Differential effect of ABCB1 haplotypes on promoter activity.

OBJECTIVE: Promoter single-nucleotide polymorphisms (SNPs) of the ABCB1 gene, encoding the placental efflux transporter P-glycoprotein, can affect its expression and alter xenobiotic transfer from the maternal to the fetal circulation. Because SNPs are arranged in specific combinations as defined haplotypes, the aims of this study were to: (i) determine the placental haplotype structure of the ABCB1 promoter and (ii) determine the differential effect of these haplotypes on placental ABCB1 promoter activity. MATERIALS AND METHODS: DNA samples from 100 healthy placentas were PCR-amplified and sequenced to identify existing SNPs in the proximal ABCB1 promoter. The haplotype structure encompassing these SNPs was inferred by PHASE analysis. Luciferase reporter constructs representing these haplotypes were generated and transfected into human placental 3A cells and their effect on ABCB1 promoter activity was determined using a dual-luciferase assay. RESULTS: We identified 12 ABCB1 promoter SNPs. These SNPs were predicted by PHASE to segregate into 28 haplotypes with frequencies ranging between 0.019 and 0.88. We found 12 of these haplotypes in our population in addition to two haplotypes not predicted by PHASE. We also generated two haplotypes to determine individual SNP effects for a total of 16 studied. Compared with the ancestral haplotype, three haplotypes significantly up-regulated (107-266% increase; P<0.05), one significantly down-regulated (95.4% decrease; P<0.01), and 12 had no statistically significant effect on ABCB1 promoter activity. DISCUSSION AND CONCLUSION: Our data show that the effect of SNPs on promoter activity depends on their presence in a specific haplotype. This indicates that haplotypes, rather than individual SNPs, could play a significant role in regulating placental P-glycoprotein expression and affect placental transfer and fetal exposure to xenobiotics.

Bisphenol A (BPA) and bisphenol S (BPS) alter the promoter activity of the ABCB1 gene encoding P-glycoprotein in the human placenta in a haplotype-dependent manner.

Exposure to bisphenols (BPA and BPS) during pregnancy can significantly affect fetal development and increase risk of adverse health consequences, however the underlying mechanisms are not fully elucidated. In human placenta, the efflux transporter P-glycoprotein (P-gp), encoded by the ABCB1 gene, extrudes its substrates from the trophoblasts back into the maternal circulation. Alterations in levels of placental P-gp could therefore significantly affect fetal exposure to xenobiotics that are P-gp substrates. The ABCB1 promoter contains many single nucleotide polymorphisms (SNPs). In the genome, SNPs are not arrayed as independent variants but as combinations forming defined haplotypes. Recently, we determined the haplotype sequences encompassing the ABCB1 promoter SNPs and found that promoter haplotypes differentially affect ABCB1 promoter activity. Here we investigate the effect of BPA and BPS on ABCB1 promoter activity by testing the hypothesis that BPA and BPS exposure affect ABCB1 promoter activity in a haplotype-dependent manner. Our data indicate that acute exposure to 50 nM BPA induced a significant haplotype-dependent increase in ABCB1 promoter activity (P < .05). However, acute exposure to 0.5 nM BPS induced a significant decrease (P < .05) in promoter activity that was haplotype-dependent. Chronic exposure to BPA and BPS individually (5 nM and 0.3 nM, respectively) or as a mixture (5 nM BPA:1.5 nM BPS) induced significant haplotype-dependent increases (P < .01) in ABCB1 promoter activity. Our data indicate that BPA and BPS significantly alter ABCB1 promoter activity in a haplotype- and exposure type- dependent manners. Such alteration could significantly impact placental P-gp levels and alter fetal exposure to many therapeutic and environmental xenobiotics.

A pharmacogenetic versus a clinical algorithm for warfarin dosing.

