile of rufinamide (CGP 33101) in rodent seizure models.

PURPOSE: To evaluate the anticonvulsant profile and behavioral toxicity of rufinamide in animal seizure models compared to the established antiepileptic drugs (AEDs): phenytoin, phenobarbital, valproate, and ethosuximide, or vehicle. METHODS: In acute studies of anticonvulsant efficacy, the AEDs were administered via oral (CF1 mice and Sprague-Dawley rats) and intraperitoneal (CF1 mice) routes. The AEDs were assessed for their ability to inhibit seizures induced by maximal electroshock (MES) or subcutaneous pentylenetetrazol, and ability to block seizures induced by subcutaneous strychnine, bicuculline, or picrotoxin. Tolerance of oral rufinamide was assessed in rats following 5-day (versus single-dose) treatment with oral rufinamide using the dose equivalent necessary to achieve a 50% decrease in seizure frequency (ED(50)). Metabolic tolerance was also evaluated using an in vitro liver microsomal assay. RESULTS: Oral rufinamide suppressed pentylenetetrazol-induced seizures in mice (ED(50) 45.8 mg/kg) but not rats, and was active against MES-induced tonic seizures in mice (ED(50) 23.9 mg/kg) and rats (ED(50) 6.1 mg/kg). Intraperitoneal rufinamide suppressed pentylenetetrazol-, bicuculline-, and picrotoxin-induced clonus in mice (ED(50) 54.0, 50.5, and 76.3 mg/kg, respectively). Rufinamide was partially effective in the mouse strychnine test. The behavioral toxicity of rufinamide was similar to or better than established AEDs tested in this study. In general, the protective index of rufinamide was greater than that of the other AEDs. CONCLUSIONS: The efficacy and behavioral toxicity profiles in these animal models suggest that rufinamide may be effective in the treatment of generalized and partial seizures.

Modulation of redox status in human lung cell lines by organoselenocompounds: selenazolidines, selenomethionine, and methylseleninic acid.

Cancer prevention strategies utilizing selenium-containing compounds have demonstrated reduced cancer mortality and efficacy for some cancer types but considerable differences in cellular effects exist among the selenocompounds employed. The variability of the effects on cell viability, redox modulation, and disruption of subcellular compartments by the conventional selenium-containing amino acid, selenomethionine, the oxidized selenosugar metabolite, methylseleninic acid, and selenazolidines was investigated in A549 and BEAS-2B human lung cell lines. Selenomethionine had little effect whereas methylseleninic acid increased cellular thiols and stress in the endoplasmic reticulum. The cyclohexylselenazolidine increased mild oxidative stress in the adenocarcinoma cell line, A549, but the effects were attenuated in the normal, but virally transformed cell line, BEAS-2B. These data demonstrate that all selenocompounds are not equal and that the form of the organic selenocompound is a major determinant in the expected cellular response.

Pre- and post-initiation chemoprevention activity of 2-alkyl/aryl selenazolidine-4(R)-carboxylic acids against tobacco-derived nitrosamine (NNK)-induced lung tumors in the A/J mouse.

The efficacy of a series of 2-aryl/alkyl selenazolidine-4(R)-carboxylic acids (SCAs) in reducing NNK [4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone]-induced lung adenomas in female A/J mice, a model for tobacco-related lung tumorigenesis, has been investigated. With selenazolidines in the diet for 1 month prior to carcinogen administration and during the subsequent 4 months of tumor development, 2-butylSCA, 2-cyclohexylSCA, 2-phenylSCA and 2-oxoSCA were chemopreventive, significantly reducing mean lung tumor numbers from the 10.9 of unsupplemented controls to 4.7, 5.3, 2.8 and 4.7, respectively. When selenazolidine supplementation began three days after carcinogen administration (i.e., post-initiation), 2-butylSCA, 2-cyclohexylSCA, and 2-oxoSCA were chemopreventive. In both regimens, selenocystine was also chemopreventive. In the post-initiation protocol, but with intervention at a precancerous stage (13 days), whole genome expression analysis of lung RNA identified six gene transcripts that weakly correlated with the efficacy of tumor reduction by the four selenocompounds at 4 months. None of these genes were among those identified to be influenced by chemopreventive selenium compounds in human lung cancer cell lines. When supplementation was for 1 month-prior until 3 days-after carcinogen administration, 2-butylSCA, and 2-phenylSCA were chemopreventive but selenocystine was ineffective. Both 2-butylSCA and 2-phenylSCA retained their chemopreventive activity (44% and 40% tumor number reduction, respectively), when the supplementation was shortened and restricted to a pre-initiation period (days -9 to -2). With supplementation spanning 2 days-prior until 3 days-after NNK, reductions in tumor numbers by 2-phenylSCA (26%) and 2-butylSCA (17%) did not achieve statistical significance. Thus, several 2-aryl/alkyl selenazolidines possess chemopreventive activity against NNK-induced lung tumors, and variously demonstrate pre-initiation and post-initiation efficacy.

