Supplementary Figure 1 from Determination of the Role of Target Tissue Metabolism in Lung Carcinogenesis Using Conditional Cytochrome P450 Reductase-Null Mice

Supplementary Information, Methods, Tables 1-4, Figure Legends 1-5 from Determination of the Role of Target Tissue Metabolism in Lung Carcinogenesis Using Conditional Cytochrome P450 Reductase-Null Mice

AIM:To evaluate the glycaemic efficacy of metformin in people with type 2 diabetes (T2D) and stage 3 chronic kidney disease (CKD3). PARTICIPANTS AND METHODS:This was a retrospective study including 145980 US veterans with T2D and an estimated glomerular filtration rate (eGFR) ≥30 mL/min/1.73 m2 who initiated metformin monotherapy between November 1999 and July 2017. Propensity-score-matched cohorts were generated based on baseline variables associated with CKD3 (eGFR 30-59 mL/min/1.73 m2 ) to evaluate the independent association between CKD3 and metformin discontinuation, the addition of a second hypoglycaemic agent, and changes in glycated haemoglobin (HbA1c) from baseline in those with and without CKD3. Associations were examined using the Kaplan-Meier method and multivariable regression models, adjusted for baseline and 12-month average metformin dose. RESULTS:The mean age of the entire cohort was 60.7 years, and 95% of the cohort were men, 21% were African American and 9% had CKD3. In the adjusted analyses, patients with CKD3 had a higher risk of metformin discontinuation or addition of a second hypoglycaemic agent, as compared with patients without CKD (hazard ratio [HR] 1.22, 95% confidence interval [CI] 1.19-1.26, and HR 1.26, 95% CI 1.13-1.40, respectively). Among metformin monotherapy users, there were no differences in the average HbA1c reduction from baseline to 12 or 24 months between patients with and without CKD3. CONCLUSIONS:Individuals with CKD3 and T2D were at increased risk of metformin monotherapy failure. However, the HbA1c-lowering efficacy of metformin was similar in patients with and without CKD3, highlighting that metformin is a valuable treatment option for newly treated individuals with T2D and CKD3.

Background: Metformin (MET) is approved for treatment of type 2 diabetes (T2D) in patients with chronic kidney disease stage 3 (CKD3); however, data on its glycemic efficacy in CKD3 is lacking. Methods: This was a retrospective study including 85,997 U.S. veterans with T2D and estimated glomerular filtration rate (eGFR)≥30 ml/min/1.73m2 treated with MET monotherapy with adequate adherence (defined by proportion of days covered ≥80%) between 01/2010 and 08/2018. CKD3 was defined as eGFR 30-59 ml/min/1.73m2. Twelve-month time-averaged A1c changes from baseline were compared in patients without and with CKD3 using univariate and multivariate linear regression analyses adjusted for case-mix. Results: The mean±SD age for the total cohort was 60.8±9.9 yrs, 95.2% were males, 76.3 and 13.0% were Whites and Blacks, respectively. Average daily mean±SD MET dose was 1341±202 mg. Pre-existing CKD3 was present in 7,566 (9.7%) of individuals. Baseline characteristics by CKD3 status are shown in Table 1. In unadjusted analysis, mean A1c reduction was 0.59±1.0 vs. 0.52±0.9 % in patients without and with CKD3 (p<0.001). The difference in A1C between two groups was lost after adjustments for baseline characteristics and MET dose (p=0.058). Conclusions: In this large retrospective study, presence of preexisting CKD stage 3 did not affect glycemic efficacy of metformin monotherapy in T2D patients. Disclosure E. Gosmanova: None. D.E. Gemoets: None. L.S. Kaminsky: None. C.P. Kovesdy: Consultant; Self; Amgen, AstraZeneca, Bayer AG, Cara Therapeutics, Reata, Takeda Pharmaceutical Company Limited, Tricida. A.R. Gosmanov: None.