BACKGROUND: The clinical utility of genotype-guided (pharmacogenetically based) dosing of warfarin has been tested only in small clinical trials or observational studies, with equivocal results. METHODS: We randomly assigned 1015 patients to receive doses of warfarin during the first 5 days of therapy that were determined according to a dosing algorithm that included both clinical variables and genotype data or to one that included clinical variables only. All patients and clinicians were unaware of the dose of warfarin during the first 4 weeks of therapy. The primary outcome was the percentage of time that the international normalized ratio (INR) was in the therapeutic range from day 4 or 5 through day 28 of therapy. RESULTS: At 4 weeks, the mean percentage of time in the therapeutic range was 45.2% in the genotype-guided group and 45.4% in the clinically guided group (adjusted mean difference, [genotype-guided group minus clinically guided group], -0.2; 95% confidence interval, -3.4 to 3.1; P=0.91). There also was no significant between-group difference among patients with a predicted dose difference between the two algorithms of 1 mg per day or more. There was, however, a significant interaction between dosing strategy and race (P=0.003). Among black patients, the mean percentage of time in the therapeutic range was less in the genotype-guided group than in the clinically guided group. The rates of the combined outcome of any INR of 4 or more, major bleeding, or thromboembolism did not differ significantly according to dosing strategy. CONCLUSIONS: Genotype-guided dosing of warfarin did not improve anticoagulation control during the first 4 weeks of therapy. (Funded by the National Heart, Lung, and Blood Institute and others; COAG ClinicalTrials.gov number, NCT00839657.).

Effect of vildagliptin and pravastatin combination on cholesterol efflux in adipocytes.

Many reports suggested that some statins are almost ineffective in reducing triglycerides or enhancing HDL-C plasma levels, although statin treatment was still efficacious in reducing LDL-C. In diabetic dyslipidemic patients, it may therefore be necessary to use a combination therapy with other drugs to achieve either LDL-C- and triglyceride-lowering or HDL-C-enhancing goals. Such ineffectiveness of statins can be attributed to their effect on the liver X receptor (LXR) which regulates the expression of the ATP-binding cassette (ABC) transporters ABCA1 and ABCG1. A decrease in the expression of these transporters eventually leads to decreased cholesterol efflux from peripheral tissues leading to low levels of HDL-C. Although manipulating the LXR pathway may complement the effects of statins, LXR synthetic ligands as T091317 have shown significant hypertriglyceridemic action which limits their use. We recently found that the antidiabetic drug vildagliptin stimulates LXR expression leading to increased ABCB1/ABCG1 expression which improves cholesterol efflux from adipocytes. Therefore, a combination of vildagliptin and statin may provide a solution without the hypertriglyceridemic action observed with LXR agonist. We hypothesize that a combination of vildagliptin and pravastatin will improve cholesterol efflux in adipocytes. Statin-treated 3T3-L1 adipocytes were treated with vildagliptin, and the expression of LXR-ABCA1/ABCG1 cascade and the cholesterol efflux were then determined. Our data indicate that a combination of vildagliptin and pravastatin significantly induces the expression of LXR-ABCA1/ABCG1 cascade and improves cholesterol efflux (P > 0.05) in adipocytes. Our data may explain, at least in part, the improvement in HDL-C levels observed in patients receiving both medications. © 2016 IUBMB Life, 68(7):535-543, 2016.

Pharmacokinetics of Bupropion and Its Pharmacologically Active Metabolites in Pregnancy.

Bupropion sustained release is used to promote smoking cessation in males and nonpregnant females. However, its efficacy as a smoking cessation aid during pregnancy is not reported. The pregnancy-associated changes in maternal physiology may alter the pharmacokinetics and pharmacodynamics of bupropion and consequently its efficacy in pregnant smokers. Therefore, the aims of this study were to determine the steady-state pharmacokinetics of bupropion during pregnancy and the effect of functional genetic variants of CYP2B6 and CYP2C19 on bupropion pharmacokinetics in pregnant women. Plasma and urine concentrations of bupropion and its metabolites hydroxybupropion (OHBUP), threohydrobupropion, and erythrohydrobupropion were determined by liquid chromatography-mass spectrometry. Subjects were genotyped for five nonsynonymous single-nucleotide polymorphisms that result in seven CYP2B6 alleles, namely *2, *3, *4, *5, *6, *7, and *9, and for CYP2C19 variants *2, *3, and *17 The present study reports that the isoform-specific effect of pregnancy on bupropion-metabolizing enzymes along with the increase of renal elimination of the drug could collectively result in a slight decrease in exposure to bupropion in pregnancy. In contrast, pregnancy-induced increase in CYP2B6-catalyzed bupropion hydroxylation did not impact the plasma levels of OHBUP, probably due to a higher rate of OHBUP glucuronidation, and renal elimination associated with pregnancy. Therefore, exposure to OHBUP, a pharmacologically active metabolite of the bupropion, appears to be similar to that of the nonpregnant state. The predicted metabolic phenotypes of CYP2B6*6 and variant alleles of CYP2C19 in pregnancy are similar to those in the nonpregnant state.

