The Cytochrome P450 (CYP) 2C8*3 Variant (rs11572080) is Associated with Improved Asthma Symptom Control in Children and Altered Lipid Mediator Production and Inflammatory Response in Human Bronchial Epithelial Cells.

This study investigated an association between the cytochrome P450 (CYP) 2C8*3 polymorphism with asthma symptom control in children and changes in lipid metabolism and pro-inflammatory signaling by human bronchial epithelial cells (HBECs) treated with cigarette smoke condensate (CSC). CYP genes are inherently variable in sequence and while such variations are known to produce clinically relevant effects on drug pharmacokinetics and pharmacodynamics, the effects on endogenous substrate metabolism and associated physiological processes are less understood. In this study, CYP2C8*3 was associated with improved asthma symptom control among children: Mean asthma control scores were 3.68 [n=207] for patients with one or more copies of the CYP2C8*3 allele vs. 4.42 [n=965] for CYP2C8*1/*1 (p=0.0133). In vitro, CYP2C8*3 was associated with an increase in montelukast 36-hydroxylation and a decrease in linoleic acid (LA) metabolism despite lower mRNA and protein expression. Additionally, CYP2C8*3 was associated with reduced mRNA expression of interleukin-6 (IL-6) and C-X-C motif chemokine ligand 8 (CXCL-8) by HBECs in response to CSC, which was replicated using the soluble epoxide hydrolase inhibitor, AUDA. Interestingly, 9(10)- and 12(13)-DiHOME, the hydrolyzed metabolites of 9(10)- and 12(13)-EpOME, increased the expression of IL-6 and CXCL-8 mRNA by HBECs. This study reveals previously undocumented effects of the CYP2C8*3 variant on the response of HBECs to exogenous stimuli. Significance Statement These findings suggest a role for CYP2C8 in regulating the EpOME:DiHOME ratio leading to a change in cellular inflammatory responses elicited by environmental stimuli that exacerbate asthma.

Dynamic Expression of Transient Receptor Potential Vanilloid-3 and Integrated Signaling with Growth Factor Pathways during Lung Epithelial Wound Repair following Wood Smoke Particle and Other Forms of Lung Cell Injury.

Prior studies revealed increased expression of the transient receptor potential vanilloid-3 (TRPV3) ion channel after wood smoke particulate matter (WSPM) treatment of human bronchial epithelial cells (HBECs). TRPV3 attenuated pathologic endoplasmic reticulum stress and cytotoxicity mediated by transient receptor potential ankyrin-1. Here, the basis for how TRPV3 expression is regulated by cell injury and the effects this has on HBEC physiology and WSPM-induced airway remodeling in mice was investigated. TRPV3 mRNA was rapidly increased in HBECs treated with WSPM and after monolayer damage caused by tryptic disruption, scratch wounding, and cell passaging. TRPV3 mRNA abundance varied with time, and stimulated expression occurred independent of new protein synthesis. Overexpression of TRPV3 in HBECs reduced cell migration and wound repair while enhancing cell adhesion. This phenotype correlated with disrupted mRNA expression of ligands of the epidermal growth factor, tumor growth factor-ß, and frizzled receptors. Accordingly, delayed wound repair by TRPV3 overexpressing cells was reversed by growth factor supplementation. In normal HBECs, TRPV3 upregulation was triggered by exogenous growth factor supplementation and was attenuated by inhibitors of growth factor receptor signaling. In mice, subacute oropharyngeal instillation with WSPM also promoted TRPV3 mRNA expression and epithelial remodeling, which was attenuated by TRPV3 antagonist pre- and cotreatment. This latter effect may be the consequence of antagonist-induced TRPV3 expression. These findings provide insights into the roles of TRPV3 in lung epithelial cells under basal and dynamic states, as well as highlight potential roles for TRPV3 ligands in modulating epithelial damage/repair. SIGNIFICANCE STATEMENT: Coordinated epithelial repair is essential for the maintenance of the airways, with deficiencies and exaggerated repair associated with adverse consequences to respiratory health. This study shows that TRPV3, an ion channel, is involved in coordinating repair through integrated repair signaling pathways, wherein TRPV3 expression is upregulated immediately after injury and returns to basal levels as cells complete the repair process. TRPV3 may be a novel target for understanding and/or treating conditions in which airway/lung epithelial repair is not properly orchestrated.

