Rifampin modulation of xeno- and endobiotic conjugating enzyme mRNA expression and associated microRNAs in human hepatocytes

Rifampin is a pleiotropic inducer of multiple drug metabolizing enzymes and transporters. This work utilized a global approach to evaluate rifampin effects on conjugating enzyme gene expression with relevance to human xeno- and endo-biotic metabolism. Primary human hepatocytes from 7 subjects were treated with rifampin (10 μmol/L, 24 hours). Standard methods for RNA-seq library construction, EZBead preparation, and NextGen sequencing were used to measure UDP-glucuronosyl transferase UGT, sulfonyltransferase SULT, N acetyltransferase NAT, and glutathione-S-transferase GST mRNA expression compared to vehicle control (0.01% MeOH). Rifampin-induced (>1.25-fold) mRNA expression of 13 clinically important phase II drug metabolizing genes and repressed (>1.25-fold) the expression of 3 genes (P < .05). Rifampin-induced miRNA expression changes correlated with mRNA changes and miRNAs were identified that may modulate conjugating enzyme expression. NAT2 gene expression was most strongly repressed (1.3-fold) by rifampin while UGT1A4 and UGT1A1 genes were most strongly induced (7.9- and 4.8-fold, respectively). Physiologically based pharmacokinetic modeling (PBPK) was used to simulate the clinical consequences of rifampin induction of CYP3A4- and UGT1A4-mediated midazolam metabolism. Simulations evaluating isolated UGT1A4 induction predicted increased midazolam N-glucuronide exposure (~4-fold) with minimal reductions in parent midazolam exposure (~10%). Simulations accounting for simultaneous induction of both CYP3A4 and UGT1A4 predicted a ~10-fold decrease in parent midazolam exposure with only a ~2-fold decrease in midazolam N-glucuronide metabolite exposure. These data reveal differential effects of rifampin on the human conjugating enzyme transcriptome and potential associations with miRNAs that form the basis for future mechanistic studies to elucidate the interplay of conjugating enzyme regulatory elements

Effects of exemestane and letrozole therapy on plasma concentrations of estrogens in a randomized trial of postmenopausal women with breast cancer

PURPOSE: Inter-individual differences in estrogen concentrations during treatment with aromatase inhibitors (AIs) may contribute to therapeutic response and toxicity. The aim of this study was to determine plasma concentrations of estradiol (E2), estrone (E1), and estrone sulfate (E1S) in a large cohort of AI-treated breast cancer patients. METHODS: In a randomized, multicenter trial of postmenopausal women with early-stage breast cancer starting treatment with letrozole (n = 241) or exemestane (n = 228), plasma estrogen concentrations at baseline and after 3 months were quantitated using a sensitive mass spectrometry-based assay. Concentrations and suppression below the lower limit of quantification (LLOQ) were compared between estrogens and between drugs. RESULTS: The ranges of baseline estrogen concentrations were <LLOQ-361 pg/mL for E2, <LLOQ-190 pg/mL for E1, and 8.3-4060 pg/mL for E1S. For E2, the frequency of suppression below the LLOQ was not statistically significantly different between AIs (exemestane: 89.0%, letrozole: 86.9%, p = 0.51). However, patients on letrozole were more likely to achieve suppression below the LLOQ of both E1 (exemestane: 80.1%, letrozole: 90.1%, p = 0.005) and E1S (exemestane: 17.4%, letrozole: 54.9%, p = 4.34e-15). After 3 months of AI therapy, the ranges of estrogen concentrations were <LLOQ-63.8 pg/mL, <LLOQ-36.7 pg/mL, and <LLOQ-1090 pg/mL for E2, E1, and E1S, respectively. During treatment, 16 patients had an increased concentration compared to the baseline concentration of at least one estrogen. CONCLUSIONS: Letrozole had greater suppression of plasma E1 and E1S than exemestane, though the response was highly variable among patients. Additional research is required to examine the clinical relevance of differential estrogen suppression

Aromatase inhibitor-induced modulation of breast density: clinical and genetic effects

Background: Change in breast density may predict outcome of women receiving adjuvant hormone therapy for breast cancer. We performed a prospective clinical trial to evaluate the impact of inherited variants in genes involved in oestrogen metabolism and signalling on change in mammographic percent density (MPD) with aromatase inhibitor (AI) therapy. Methods: Postmenopausal women with breast cancer who were initiating adjuvant AI therapy were enrolled onto a multicentre, randomised clinical trial of exemestane vs letrozole, designed to identify associations between AI-induced change in MPD and single-nucleotide polymorphisms in candidate genes. Subjects underwent unilateral craniocaudal mammography before and following 24 months of treatment. Results: Of the 503 enrolled subjects, 259 had both paired mammograms at baseline and following 24 months of treatment and evaluable DNA. We observed a statistically significant decrease in mean MPD from 17.1 to 15.1% (P<0.001), more pronounced in women with baseline MPD ⩾20%. No AI-specific difference in change in MPD was identified. No significant associations between change in MPD and inherited genetic variants were observed. Conclusion: Subjects with higher baseline MPD had a greater average decrease in MPD with AI therapy. There does not appear to be a substantial effect of inherited variants in biologically selected candidate genes

Hedgehog pathway mutations drive oncogenic transformation in high-risk T-cell acute lymphoblastic leukemia.