Caffeic Acid Phenethyl Ester: A Review of Its Antioxidant Activity, Protective Effects against Ischemia-reperfusion Injury and Drug Adverse Reactions.

Propolis, a honey bee product, has been used in folk medicine for centuries for the treatment of abscesses, canker sores and for wound healing. Caffeic acid phenethyl ester (CAPE) is one of the most extensively investigated active components of propolis which possess many biological activities, including antibacterial, antiviral, antioxidant, anti-inflammatory, and anti-cancer effects. CAPE is a polyphenolic compound characterized by potent antioxidant and cytoprotective activities and protective effects against ischemia-reperfusion (I/R)-induced injury in multiple tissues such as brain, retina, heart, skeletal muscles, testis, ovaries, intestine, colon, and liver. Furthermore, several studies indicated the protective effects of CAPE against chemotherapy-induced adverse drug reactions (ADRs) including several antibiotics (streptomycin, vancomycin, isoniazid, ethambutol) and chemotherapeutic agents (mitomycin, doxorubicin, cisplatin, methotrexate). Due to the broad spectrum of pharmacological activities of CAPE, this review makes a special focus on the recently published data about CAPE antioxidant activity as well as its protective effects against I/R-induced injury and many adverse drug reactions.

Glucagon-like peptide 1 (GLP-1)-based therapy upregulates LXR-ABCA1/ABCG1 cascade in adipocytes.

A promising treatment for obesity involves the use of therapeutic agents that increase the level of the glucagon-like peptide (GLP-1) which reduces appetite and food intake. Native GLP-1 is rapidly metabolized by the dipeptidyl peptidase-4 (DPP-4) enzyme and, as such, GLP-1 mimetics or DPP-4 inhibitors represent promising treatment approaches. Interestingly, obese patient receiving such medications showed improved lipid profiles and cholesterol homeostasis, however the mechanism(s) involved are not known. Members of the ATP-binding cassette (ABC) transporters, including ABCA1 and ABCG1, play essential roles in reverse cholesterol transport and in high density lipoprotein (HDL) formation. These transporters are under the transcriptional regulation of liver X receptor alpha (LXR-α). We hypothesize that GLP-1 mimetics and/or DPP-4 inhibitors modulate ABCA1/ABCG1 expression in adipocytes through an LXR-α mediated process and thus affecting cholesterol homeostasis. 3T3-L1 adipocytes were treated with the DPP-4 inhibitor vildagliptin (2 nM) or the GLP-1 mimetic exendin-4 (5 nM). Gene and protein expression of ABCA1, ABCG1 and LXR-α were determined and correlated with cholesterol efflux. Expression levels of interleukin-6 (IL-6), leptin and the glucose transporter-4 (GLUT-4) were also determined. Treatment with both medications significantly increased the expression of ABCA1, ABCG1, LXR-α and GLUT-4, decreased IL-6 and leptin, and improved cholesterol efflux from adipocytes (P < 0.05). Our data suggest that GLP-1-based therapy modulate ABCA1/ABCG1 expression in adipocytes potentially through an LXR-α mediated process.

Influence of promoter/enhancer region haplotypes on MGMT transcriptional regulation: a potential biomarker for human sensitivity to alkylating agents.