A Phase I Study of Neoadjuvant Chemotherapy With Nab-Paclitaxel, Doxorubicin, and Cyclophosphamide in Patients With Stage II to III Breast Cancer.

BACKGROUND: The aims of this study were to assess the safety and tolerability of nanoparticle albumin bound paclitaxel (nab-paclitaxel), doxorubicin, and cyclophosphamide as combination therapy for breast cancer patients in the neoadjuvant setting and to assess the overall clinical response and pathologic complete response (pCR). PATIENTS AND METHODS: Twenty-six women with newly diagnosed stage II to III histologically or cytologically proven adenocarcinoma of the breast with negative HER2 status were enrolled. Patients were treated with nab-paclitaxel 100 mg/m2, doxorubicin 50 mg/m2, and cyclophosphamide 500 mg/m2 on day 1 and nab-paclitaxel 100 mg/m2 on day 8 in a 21-day cycle for 6 cycles total. RESULTS: Most adverse events attributed to treatment were decreased white blood cell count, neutropenia, anemia, thrombocytopenia, and lymphopenia with a median duration of 8 days. Fifteen of 23 (65.2%; 95% confidence interval [CI], 45.7%-84.6%) had a complete clinical response and 8 of 23 (34.7%; 95% CI, 15.2%-54.1%) had a partial clinical response for an overall clinical response rate of 100%. Thirteen of 23 patients (56.5%; 95% CI, 36.2%-76.7%) had a pCR. All 10 triple-negative breast cancer (TNBC) patients (100%) achieved a pCR. CONCLUSION: The regimen of nab-paclitaxel, doxorubicin, and cyclophosphamide chemotherapy was well tolerated and resulted in high clinical as well as pathologic responses, particularly in TNBC.

Window-of-Opportunity Study of Valproic Acid in Breast Cancer Testing a Gene Expression Biomarker.

PURPOSE: The anticancer activity of valproic acid (VPA) is attributed to the inhibition of histone deacetylase. We previously published the genomically derived sensitivity signature for VPA (GDSS-VPA), a gene expression biomarker that predicts breast cancer sensitivity to VPA in vitro and in vivo. We conducted a window-of-opportunity study that examined the tolerability of VPA and the ability of the GDSS-VPA to predict biologic changes in breast tumors after treatment with VPA. PATIENTS AND METHODS: Eligible women had untreated breast cancer with breast tumors larger than 1.5 cm. After a biopsy, women were given VPA for 7 to 12 days, increasing from 30 mg/kg/d orally divided into two doses per day to a maximum of 50 mg/kg/d. After VPA treatment, serum VPA level was measured and then breast surgery or biopsy was performed. Tumor proliferation was assessed by using Ki-67 immunohistochemistry. Histone acetylation of peripheral blood mononuclear cells was assessed by Western blot. Dynamic contrast-enhanced magnetic resonance imaging scans were performed before and after VPA treatment. RESULTS: Thirty women were evaluable. The median age was 54 years (range, 31-73 years). Fifty-two percent of women tolerated VPA at 50 mg/kg/d, but 10% missed more than two doses as a result of adverse events. Grade 3 adverse events included vomiting and diarrhea (one patient) and fatigue (one patient). The end serum VPA level correlated with a change in histone acetylation of peripheral blood mononuclear cells (ρ = 0.451; P = .024). Fifty percent of women (three of six) with triple-negative breast cancer had a Ki-67 reduction of at least 10% compared with 17% of other women. Women whose tumors had higher GDSS-VPA were more likely to have a Ki-67 decrease of at least 10% (area under the curve, 0.66). CONCLUSION: VPA was well tolerated and there was a significant correlation between serum VPA levels and histone acetylation. VPA treatment caused a decrease in proliferation of breast tumors. The genomic biomarker correlated with decreased proliferation. Inhibition of histone deacetylase is a valid strategy for drug development in triple-negative breast cancer using gene expression biomarkers.