Cytochrome P450 oxidoreductase (POR) is required for drug metabolism by all microsomal cytochrome P450 enzymes. The aim of this study was to investigate whether any of the common single nucleotide polymorphisms (SNPs) in the POR gene and its flanking intergenic sequences correlate with interindividual variations in the warfarin maintenance dose (which is determined partly by rates of warfarin metabolism) in patients undergoing anticoagulation therapy. Warfarin dose and patients' demographic and clinical information were collected from 124 patients, who had attained a stable warfarin dose while receiving treatment at the Stratton VA Medical Center. Genomic DNAs were isolated from blood samples and were genotyped for 15 SNPs (including 10 SNPs on the POR gene). Association analysis was performed on 122 male patients by linear regression. Simple regression analysis revealed that vitamin K epoxide reductase complex subunit 1 (VKORC1) -1639A>G, CYP2C9*2, CYP2C9*3, age, and chronic aspirin therapy were significantly associated with warfarin dose. In contrast, multiple regression analysis revealed that, in addition to several known factors contributing to the variations in warfarin maintenance dose (VKORC1 -1639A>G, CYP2C9*2, CYP2C9*3, CYP4F2 rs2108622, and chronic aspirin therapy), three common POR SNPs (-173C>A, -208C>T, and rs2868177) were also significantly associated with variations in warfarin maintenance dose. These results indicate, for the first time, that three common SNPs in the POR gene may contribute to the interindividual variability in warfarin maintenance dose. Further studies on functional characterization of the POR SNPs identified, including their impact on the in vivo metabolism of additional drugs, are needed.

To better understand androgen function in the mammalian nose, we have determined the levels of testosterone (T) in the olfactory mucosa (OM, which harbors the olfactory receptor neurons) and the lateral nasal gland (LNG, which is the largest anterior nasal gland) of C57BL/6 mice. The results indicated that, in adult male mice, T levels in the LNG were substantially higher than those in the OM and other non-reproductive or non-endocrine tissues examined, including liver, kidney, and brain. Furthermore, in the LNG, the high T levels were accompanied by high levels of salivary androgen-binding protein (sABP) and low microsomal T-hydroxylase activities. The high abundance of T and sABP in the LNG suggests not only that the LNG is a storage site for androgen, but also the possibility that unusually high T levels may occur in other organs that have abundant expression of sABP but low expression of steroid-metabolizing enzymes. Our findings suggest a critical need to determine androgen levels in various organs, as well as to establish the functional significance of an unusually high T level in the LNG, a gland known for its secretion of biologically active molecules, such as odorant binding proteins and immunoglobulin A, to the nasal cavity.

To determine the in vivo function of intestinal cytochrome P450 (P450) enzymes, we have generated an intestinal epithelium (IE)-specific P450 reductase gene (Cpr) knockout mouse model (designated IE-Cpr-null). In the IE-Cpr-null mouse, CPR expression was abolished in IE cells; however, CPR expression was not altered in other tissues examined. The loss of CPR expression in the small intestine (SI) led to increased expression of several P450 proteins examined, including CYP1A1, CYP2B, CYP2C, and CYP3A. It is interesting to note that the expression of CYP1A1 was also increased in the liver, kidney, and lung of the IE-Cpr-null mice compared with wild-type (WT) littermates, a result strongly supporting the notion that SI metabolism of putative dietary CYP1A1 inducers can influence the systemic bioavailability of these inducers. The rates of SI microsomal metabolism of nifedipine (NFP) in the IE-Cpr-null mice were approximately 10% of the rates in WT littermates, despite the compensatory expression of multiple P450 enzymes in the SI. Furthermore, the area under the concentration-time curve (AUC) values for blood NFP (dosed at 10 mg/kg) levels were 1.6-fold higher in IE-Cpr-null mice than in WT littermates when NFP was given orally; in contrast, the AUC values were comparable for the two strains when NFP was given intravenously. This result directly showed that P450-catalyzed NFP metabolism in the SI plays an important role in the first-pass clearance of oral NFP. Our findings indicate that the IE-Cpr-null mouse model can be used to study the in vivo function of intestinal P450 enzymes in the clearance of oral drugs and other xenobiotics.