The antimutagenicity of 2-substituted selenazolidine-4-(R)-carboxylic acids.

Selenium can have cancer chemopreventive activity, although the mechanism of action has not been well defined. Selenazolidine-4-(R)-carboxylic acids (SCAs) were devised as prodrugs of L-selenocysteine, to provide selenium in a form and at a concentration commensurate with cancer chemopreventive activity. In the present study, a series of selenazolidines has been evaluated in the Salmonella typhimurium TA98 tester strain and all were found to possess antimutagenic activity. There was little difference between the seven selenazolidines in their effectiveness against either benzo[a]pyrene (B[a]P) or 3,6-bis(dimethylamino)acridine (acridine orange), agents which differ in their requirement for mammalian enzyme bioactivation for mutagenicity. Antimutagenic activity against acridine orange was dependent on selenazolidine concentration, and EC50 values were in the 5-10 microM range. At 25 microM, the concentration tested in common for the two mutagens, the selenazolidines were more effective antimutagens against acridine orange than against B[a]P, with reductions in mutant frequency ranging from 54 to 71% for B[a]P and 79 to 93% for acridine orange. Efficacy against B[a]P was not enhanced when the concentration was increased to 50 microM. The similarity in efficacy among the selenazolidines against B[a]P mutagenicity, contrasted with inter-compound differences in their ability to inhibit S9 CYP1A activity. The CYP1A Ki values ranged from a low of 63 microM (2-[2'-hydroxyphenyl]SCA) to a high of 1.1mM (2-cyclohexylSCA), but all were above the concentration required to inhibit mutagenicity by 50%. Thus, all the SCAs possess antimutagenic activity against both B[a]P and acridine orange, the efficacy varies little between the individual selenazolidines, and for B[a]P, the efficacy is not proportional to the inhibitory effect on the mutagen bioactivating enzyme.

Murine hepatoma (Hepa1c1c7) cells: a responsive in vitro system for chemoprotective enzyme induction by organoselenium compounds.

Murine (Hepa1c1c7) hepatoma cells are a suitable in vitro system for investigating the regulation of chemoprotective enzymes by selenazolidines, novel l-selenocysteine prodrugs developed as potential chemopreventive agents. They are less sensitive to the cytotoxic effects of both selenite and the less toxic selenazolidines than rat hepatoma (H4IIE) cells. All four selenazolidine 4-carboxylic acid (SCA) derivatives examined elevated thioredoxin reductase (Txnrd1), alpha-class glutathione transferases (Gsta), and UDP-glucuronosyltransferase (Ugt)1a6 mRNAs. NAD(P)H-quinone oxidoreductase (Nqo1) was induced by the three 2-alkyl derivatives (2-cyclohexylSCA, 2-butylSCA, and 2-methylSCA) but not SCA itself. Transcripts of mu- and pi-class glutathione transferases were induced only by 2-cyclohexylSCA and 2-butylSCA. Only Gsta and Txnrd1 transcripts were elevated by l-selenomethionine, l-selenocystine, or Se-methyl-l-selenocysteine. Txnrd1, Gsta, Nqo1, and Gstp responses to selenazolidines were all abolished by actinomycin D while Ugt1a6 responses were not. Induction responses to the selenazolidines were also eliminated (most) or reduced (Txnrd1 by 2-methylSCA) by cycloheximide, with the exception of Ugt1a6. The Ugt1a6 mRNA levels in the presence of SCAs and cycloheximide were similar to those with cycloheximide alone, and were almost double those of vehicle-treated cells. Thus, Hepa1c1c7 cells appear to provide a viable platform for the study of protective enzyme regulation by selenocompounds, and with the exception of Ugt1a6, the mRNA elevations from selenazolidines are transcriptionally dependent.