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.

Mushroom Ganoderma lucidum prevents colitis-associated carcinogenesis in mice.

BACKGROUND: Epidemiological studies suggest that mushroom intake is inversely correlated with gastric, gastrointestinal and breast cancers. We have recently demonstrated anticancer and anti-inflammatory activity of triterpene extract isolated from mushroom Ganoderma lucidum (GLT). The aim of the present study was to evaluate whether GLT prevents colitis-associated carcinogenesis in mice. METHODS/PRINCIPAL FINDINGS: Colon carcinogenesis was induced by the food-borne carcinogen (2-Amino-1-methyl-6-phenylimidazol[4,5-b]pyridine [PhIP]) and inflammation (dextran sodium sulfate [DSS]) in mice. Mice were treated with 0, 100, 300 and 500 mg GLT/kg of body weight 3 times per week for 4 months. Cell proliferation, expression of cyclin D1 and COX-2 and macrophage infiltration was assessed by immunohistochemistry. The effect of GLT on XRE/AhR, PXR and rPXR was evaluated by the reporter gene assays. Expression of metabolizing enzymes CYP1A2, CYP3A1 and CYP3A4 in colon tissue was determined by immunohistochemistry. GLT treatment significantly suppressed focal hyperplasia, aberrant crypt foci (ACF) formation and tumor formation in mice exposed to PhIP/DSS. The anti-proliferative effects of GLT were further confirmed by the decreased staining with Ki-67 in colon tissues. PhIP/DSS-induced colon inflammation was demonstrated by the significant shortening of the large intestine and macrophage infiltrations, whereas GLT treatment prevented the shortening of colon lengths, and reduced infiltration of macrophages in colon tissue. GLT treatment also significantly down-regulated PhIP/DSS-dependent expression of cyclin D1, COX-2, CYP1A2 and CYP3A4 in colon tissue. CONCLUSIONS: Our data suggest that GLT could be considered as an alternative dietary approach for the prevention of colitis-associated cancer.

Hippocampal betaine/GABA transporter mRNA expression is not regulated by inflammation or dehydration post-status epilepticus.

Seizure activity can alter GABA transporter and osmoprotective gene expression, which may be involved in the pathogenesis of epilepsy. However, the response of the betaine/GABA transporter (BGT1) is unknown. The goal of the present study was to compare the expression of BGT1 mRNA to that of other osmoprotective genes and GABA transporters following status epilepticus (SE). The possible contributory role of dehydration and inflammation was also investigated because both have been shown to be involved in the regulation of GABA transporter and/or osmoprotective gene expression. BGT1 mRNA was increased 24 h post-SE, as were osmoprotective genes. BGT1 was decreased 72 h and 4 weeks post-SE, as were the GABA transporter mRNAs. The mRNA values for osmoprotective genes following 24-h water withdrawal were significantly lower than the values obtained 24 h post-SE despite similarities in their plasma osmolality values. BGT1 mRNA was not altered by lipopolysaccharide-induced inflammation while the transcription factor tonicity-responsive enhancer binding protein and the GABA transporters 1 and 3 were. These results suggest that neither plasma osmolality nor inflammation fully account for the changes seen in BGT1 mRNA expression post-SE. However, it is evident that BGT1 mRNA expression is altered by SE and displays a temporal pattern with similarities to both GABA and osmolyte transporters. Further investigation of BGT1 regulation in the brain is warranted.

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.

Potent mutagenicity of 3-methylindole requires pulmonary cytochrome P450-mediated bioactivation: a comparison to the prototype cigarette smoke mutagens B(a)P and NNK.