The O6-methylguanine-DNA methyltransferase gene (MGMT) encodes the direct reversal DNA repair protein that removes alkyl adducts from the O6 position of guanine. Several single-nucleotide polymorphisms (SNPs) exist in the MGMT promoter/enhancer (P/E) region. However, the haplotype structure encompassing these SNPs and their functional/biological significance are currently unknown. We hypothesized that MGMT P/E haplotypes, rather than individual SNPs, alter MGMT transcription and can thus alter human sensitivity to alkylating agents. To identify the haplotype structure encompassing the MGMT P/E region SNPs, we sequenced 104 DNA samples from healthy individuals and inferred the haplotypes using the data generated. We identified eight SNPs in this region, namely T7C (rs180989103), T135G (rs1711646), G290A (rs61859810), C485A (rs1625649), C575A (rs113813075), G666A (rs34180180), C777A (rs34138162) and C1099T (rs16906252). Phylogenetics and Sequence Evolution analysis predicted 21 potential haplotypes that encompass these SNPs ranging in frequencies from 0.000048 to 0.39. Of these, 10 were identified in our study population as 20 paired haplotype combinations. To determine the functional significance of these haplotypes, luciferase reporter constructs representing these haplotypes were transfected into glioblastoma cells and their effect on MGMT promoter activity was determined. Compared with the most common (reference) haplotype 1, seven haplotypes significantly upregulated MGMT promoter activity (18-119% increase; P < 0.05), six significantly downregulated MGMT promoter activity (29-97% decrease; P < 0.05) and one haplotype had no effect. Mechanistic studies conducted support the conclusion that MGMT P/E haplotypes, rather than individual SNPs, differentially regulate MGMT transcription and could thus play a significant role in human sensitivity to environmental and therapeutic alkylating agents.

Indomethacin Pharmacokinetics and Pharmacodynamics in Pregnancies With Preterm Labor: The Need for Dose-Ranging Trials.

The use of indomethacin to delay delivery in preterm labor (PTL) is widely accepted; however, the optimal dosage of indomethacin in pregnancy is unknown. Here, we perform population pharmacokinetic (PK) and pharmacodynamic (PD) analyses, characterize the plasma disposition of indomethacin in pregnant women with PTL, and relate indomethacin exposure to delayed delivery and maternal/neonatal safety. We analyzed plasma and urine samples collected from a multicenter, prospective, opportunistic PK/PD study of indomethacin in pregnant women 12-32 weeks gestation admitted with PTL. Ninety-four participants with 639 plasma concentrations for indomethacin were included in the analysis. The final population PK (popPK) model for indomethacin was a 2-compartment structural model with first-order absorption and elimination and a covariate effect of body mass index on apparent oral clearance. We observed a 21%-60% increase in apparent oral clearance observed during pregnancy. There was no clear association between indomethacin exposure and maternal or neonatal safety outcomes, or with the magnitude of delayed delivery; however, 96.7% of women treated with indomethacin had a delivery that was delayed at least 48 hours. Given the changes to indomethacin apparent oral clearance during pregnancy, and the lack of relationship between indomethacin exposure and safety, dose-finding studies of indomethacin in pregnant women with PTL may help clarify the most safe and efficacious dosage and duration of indomethacin.

Caffeic acid phenethyl ester, a promising component of propolis with a plethora of biological activities: a review on its anti-inflammatory, neuroprotective, hepatoprotective, and cardioprotective effects.

Caffeic acid phenethyl ester (CAPE) is an important active component of honey bee propolis that possesses a plethora of biological activities. Propolis is used safely in traditional medicine as a dietary supplement for its therapeutic benefits. This review highlights the recently published data about CAPE bioavailability, anti-inflammatory, neuroprotective; hepatoprotective and cardioprotective activities. CAPE showed promising efficacy both in vitro and in vivo studies in animal models with minimum adverse effects. Its effectiveness was demonstrated in multiple target organs. Despite this fact, it has not been yet investigated as a protective agent or a potential therapy in humans. Investigation of CAPE efficacy in clinical trials is strongly encouraged to elucidate its therapeutic benefit for different human diseases after performing full preclinical toxicological studies and gaining more insights into its pharmacokinetics.

Caffeic acid phenethyl ester synergistically enhances docetaxel and paclitaxel cytotoxicity in prostate cancer cells.