Effect of selenium-containing compounds on hepatic chemoprotective enzymes in mice.

Selenite and organoselenium compounds have been examined at supranutritional levels for their ability to influence the activity and mRNA levels of chemoprotective enzymes in the livers of selenium-sufficient mice and the changes compared to those elicited by oltipraz. Compounds investigated included novel selenocysteine prodrugs that have previously been evaluated for their ability to reduce the tumorigenicity of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in mice. Following seven daily doses (i.g.), all compounds except 2-methylselenazolidine-4(R)-carboxylic acid (MSCA) increased thioredoxin reductase activity (43-92%) but only for 2-oxoselenazolidine-4(R)-carboxylic acid (OSCA) was there an accompanying increase in mRNA. No compound enhanced glutathione peroxidase activity, although sodium selenite significantly elevated the mRNA of this enzyme. Oltipraz was an efficacious inducer of both thioredoxin reductase and glutathione peroxidase mRNAs. Sodium selenite, selenazolidine-4(R)-carboxylic acid (SCA), and OSCA elevated NAD(P)H-quinone oxidoreductase mRNA but only for OSCA was the elevation in mRNA accompanied by an increase in enzyme activity. L-Selenocystine significantly increased this activity without increasing mRNA levels. Sodium selenite, L-selenocystine, L-selenomethionine, and Se-methyl-L-selenocysteine all enhanced glutathione S-transferase activity. The increased activity with sodium selenite was accompanied by increases in mRNAs of Gst alpha, Gst mu and Gst pi classes, while for L-selenocystine and Se-methyl-L-selenocysteine, only an elevation in the mRNA for the Gst alpha class was observed. Gst alpha and Gst mu class mRNAs were elevated by OSCA without a significant elevation in enzyme activity. SCA and MSCA both elevated a Gst pi mRNA and MSCA elevated Gst mu in addition. By comparison, oltipraz only significantly elevated the mRNA of Gst mu, adding to the conclusion that across the entire study, no selenium compound appears to be acting purely through the antioxidant response typified by oltipraz. Despite their chemical similarity, the three cysteine prodrugs, SCA, MSCA, and OSCA, each produced its own unique pattern of effects on protective enzymes and none was identical to the pattern elicited by sodium selenite, L-selenocystine, L-selenomethionine, and Se-methyl-L-selenocysteine. The study also shows that after 7 days of administration, there was only occasional concordance between elevations in mRNA and enzyme activity for any selenium compound and for any protective enzyme, there was no response in common for all selenium compounds.

Acute effects of novel selenazolidines on murine chemoprotective enzymes.