3-Methylindole (3MI) is a preferential pneumotoxicant found in cigarette smoke. A number of lung-expressed human cytochrome P450 enzymes, including 1A1, 2F1, and 2A13, catalyze the metabolism of 3MI to reactive intermediates that fragment DNA, measured with the Comet assay to assess DNA damage, in a cytochrome P450-dependent manner in primary normal human lung cells in culture, but the mutagenesis of 3MI has been controversial. In the present study, the mutagenic potential of 3MI was compared to the prototypical cigarette smoke carcinogens benzo(a)pyrene (B(a)P) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). 3MI, B(a)P, and NNK were incubated with the Salmonella typhimurium strain TA98, which is known to detect the most common subtype of cigarette smoke-induced mutagenicity, frameshift mutations in DNA, and with Salmonella typhimurium strain TA100, which detects base pair substitution mutants, with five sources of P450-mediated bioactivation: rat liver S9, human lung microsomes, recombinant CYP2A13, purified CYP2F3, and recombinant CYP1A1. Only B(a)P was mutagenic in TA100, and it was bioactivated by human lung microsomes and rat liver S9 sources of P450s. However, with the TA98 strain, CYP1A1, CYP2A13, CYP2F3, and human lung microsomes bioactivated 3MI to highly mutagenic intermediates, whereas neither human nor rat liver S9 subcellular fractions formed mutagenic intermediates from 3MI. Quantitative Western blot analysis verified that all three respiratory enzymes were present in human lung microsomes in widely varying amounts. These results indicate that metabolism of 3MI by human lung-expressed cytochrome P450 enzymes but not hepatic P450s elicits equivalent or higher mutagenicity than the prototype cigarette smoke mutagens B(a)P and NNK and indicates that 3MI is a likely human pulmonary carcinogen.

3-Methylindole is mutagenic and a possible pulmonary carcinogen.

Previous work has shown that bioactivation of the cigarette smoke pneumotoxicant 3-methylindole (3MI) by pulmonary cytochrome P450 enzymes is directly associated with formation of DNA adducts. Here, we present evidence that normal human lung epithelial cells, exposed to low micromolar concentrations of 3MI, showed extensive DNA damage, as measured by the comet assay, with similar potency to the prototypical genotoxic agents, doxorubicin and irinotecan. The DNA damage caused by 3MI was predominantly caused by single-strand breaks. Furthermore, we show that this damage decreased with time, given a subtoxic concentration, with detectable DNA fragmentation peaking 4 h after exposure and diminishing to untreated levels within 24 h. Pretreatment with an inhibitor of poly(ADP-ribose) polymerase 1 (PARP1), NU1025, nearly doubled the DNA damage produced by 5 microM 3MI, implying that PARP1, which among other activities, functions to repair single-strand breaks in DNA, repaired at least some of the 3MI-induced DNA fragmentation. A key cellular response to DNA damage, phosphorylation, and nuclear localization of p53 was seen at subtoxic levels of 3MI exposure. 3MI was highly mutagenic, with essentially the same potency as the prototype carcinogen, benzo[a]pyrene, only when a lung-expressed CYP2F3 enzyme was used to dehydrogenate 3MI to its putative DNA-alkylating intermediate. Conversely, a rat liver S9 metabolic system did not bioactivate 3MI to its mutagenic intermediate(s). Concentrations higher than 25 microM caused apoptosis, which became extensive at 100 microM, similar to the response seen with 10 microM doxorubicin. Our findings indicate that there is a low concentration window in which 3MI can cause extensive DNA damage and mutation, without triggering apoptotic defenses, reinforcing the hypothesis that inhaled 3MI from cigarette smoke may be a potent lung-selective carcinogen.

Xenobiotic metabolism of plant secondary compounds in oak (Quercus agrifolia) by specialist and generalist woodrat herbivores, genus Neotoma.