244 Introduction: 705 Discussion: 1500 ABBREVIATIONS: P450, cytochrome P450; CPR, NADPH-cytochrome P450 reductase; HPLC, high performance liquid chromatography; AUC, area under the concentration-time curve; IE-Cpr-null, intestinal epithelium-specific Cpr-knockout; SI, small intestine; WT, wild-type; NFP, nifedipine. This article has not been copyedited and formatted. The final version may differ from this version. DMD Fast Forward. Published on December 4, 2008 as DOI: 10.1124/dmd.108.025429 at A PE T Jornals on Jne 3, 2017 dm d.aspurnals.org D ow nladed from

Abstract Critical to mechanisms of chemical carcinogenesis and the design of chemopreventive strategies is whether procarcinogen bioactivation in an extrahepatic target tissue (e.g., the lung) is essential for tumor formation. This study aims to develop a mouse model capable of revealing the role of pulmonary microsomal cytochrome P450 (P450)–mediated metabolic activation in xenobiotic-induced lung cancer. A novel triple transgenic mouse model, with the NADPH-P450 reductase (Cpr) gene deleted in a lung-specific and doxycycline-inducible fashion (lung-Cpr-null), was generated. CPR, the obligate electron donor for microsomal P450 enzymes, is essential for the bioactivation of many procarcinogens. The lung-Cpr-null mouse was studied to resolve whether pulmonary P450 plays a major role in 4-(methylnitrosamino)-1–(3-pyridyl)-1-butanone (NNK)–induced lung cancer by producing carcinogenic metabolites in the target tissue. A liver-Cpr-null mouse was also studied to test whether hepatic P450 contributes predominantly to systemic clearance of NNK, thereby decreasing NNK-induced lung cancer. The numbers of NNK-induced lung tumors were reduced in the lung-Cpr-null mice but were increased in the liver-Cpr-null mice, relative to wild-type control mice. Decreased lung tumor multiplicity in the lung-Cpr-null mice correlated with reduced lung O6-methylguanine adduct levels, without decreases in NNK bioavailability, consistent with decreased NNK bioactivation in the lung. Moreover, lung tumors in lung-Cpr-null mice were positive for CPR expression, indicating that the tumors did not originate from Cpr-null cells. Thus, we have confirmed the essential role of pulmonary P450-mediated metabolic activation in NNK-induced lung cancer, and our mouse models should be applicable to studies on other procarcinogens that require P450-mediated metabolic activation. [Cancer Res 2007;67(16):7825–32]

Several systemic and cellular markers of 3'-azido-3'-dideoxythymidine (AZT) metabolism and AZT incorporation into nuclear DNA were measured in cord blood from uninfected infants born to HIV-1-infected mothers receiving prepartum therapies based on AZT or AZT in combination with 2',3'-dideoxy-3'-thiacytidine (3TC). In addition, the relationships among these pharmacological end points, levels of AZT-DNA incorporation, and the previously reported mutagenic responses in these infants were evaluated. AZT- and 3TC-specific radioimmunoassays (RIAs), or HPLC coupled with AZT-RIA, were used to measure plasma levels of AZT and the AZT-glucuronide, and cellular levels of AZT, phosphorylated AZT, and DNA incorporation of AZT or 3TC in cord blood mononuclear cells from treated infants compared with unexposed controls born to HIV-uninfected mothers. Fewer infants had detectable AZT-DNA incorporation levels in the group exposed to AZT (71%; n = 7) compared with those receiving AZT-3TC (100%; n = 21), and the mean AZT-DNA incorporation for AZT-exposed infants (14.6 +/- 6.3 AZT/10(6) nucleotides) was significantly lower than that in AZT-3TC exposed infants (51.6 +/- 10.2 AZT/10(6) nucleotides; P = 0.028). Low levels of 3TC-DNA incorporation found in a few AZT-3TC-exposed newborns correlated with AZT-DNA incorporation values in the same samples. Among the metabolites studied, there were positive correlations between levels of AZT-diphosphate and AZT-triphosphate, and AZT-triphosphate and AZT-DNA incorporation, in nucleoside analog-exposed infants. Levels of AZT-DNA incorporation, however, did not correlate well with the reported frequencies of somatic mutations in the same population of nucleoside analog-treated children. While these data support the continued use of AZT-based therapies during pregnancy, infants receiving prepartum AZT should be monitored long-term for adverse health effects.