Evidence is growing for the beneficial role of selective estrogen receptor modulators (SERM) in prostate diseases. Caffeic acid phenethyl ester (CAPE) is a promising component of propolis that possesses SERM activity. This study aimed at investigating the modulatory impact of CAPE on docetaxel (DOC) and paclitaxel (PTX) cytotoxicity in prostate cancer cells and exploring the possible underlying mechanisms for this chemomodulation. CAPE significantly increased DOC and PTX potency in PC-3, DU-145 and LNCaP prostate cancer cells. Combination index calculations showed synergistic interaction of CAPE/DOC and CAPE/PTX cotreatments in all the tested cell lines. Subsequent mechanistic studies in PC-3 cells indicated that cyclin D1 and c-myc were significantly reduced in the combined treatment groups with concurrent increase in p27kip. DNA-ploidy analysis indicated a significant increase in the percentage of cells in pre-G1 in CAPE/DOC and CAPE/PTX cotreatments. Decreased Bcl-2/Bax ratio together with increased caspase-3 activity and protein abundance were observed in the same groups. Estrogen receptor-ß (ER-ß) and its downstream tumor suppressor forkhead box O1 levels were significantly elevated in CAPE and combination groups compared to DOC or PTX-alone. ER-α and insulin-like growth factor-1 receptor protein abundance were reduced in the same groups. CAPE significantly reduced AKT, ERK and ER-α (Ser-167) phosphorylation in PC-3 cells. CAPE-induced inhibition of AKT phosphorylation was more prominent (1.7-folds higher) in cells expressing ER-α such as PC-3 compared to LNCaP. In conclusion, CAPE enhances the antiproliferative and cytotoxic effects of DOC and PTX in prostate cancer cells. This can be, at least partly, attributed to CAPE augmentation of DOC and PTX proapoptotic effects in addition to CAPE-induced alterations in ER-α and ER-ß abundance.

Induction of base excision repair enzymes NTH1 and APE1 in rat spleen following aniline exposure.

Mechanisms by which aniline exposure elicits splenotoxicity, especially a tumorigenic response, are not well-understood. Earlier, we have shown that aniline exposure leads to oxidative DNA damage and up-regulation of OGG1 and NEIL1/2 DNA glycosylases in rat spleen. However, the contribution of endonuclease III homolog 1 (NTH1) and apurinic/apyrimidinic endonuclease 1 (APE1) in the repair of aniline-induced oxidative DNA damage in the spleen is not known. This study was, therefore, focused on examining whether NTH1 and APE1 contribute to the repair of oxidative DNA lesions in the spleen, in an experimental condition preceding tumorigenesis. To achieve this, male SD rats were subchronically exposed to aniline (0.5 mmol/kg/day via drinking water for 30 days), while controls received drinking water only. By quantitating the cleavage products, the activities of NTH1 and APE1 were assayed using substrates containing thymine glycol (Tg) and tetrahydrofuran, respectively. Aniline treatment led to significant increases in NTH1- and APE1-mediated BER activity in the nuclear extracts of spleen of aniline-treated rats compared to the controls. NTH1 and APE1 mRNA expression in the spleen showed 2.9- and 3.2-fold increases, respectively, in aniline-treated rats compared to the controls. Likewise, Western blot analysis showed that protein expression of NTH1 and APE1 in the nuclear extracts of spleen from aniline-treated rats was 1.9- and 2.7-fold higher than the controls, respectively. Immunohistochemistry indicated that aniline treatment also led to stronger immunoreactivity for both NTH1 and APE1 in the spleens, confined to the red pulp areas. These results, thus, show that aniline exposure is associated with induction of NTH1 and APE1 in the spleen. The increased repair activity of NTH1 and APE1 could be an important mechanism for the removal of oxidative DNA lesions. These findings thus identify a novel mechanism through which NTH1 and APE1 may regulate the repair of oxidative DNA damage in aniline-induced splenic toxicity.

CYP1A2*1F and GSTM1 alleles are associated with susceptibility to porphyria cutanea tarda.