The challenge of consuming plant compounds that are recognized to have toxic physiological effects is an unavoidable consequence of an herbivorous diet and requires mechanisms to metabolize and eliminate them after consumption. We took a pharmacological approach to understanding how an oak (Quercus agrifolia) specialist (Neotoma macrotis) and generalist (N. lepida) herbivores process the same dietary toxins. Oak contains polyphenolic compounds considered toxic to most other mammals. N. macrotis includes up to 85% of oak in their diet. N. lepida includes oak as a portion of the diet but is considered a generalist in areas where sympatric with N. macrotis. Xenobiotic metabolizing enzyme activities of N. macrotis and N. lepida were investigated after animals were fed a 70% oak diet and a toxin-free control diet. Biotransformation activities of five major enzymes [cytochrome P450s (CYP), NAD(P)H/quinone oxidoreductase (QOR), UDP-glucuronosyltransferase (UGT), sulfotransferase (SULT), and glutathione S-transferase (GST)] and three specific CYP isozymes (CYP1A, CYP2B, and CYP3A) were investigated. The results indicate that, with the exception of CYP2B induction, N. macrotis and N. lepida enzyme activities are not changed by an oak diet. The major differences in enzyme activities were constitutive. The specialist, N. macrotis, had higher constitutive activity of QOR, UGT, and GST. The generalist, N. lepida, had higher constitutive activity levels of CYP1A and SULT.

Anticonvulsant activity, neural tube defect induction, mutagenicity and pharmacokinetics of a new potent antiepileptic drug, N-methoxy-2,2,3,3-tetramethylcyclopropane carboxamide.

N-methoxy-2,2,3,3-tetramethylcyclopropane carboxamide (OM-TMCD) is a methoxyamide derivative of a cyclopropyl analogue of valproic acid (VPA). The structural considerations used in the design of OM-TMCD were aimed to enhance OM-TMCD anticonvulsant potency (compared to VPA) and to prevent VPA's two life-threatening side effects, i.e., induction of neural tube defects (NTDs) and hepatotoxicity. Following i.p. administration to rats OM-TMCD demonstrated a broad spectrum of anticonvulsant activity and showed better potency than VPA in the maximal electroshock seizure and subcutaneous pentylenetetrazole tests as well as in the hippocampal kindling model. OM-TMCD was inactive in the mouse 6-Hz test at 100 mg/kg dose. Teratogenicity studies performed in a SWV/Fnn-mouse model for VPA-induced-exencephaly showed that on the equimolar basis OM-TMCD possesses the same fetal toxicity and ability to induce NTDs as VPA, but since OM-TMCD is a much more potent anticonvulsant its activity/exencephaly formation ratio appears to be much more beneficial than that of VPA. OM-TMCD was found to be non-mutagenic and non-pro-mutagenic in the Ames test. It showed a beneficial pharmacokinetic profile in rats, having a high oral bioavailability of 75% and satisfactory values of clearance and volume of distribution. These results support further studies to fully characterize the therapeutic potential of OM-TMCD.

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.

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.

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.

Cell-based studies reveal differences in glutathione S-transferase induction between oltipraz and tert-butylhydroquinone.

Selective induction of Phase II over Phase I drug-metabolizing enzymes has been proposed as a mechanism for reduction of chemical carcinogenesis. Enzymes likely to play a role in this amelioration include the glutathione S-transferases (GSTs) and among compounds that selectively induce key GSTs are tert-butylhydroquinone (tBHQ) and oltipraz [4-methyl-5-(2-pyrazinyl)-3H-1,2-dithiole-3-thione]. In vivo, and in hepatoma cells (H4IIE), these two agents induce rat GSTA2 mRNA to a similar extent. However, with a luciferase reporter construct containing 1651 bp of the proximal 5' flanking region of the rGSTA2 gene in the same cell line and under similar conditions, luciferase activity was induced to a much greater extent by tBHQ than by oltipraz. A similar large intercompound differential was seen with reporter constructs containing either the rGSTA2 ARE enhancer and HNF1 site (-872 to -582) or XRE enhancer and HNF1 site (-1110 to -812). In H4IIE cells, the rGSTA2 mRNA response to each agent was completely inhibited by 1 microM actinomycin-D cotreatment. With 1 microM cycloheximide cotreatment however, some induction by tBHQ remained, while induction by oltipraz was completely abolished. The induction response to tBHQ but not oltipraz was augmented by pretreatment with PD98059, a MEK1/2 specific inhibitor. Notwithstanding induction characteristics in common, oltipraz, and tBHQ have sufficient dissimilarities to indicate that rGSTA2 upregulation by the two agents is not identical.