The role of Hedgehog signaling in normal and malignant T-cell development is controversial. Recently, Hedgehog pathway mutations have been described in T-ALL, but whether mutational activation of Hedgehog signaling drives T-cell transformation is unknown, hindering the rationale for therapeutic intervention. Here, we show that Hedgehog pathway mutations predict chemotherapy resistance in human T-ALL, and drive oncogenic transformation in a zebrafish model of the disease. We found Hedgehog pathway mutations in 16% of 109 childhood T-ALL cases, most commonly affecting its negative regulator PTCH1. Hedgehog mutations were associated with resistance to induction chemotherapy (P = 0.009). Transduction of wild-type PTCH1 into PTCH1-mutant T-ALL cells induced apoptosis (P = 0.005), a phenotype that was reversed by downstream Hedgehog pathway activation (P = 0.007). Transduction of most mutant PTCH1, SUFU, and GLI alleles into mammalian cells induced aberrant regulation of Hedgehog signaling, indicating that these mutations are pathogenic. Using a CRISPR/Cas9 system for lineage-restricted gene disruption in transgenic zebrafish, we found that ptch1 mutations accelerated the onset of notch1-induced T-ALL (P = 0.0001), and pharmacologic Hedgehog pathway inhibition had therapeutic activity. Thus, Hedgehog-activating mutations are driver oncogenic alterations in high-risk T-ALL, providing a molecular rationale for targeted therapy in this disease

Association of Variants in Candidate Genes with Lipid Profiles in Women with Early Breast Cancer on Adjuvant Aromatase Inhibitor Therapy

Purpose: Aromatase inhibitors can exert unfavorable effects on lipid profiles; however, previous studies have reported inconsistent results. We describe the association of single-nucleotide polymorphisms (SNP) in candidate genes with lipid profiles in women treated with adjuvant aromatase inhibitors. Experimental design: We conducted a prospective observational study to test the associations between SNPs in candidate genes in estrogen signaling and aromatase inhibitor metabolism pathways with fasting lipid profiles during the first 3 months of aromatase inhibitor therapy in postmenopausal women with early breast cancer randomized to adjuvant letrozole or exemestane. We performed genetic association analysis and multivariable linear regressions using dominant, recessive, and additive models. Results: A total of 303 women had complete genetic and lipid data and were evaluable for analysis. In letrozole-treated patients, SNPs in CYP19A1, including rs4646, rs10046, rs700518, rs749292, rs2289106, rs3759811, and rs4775936 were significantly associated with decreases in triglycerides by 20.2 mg/dL and 39.3 mg/dL (P < 0.00053), respectively, and with variable changes in high-density lipoprotein (HDL-C) from decreases by 4.2 mg/dL to increases by 9.8 mg/dL (P < 0.00053). Conclusions: Variants in CYP19A1 are associated with decreases in triglycerides and variable changes in HDL-C in postmenopausal women on adjuvant aromatase inhibitors. Future studies are needed to validate these findings, and to identify breast cancer survivors who are at higher risk for cardiovascular disease with aromatase inhibitor therapy

Processes of ammonia air-surface exchange in a fertilized Zea mays canopy

Recent incorporation of coupled soil biogeochemical and bi-directional NH3 air–surface exchange algorithms into regional air quality models holds promise for further reducing uncertainty in estimates of NH3 emissions from fertilized soils. While this represents a significant advancement over previous approaches, the evaluation and improvement of such modeling systems for fertilized crops requires process-level field measurements over extended periods of time that capture the range of soil, vegetation, and atmospheric conditions that drive short-term (i.e., post-fertilization) and total growing season NH3 fluxes. This study examines the processes of NH3 air–surface exchange in a fertilized corn (Zea mays) canopy over the majority of a growing season to characterize soil emissions after fertilization and investigate soil–canopy interactions. Micrometeorological flux measurements above the canopy, measurements of soil, leaf apoplast and dew/guttation chemistry, and a combination of in-canopy measurements, inverse source/sink, and resistance modeling were employed. Over a period of approximately 10 weeks following fertilization, daily mean and median net canopy-scale fluxes yielded cumulative total N losses of 8.4% and 6.1%, respectively, of the 134 kg N ha−1 surface applied to the soil as urea ammonium nitrate (UAN). During the first month after fertilization, daily mean emission fluxes were positively correlated with soil temperature and soil volumetric water. Diurnally, maximum hourly average fluxes of ≈ 700 ng N m−2 s−1 occurred near mid-day, coincident with the daily maximum in friction velocity. Net emission was still observed 5 to 10 weeks after fertilization, although mid-day peak fluxes had declined to ≈ 125 ng N m−2 s−1. A key finding of the surface chemistry measurements was the observation of high pH (7.0–8.5) in leaf dew/guttation, which reduced the ability of the canopy to recapture soil emissions during wet periods. In-canopy measurements near peak leaf area index (LAI) indicated that the concentration of NH3 just above the soil surface was highly positively correlated with soil volumetric water, which likely reflects the influence of soil moisture on resistance to gaseous diffusion through the soil profile and hydrolysis of remaining urea. Inverse source/sink and resistance modeling indicated that the canopy recaptured ≈ 76% of soil emissions near peak LAI. Stomatal uptake may account for 12–34% of total uptake by foliage during the day compared to 66–88% deposited to the cuticle. Future process-level NH3 studies in fertilized cropping systems should focus on the temporal dynamics of net emission to the atmosphere from fertilization to peak LAI and improvement of soil and cuticular resistance parameterizations