Porphyria cutanea tarda (PCT) is a cutaneous porphyria with sporadic (type 1) and familial (type 2) subtypes, both resulting from decreased hepatic uroporphyrinogen decarboxylase (UROD) activity. Environmental and genetic factors are involved in the development of PCT, and genetic variants in the cytochrome P450 (CYP ) genes, CYP1A1 and CYP1A2, have been implicated. We investigated the association between PCT and variants in CYP1A1, CYP1A2 and CYP2E1, and the glutathione-S-transferase (GST ) genes, GSTM1 and GSTT1. PCT diagnosis was based on urinary or plasma porphyrin profiles. Patients were classified as type 1 or 2 PCT based on UROD mutation analysis. The CYP1A2*1F promoter A allele frequency was significantly higher (P < 0.022) and the A/A genotype frequency marginally higher in PCT patients overall (P < 0.057), with the A/A genotype significantly more common in type 1 PCT (P < 0.043). The presence of the wild-type GSTM1 allele also was associated significantly with PCT (P < 0.019). Neither hemochromatosis (HFE) mutations, tobacco smoking, hepatitis C and HIV infection, ethanol consumption, nor estrogen use were associated with these allelic variants. Age at onset was significantly lower in type 2 PCT patients (P < 0.001), as observed previously. Thus, positive associations between PCT and the CYP1A2*1F promoter A allele and A/A genotype and the wild-type GSTM1 allele indicates that these functional hepatic biotransformation enzymes are risk factors for the development of this disease.

Induction of NEIL1 and NEIL2 DNA glycosylases in aniline-induced splenic toxicity.

The mechanisms by which aniline exposure elicits splenotoxic response, especially the tumorigenic response, are not well-understood. Earlier, we have shown that aniline-induced oxidative stress is associated with increased oxidative DNA damage in rat spleen. The base excision repair (BER) pathway is the major mechanism for the repair of oxidative DNA base lesions, and we have shown an up-regulation of 8-oxoguanine glycosylase 1 (OGG1), a specific DNA glycosylase involved in the removal of 8-hydroxy-2'-deoxyguanosine (8-OHdG) adducts, following aniline exposure. Nei-like DNA glycosylases (NEIL1/2) belong to a family of BER proteins that are distinct from other DNA glycosylases, including OGG1. However, contribution of NEIL1/2 in the repair of aniline-induced oxidative DNA damage in the spleen is not known. This study was, therefore, focused on evaluating if NEILs also contribute to the repair of oxidative DNA lesions in the spleen following aniline exposure. To achieve that, male SD rats were subchronically exposed to aniline (0.5 mmol/kg/day via drinking water for 30 days), while controls received drinking water only. The BER activity of NEIL1/2 was assayed using a bubble structure substrate containing 5-OHU (preferred substrates for NEIL1 and NEIL2) and by quantitating the cleavage products. Aniline treatment led to a 1.25-fold increase in the NEIL1/2-associated BER activity in the nuclear extracts of spleen compared to the controls. Real-time PCR analysis for NEIL1 and NEIL2 mRNA expression in the spleen revealed 2.7- and 3.9-fold increases, respectively, in aniline-treated rats compared to controls. Likewise, Western blot analysis showed that protein expression of NEIL1 and NEIL2 in the nuclear extract of spleens from aniline-treated rats was 2.0- and 3.8-fold higher than controls, respectively. Aniline treatment also led to stronger immunoreactivity for NEIL1 and NEIL2 in the spleens, confined to the red pulp areas. These studies, thus, show that aniline-induced oxidative stress is associated with an induction of NEIL1/2. The increased NIEL-mediated BER activity is another indication of aniline-induced oxidative damage in the spleen and could constitute another important mechanism of removal of oxidative DNA lesions, especially in transcribed DNA following aniline insult.

Evaluating the effects of genetic variants of DNA repair genes using cytogenetic mutagen sensitivity approaches.

Mutagen sensitivity, measured in short-term cultures of peripheral blood lymphocytes by cytogenetic endpoints, is an indirect measure for DNA repair capacity and has been used for many years as a biomarker for intrinsic susceptibility for cancer. In this article, we briefly give an overview of the different cytogenetic mutagen sensitivity approaches that have been used successfully to evaluate the biological effects of polymorphisms in DNA repair genes based on a current review of the literature and based on the need for biomarkers that would allow the characterization of the biological and functional significance of such polymorphisms. We also address some of the future challenges facing this emerging area of research.

Regulatory regions responsive to oxidative stress in the promoter of the human DNA glycosylase gene NEIL2.