Novel selenazolidines, designed as l-selenocysteine prodrugs and potential cancer chemopreventive agents, were examined for their ability to affect the transcription of murine hepatic chemoprotective enzymes. Compounds investigated were selenazolidine-4(R)-carboxylic acid (SCA) and six 2-substituted derivatives that cover a C log P range of -0.512 to -3.062. Their biological effects were compared with those of L-selenocystine. Gene transcripts were examined 24 h after a single dose, administered i.p. and i.g., and covered a range of chemoprotective enzymes; alpha, mu and pi class glutathione transferases (Gsts), UDP-glucuronosyltransferases (Ugts) 1a1, 1a6, 1a9, and 2b5, glutathione peroxidase 1 (Gpx), thioredoxin reductase (Tr), NAD(P)H-quinone oxidoreductase 1 (Nqo), and microsomal epoxide hydrolase (Meh). When given i.g., 2-butyl SCA (BSCA) resulted in elevations in alpha, mu and pi class Gsts, Ugt1a6, Tr, and Gpx, and 2-phenyl SCA (PhSCA) elevated GstP, Ugt1a9, Tr, Gpx (3 kb), and Meh. Other derivatives with C log P values both lower [2-(2'-hydroxy)phenyl SCA (PhOHSCA) and 2-methyl SCA (MSCA)] and higher [2-cyclohexyl SCA (ChSCA) and 2-oxo SCA (OSCA)] than BSCA and PhSCA elevated far fewer transcripts; PhOHSCA (Ugt1a1, Gpx), MSCA (Ugt1a1, Meh), ChSCA (Ugt1a1, Ugt1a9), and OSCA (Ugt1a6, Ugt1a9, GstM). When given i.p., the most pervasive transcript changes were parallel increases in Nqo and Tr transcripts which occurred with BSCA, PhSCA, MSCA, and OSCA. PhSCA also increased GstP, and PhOHSCA increased Ugt1a1 and Ugt1a6 levels. Unique among the compounds, PhSCA reduced the transcript levels of GstA, and the 1.6 kb transcript of Gpx although only when given i.p. Neither l-selenocystine nor SCA affected the level of any transcript and no compound altered the amount of Ugt2b5 mRNA. Despite chemical similarity and common ability to potentially serve as a source of l-selenocysteine, each selenazolidine compound appeared to elicit a unique pattern of mRNA responses and by either route of administration, there was no correlation between the magnitude of response of any gene and the calculated C log P values of the organoselenium compounds.

Traditional Preparations and Methanol Extracts of Medicinal Plants from Papua New Guinea Exhibit Similar Cytochrome P450 Inhibition.

The hypothesis underlying this current work is that fresh juice expressed from Papua New Guinea (PNG) medicinal plants (succus) will inhibit human Cytochrome P450s (CYPs). The CYP inhibitory activity identified in fresh material was compared with inhibition in methanol extracts of dried material. Succus is the most common method of traditional medicine (TM) preparation for consumption in PNG. There is increasing concern that TMs might antagonize or complicate drug therapy. We have previously shown that methanol extracts of commonly consumed PNG medicinal plants are able to induce and/or inhibit human CYPs in vitro. In this current work plant succus was prepared from fresh plant leaves. Inhibition of three major CYPs was determined using human liver microsomes and enzyme-selective model substrates. Of 15 species tested, succus from 6/15 was found to inhibit CYP1A2, 7/15 inhibited CYP3A4, and 4/15 inhibited CYP2D6. Chi-squared tests determined differences in inhibitory activity between succus and methanol preparations. Over 80% agreement was found. Thus, fresh juice from PNG medicinal plants does exhibit the potential to complicate drug therapy in at risk populations. Further, the general reproducibility of these findings suggests that methanol extraction of dried material is a reasonable surrogate preparation method for fresh plant samples.

Attenuation of polychlorinated biphenyl induced uroporphyria by iron deprivation.

A toxic sequel to polyhalogenated aromatic hydrocarbon exposure in humans is the development of porphyria cutanea tarda. In a mouse model (experimental uroporphyria) utilizing an environmentally relevant polychlorinated biphenyl (PCB) mixture, we show that the toxicity can be markedly influenced by nutritional status. In mice made susceptible to uroporphyria through a targeted deletion of one allele of uroporphyrinogen decarboxylase (Uro-D+/-), an iron deficient diet prevented the development of the uroporphyria and the changes in associated parameters normally seen within three weeks following a single exposure to Aroclor 1254. Iron deprivation also completely prevented PCB-induced uroporphyria in mice wild-type at the Uro-D locus (Uro-D+/+), a model that requires δ-aminolevulinic acid administration for the development of uroporphyria. In Uro-D+/- mice consuming δ-aminolevulinic acid, PCB exposure produced a severe uroporphyria that was attenuated, not prevented, by iron deficiency. This attenuation moderated hepatic uroporphyrin and uroporphyrinogen decarboxylase inhibitor levels, but not the depression of cytosolic uroporphyrinogen decarboxylase activity.

A method for determining δ-aminolevulinic acid synthase activity in homogenized cells and tissues.

OBJECTIVE: In mammalian cells the rate-limiting step in heme biosynthesis is the formation of δ-aminolevulinic acid (ALA). The reaction intermediates, porphyrins and iron and the final product, heme can be highly cytotoxic if allowed to accumulate. The importance of maintaining the levels of metabolic intermediates and heme within a narrow range is apparent based on the complex homeostatic system(s) that have developed. Ultimately, determining the enzymatic activity of ALA synthase (ALAS) present in the mitochondria is highly beneficial to confirm the effects of the transcriptional, translational and post-translational events. The aim of this study was to develop a highly sensitive assay for ALAS that could be used on whole tissue or cellular homogenates. DESIGN AND METHODS: A systematic approach was used to optimize steps in formation of ALA by ALAS. Reducing the signal to noise ratio for the assay was achieved by derivatizing the ALA formed into a fluorescent product that could be efficiently separated by ultra performance liquid chromatography (UPLC) from other derivatized primary amines. The stability of ALAS activity in whole tissue homogenate and cellular homogenate was determined after extended storage at -80 °C. CONCLUSIONS: A method for assaying ALAS has been developed that can be used with tissue homogenates or cellular lysates. There is no need to purify mitochondria and radiolabeled substrates are not needed for this assay. General laboratory reagents can be used to prepare the samples. Standard UPLC chromatography will resolve the derivatized ALA peak. Samples of tissue homogenate can be stored for approximately one year without significant loss of enzymatic activity.

Modulation of phase II drug metabolizing enzyme activities by N-heterocycles.

Drugs and other chemicals that contain one or more heterocyclic nitrogen atoms are most widely known for their ability to both inhibit and induce cytochrome p450s. Their ability to affect a wide range of Phase II drug metabolizing enzyme activities has received much less attention and exposure. For some N-heterocycles, induction of Phase II metabolism occurs in the absence of induction effects on cytochrome p450, a property with potential utility in reducing drug and chemical toxicity (chemoprotection). The mechanism of this induction, and an accounting of the Phase II selectivity, has not been rigorously investigated. This review gathers and documents existing information on the simultaneous inductive effects of N-heterocycles on glucuronidation, sulfation (sulfonation) and glutathione conjugation reactions, as well epoxide hydrolase and quinone oxidoreductase activities. Basic information on cytochrome p450 induction is provided for comparative purposes. The review will serve as a base for any future interest in the phenomenon. The existing information on enzyme changes derives largely from laboratory animal studies. Chemicals investigated range from both simple and complex pyridines and imidazoles to benzoquinolines and phenanthrolines. Isomeric differences can have a major impact on induction characteristics and Phase II selectivity. Promising indications for the effectiveness of N-heterocycle elicited enzyme changes in chemoprotection in vivo awaits investigation.

Interactions of Papua New Guinea medicinal plant extracts with antiretroviral therapy.

ETHNOPHARMACOLOGICAL RELEVANCE: A substantial proportion of the population in Papua New Guinea (PNG) lives with human immunodeficiency virus (HIV). Treatment requires lifelong use of antiretroviral therapy (ART). The majority of people in PNG use traditional medicines (TM) derived from plants for all types of health promotions. Consequently, there is a concern that herb-drug interactions may impact the efficacy of ART. Herb-drug, or drug-drug, interactions occur at the level of metabolism through two major mechanisms: enzyme induction or enzyme inhibition. In this study, extracts of commonly-used medicinal plants from PNG were screened for herb-drug interactions related to cytochrome P450s (CYPs). MATERIALS AND METHODS: Sixty nine methanol extracts of TM plants were screened for their ability to induce CYPs by human aryl hydrocarbon receptor- (hAhR-) and human pregnane X receptor- (hPXR-) dependent mechanisms, utilizing a commercially available cell-based luciferase reporter system. Inhibition of three major CYPs, CYP1A2, CYP3A4, and CYP2D6, was determined using human liver microsomes and enzyme-selective model substrates. RESULTS: Almost one third of the TM plant extracts induced the hAhR-dependent expression of CYP1A2, the hPXR-dependent expression of CYP3A4, or both. Almost two thirds inhibited CYP1A2, CYP3A4, or CYP2D6, or combinations thereof. Many plant extracts exhibited both induction and inhibition properties. CONCLUSIONS: We demonstrated that the potent and selective ability of extracts from PNG medicinal plants to affect drug metabolizing enzymes through induction and/or inhibition is a common phenomenon. Use of traditional medicines concomitantly with ART could dramatically alter the concentrations of antiretroviral drugs in the body; and their efficacy. PNG healthcare providers should counsel HIV patients because of this consequence.