Reactive oxygen species (ROS) generated endogenously or from exogenous sources produce mutagenic DNA lesions. If not repaired, these lesions could lead to genomic instability and, potentially, to cancer development. NEIL2 (EC 4.2.99.18), a mammalian base excision repair (BER) protein and ortholog of the bacterial Fpg/Nei, excises oxidized DNA lesions from bubble or single-stranded structures, suggesting its involvement in transcription-coupled DNA repair. Perturbation in NEIL2 expression may, therefore, significantly impact BER capacity and promote genomic instability. To characterize the genetic and environmental factors regulating NEIL2 gene expression, we mapped the human NEIL2 transcriptional start site and partially characterized the promoter region of the gene using a luciferase reporter assay. We identified a strong positive regulatory region from nucleotide -206 to +90 and found that expression from this region was contingent on its being isolated from an adjacent strong negative regulatory region located downstream (+49 to +710 bp), suggesting that NEIL2 transcription is influenced by both these regions. We also found that oxidative stress, induced by glucose oxidase treatment, reduced the positive regulatory region expression levels, suggesting that ROS may play a significant role in regulating NEIL2 transcription. In an initial attempt to characterize the underlying mechanisms, we used in silico analysis to identify putative cis-acting binding sites for ROS-responsive transcription factors within this region and then used site-directed mutagenesis to investigate their role. A single-base change in the region encompassing nucleotides -206 to +90 abolished the effect of oxidative stress that was observed in the absence of the mutation. Our study is the first to provide an initial partial characterization of the NEIL2 promoter and opens the door for future research aimed at understanding the role of genetic and environmental factors in regulating NEIL2 expression.

2-methoxyestradiol sensitizes breast cancer cells to taxanes by targeting centrosomes.

Centrosomes amplification is a hallmark of cancer. We hypothesize that 2-methoxyestradiol (2-ME) sensitizes breast cancer (BC) cells to taxanes by targeting amplified centrosomes. We assessed the extent by which 2-ME together with paclitaxel (PTX) induces centrosome alterations with subsequent mitotic catastrophe in different BC subtypes. 2-ME induced a significant reduction in PTX IC50 values in all cells tested ranging from 28-44% (P < 0.05). Treatment with both PTX and 2-ME significantly increased the number of misaligned metaphases compared to PTX alone (34%, 100% and 52% for MCF7, MDA-MB231 and SUM149, respectively; P < 0.05). The number of cells with multipolar spindle formation was significantly increased (81%, 220% and 285% for MCF7, MDA-MB231 and SUM 149, respectively; P < 0.05). PTX and 2-ME treatment significantly increased interphase declustering in cancer cells (56% for MCF7, 208% for MDA-MB231 and 218% for SUM149, respectively; P < 0.05) and metaphase declustering (1.4-fold, 1.56-fold and 2.48-fold increase for MCF7, MDA-MB231 and SUM149, respectively; P < 0.05). This report is the first to document centrosome declustering as a mechanism of action of 2-ME and provides a potential approach for reducing taxane toxicity in cancer treated patients.

Single nucleotide polymorphisms 5' upstream the coding region of the NEIL2 gene influence gene transcription levels and alter levels of genetic damage.

NEIL2 (EC 4.2.99.18), a mammalian DNA glycosylase and ortholog of the bacterial Fpg/Nei, excises oxidized DNA lesions from bubble or single-stranded structures, suggesting its involvement in transcription-coupled DNA repair. Because base excision repair (BER) proteins act collectively and in a progressive fashion, their proper balance is essential for optimal repair. Thus, inter-individual variability in transcription levels of NEIL2 may predispose to compromised DNA repair capacity and genomic instability by altering the balance of critical BER proteins. In a study of lymphocytes of 129 healthy subjects, using absolute quantitative reverse transcription PCR, we found that NEIL2 transcription varied significantly (up to 63 fold) and that this variability was influenced by certain single nucleotide polymorphisms (SNPs) located 5' of the start site. Using the mutagen sensitivity assay to characterize the biological significance of these SNPs, we observed a significant increase in mutagen-induced genetic damage associated with two SNPs in the promoter region of the NEIL2 gene. To characterize the functional significance of these SNPs, we engineered luciferase-reporter constructs of the NEIL2 promotor with mutations corresponding to these SNPs. We transfected these constructs into MRC-5 cells and evaluated their impact on NEIL2 expression levels. Our results indicate that NEIL2 expression was significantly reduced by over 50% (P < 0.01) in the presence of two SNPs, ss74800505 and rs8191518, located near the NEIL2 start site, which were in significant linkage disequilibrium (D' = 73%; P < 0.05). This first report on in vivo variability in NEIL2 expression in humans identifies SNPs in the NEIL2 promoter region that have functional effects.