Comparative analysis of human CYP3A4 and rat CYP3A1 induction and relevant gene expression by bisphenol A and diethylstilbestrol: implications for toxicity testing paradigms.

Bisphenol A (BPA) and diethylstilbestrol (DES) are endocrine-disrupting chemicals that interact with the human pregnane X receptor (PXR). CYP3A4 enzyme is essential in the hydroxylation of steroid hormones and is regulated by PXR. In the present study, human and rat hepatoma cell lines were exposed to BPA and DES. Both BPA and DES (10-50µM) caused a significant activation of the CYP3A4 promoter via the PXR in the DPX2 human hepatoma cell line. No activation of rat PXR was seen. BPA and DES treated DPX2 cells demonstrated increased expression of CYP3A4 mRNA, and increased enzyme activity. In summary, BPA, in concentrations relevant to current safety levels of human exposure, activates the human PXR and demonstrates an increase in CYP3A4 mRNA expression and enzyme activity. BPA actions in this model system occur to a greater extent than DES. This study raises concerns regarding our current toxicity testing paradigms and species utilization.

Hyper- and hypo-induction of cytochrome P450 activities with Aroclor 1254 and 3-methylcholanthrene in Cyp1a2(-/-) mice.

The response of hepatic mono-oxygenase activities to Aroclor 1254 or 3-methylcholanthrene was investigated in wild-type and Cyp1a2(-/-) mice. Cytochrome P450 concentrations were similar in naïve Cyp1a2(-/-) and wild-type mice. There was no difference between naïve wild-type and Cyp1a2(-/-) animals in 7-ethoxyresorufin and 7-ethoxy-4-trifluoromethylcoumarin dealkylase activities, nor was the induction response after 3-methylcholanthrene any different between the two genotypes. However, both activities were induced to a higher extent in Cyp1a2(-/-) mice after Aroclor 1254. In contrast, 7-pentoxyresorufin dealkylation activity was lower in Cyp1a2(-/-) mice and this differential was maintained during induction by both agents. 7-Methoxy- and 7-benzoxyresorufin dealkylation activities were also lower than wild-type in naïve Cyp1a2(-/-) animals and during 3-methylcholanthrene induction, but showed accelerated induction in Cyp1a2(-/-) mice with Aroclor 1254. Bufuralol 1'- and testosterone 6beta-hydroxylation activities, and P450 characteristics were evaluated 48h after inducer administration. Bufuralol 1'-hydroxylation, a sexual dimorphic activity (female>male) showed no genotype differences in naïve animals. Activity changes varied across gender and genotype, with 3-methylcholanthrene and Aroclor 1254 inducing in male Cyp1a2(-/-), and Aroclor 1254 inducing in female wild-type. Testosterone 6beta-hydroxylation activity was 16% higher in Cyp1a2(-/-) mice and neither 3-methylcholanthrene nor Aroclor 1254 elicited induction. After Aroclor 1254, a 24% increase in P450 concentration with a hypsochromic shift in the ferrous-CO maximum characteristic of CYP1A enzymes occurred in wild-type, compared to no change in either parameter in Cyp1a2(-/-) mice. Induction changes with 3-methylcholanthrene were greater in wild-type mice, a 60% increase in concentration and approximately 2 nm hypsochromic shift versus a 10% increase and approximately 1nm hypsochromic shift in Cyp1a2(-/-) mice. The study demonstrates that deletion of a single P450 can profoundly affect the induction response, as monitored with activities of other P450s, in a manner unrelated to the contribution of the deleted P450 to the activity.