Effect of CYP2C9 Polymorphisms on the Pharmacokinetics of Indomethacin During Pregnancy.

BACKGROUND AND OBJECTIVE: Cytochrome P450 (CYP) 2C9 catalyzes the biotransformation of indomethacin to its inactive metabolite O-desmethylindomethacin (DMI). The aim of this work was to determine the effect of CYP2C9 polymorphisms on indomethacin metabolism in pregnant women. METHODS: Plasma concentrations of indomethacin and DMI at steady state were analyzed with a validated LC-MS/MS method. DNA was isolated from subject blood and buccal smear samples. Subjects were grouped by genotype for comparisons of pharmacokinetic parameters. RESULTS: For subjects with the *1/*2 genotype, the mean steady-state apparent oral clearance (CL/Fss) of indomethacin was 13.5 ± 7.7 L/h (n = 4) and the mean metabolic ratio (AUCDMI/AUCindomethacin) was 0.291 ± 0.133. For subjects with the *1/*1 genotype, these values were 12.4 ± 2.7 L/h and 0.221 ± 0.078, respectively (n = 14). Of note, we identified one subject who was a carrier of both the *3 and *4 alleles, resulting in an amino acid change (I359P) which has not been reported previously. This subject had a metabolic ratio of 0.390 and a CL/Fss of indomethacin (24.3 L/h) that was nearly double the wild-type clearance. CONCLUSION: Although our results are limited by sample size and are not statistically significant, these data suggest that certain genetic polymorphisms of CYP2C9 may lead to an increased metabolic ratio and an increase in the clearance of indomethacin. More data are needed to assess the impact of CYP2C9 genotype on the effectiveness of indomethacin as a tocolytic agent.

Oxidative DNA damage and its repair in rat spleen following subchronic exposure to aniline.

The mechanisms by which aniline exposure elicits splenotoxic response, especially the tumorigenic response, are not well-understood. Splenotoxicity of aniline is associated with iron overload and generation of reactive oxygen species (ROS) which can cause oxidative damage to DNA, proteins and lipids (oxidative stress). 8-Hydroxy-2'-deoxyguanosine (8-OHdG) is one of the most abundant oxidative DNA lesions resulting from ROS, and 8-oxoguanine glycosylase 1 (OGG1), a specific DNA glycosylase/lyase enzyme, plays a key role in the removal of 8-OHdG adducts. This study focused on examining DNA damage (8-OHdG) and repair (OGG1) in the spleen in an experimental condition preceding a tumorigenic response. To achieve that, male Sprague-Dawley rats were subchronically exposed to aniline (0.5 mmol/kg/day via drinking water for 30 days), while controls received drinking water only. Aniline treatment led to a significant increase in splenic oxidative DNA damage, manifested as a 2.8-fold increase in 8-OHdG levels. DNA repair activity, measured as OGG1 base excision repair (BER) activity, increased by approximately 1.3 fold in the nuclear protein extracts (NE) and approximately 1.2 fold in the mitochondrial protein extracts (ME) of spleens from aniline-treated rats as compared to the controls. Real-time PCR analysis for OGG1 mRNA expression in the spleen revealed a 2-fold increase in expression in aniline-treated rats than the controls. Likewise, OGG1 protein expression in the NEs of spleens from aniline-treated rats was approximately 1.5 fold higher, whereas in the MEs it was approximately 1.3 fold higher than the controls. Aniline treatment also led to stronger immunostaining for both 8-OHdG and OGG1 in the spleens, confined to the red pulp areas. It is thus evident from our studies that aniline-induced oxidative stress is associated with increased oxidative DNA damage. The BER pathway was also activated, but not enough to prevent the accumulation of oxidative DNA damage (8-OHdG). Accumulation of mutagenic oxidative DNA lesions in the spleen following exposure to aniline could play a critical role in the tumorigenic process.