Trace metals and polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental co-contaminants and the trace metals could influence the carcinogenicity of the PAHs by altering their extent of induction of cytochromes P4501A1, 1A2, and 1B1 (CYP). Studies in cell lines from humans, rodents, chickens, and fish, and in cell culture generally indicate that trace metals diminish the inductive potency of PAHs for these CYPs. The extent of the effect is species-, metal-, PAH-, and metal dose-dependent. Both transcriptional and post-translational mechanisms are involved in the trace metal-mediated down regulation of the CYP1 forms. The latter mechanism incorporates induction of heme oxygenase-1 by the metals, with resultant heme catabolism. Thus, trace metals could diminish the carcinogenicity of PAHs.

A mouse model with a hypomorphic NADPH-cytochrome P450 reductase (Cpr) gene (designated Cpr(low) allele) was generated and characterized in this study. The Cpr gene in these mice was disrupted by the insertion of a neo gene in intron 15, which led to 74 to 95% decreases in CPR expression in all tissues examined, including olfactory mucosa, adrenal gland, brain, testis, ovary, lung, kidney, liver, and heart. In the liver, a pattern of pericentral distribution of CPR protein was preserved in the Cpr(low/low) mice, despite an overall reduction in CPR expression. Genotype distribution in F2 pups indicated limited embryonic lethality associated with the Cpr(low) allele, a finding that confirms the role of CPR-dependent enzymes in development. Adult male homozygotes had decreased body weight and decreased heart, lung, and kidney weights, whereas homozygous Cpr(low) females, which had increased serum testosterone and progesterone and decreased copulatory activities, were infertile. Furthermore, adult Cpr(low/low) mice had decreased plasma cholesterol, and some mice developed mild centrilobular hepatic lipidosis. In addition, despite apparently compensatory increases in total microsomal cytochrome P450 content in the liver and kidney, the decreases in CPR expression were accompanied by reductions in systemic clearance of pentobarbital, as well as in hepatic microsomal metabolism of acetaminophen and testosterone. These phenotypes illustrate the potential impact of a globally decreased CPR activity in human adults, and this novel knock-in mouse model provides a unique opportunity for further explorations of the in vivo roles of CPR and CPR-dependent enzymes.

Polycyclic aromatic hydrocarbons (PAHs) and heavy metals are often environmental cocontaminants that could interact to alter PAH carcinogenicity. The heavy metal, arsenite, and the PAH, benzo[k]fluoranthene, were used as prototypes to investigate, in human HepG2 cells, mechanisms whereby the bioactivation of benzo[k]fluoranthene by human CYP1A1 could be diminished by arsenite-mediated decreases in CYP1A1 induction by benzo[k]fluoranthene. To determine whether arsenite down-regulates CYP1A1 transcription, quantitative real-time reverse transcriptase-polymerase chain reaction assays and luciferase reporter gene expression assays were used with HepG2 cells treated with benzo[k]fluoranthene and arsenite, separately and as a mixture. Benzo[k]fluoranthene (0.5 μM) and arsenite (5 μM) markedly decreased benzo[k]fluoranthene-mediated induction of CYP1A1 mRNA by 45%. Plasmids containing the CYP1A1 promoter region (pHu-1A1-FL) were induced 7.4-fold over vehicle by benzo[k]fluoranthene (0.5 μM), whereas arsenite (1, 2.5, or 5 μM) decreased reporter gene expression by 46%, 45%, and 61%, respectively. The plasmid, pHu-1A1-Δ100-FL, lacked xenobiotic response element (XRE) sites at –1061 and –981 and showed greater responsiveness relative to pHu-1A1-FL, by 1.7-fold. Benzo[k]fluoranthene (0.5 μM) and arsenite (1, 2.5, or 5 μM) decreased reporter gene expression by 0%, 27%, and 39%, respectively, relative to expression levels produced by benzo[k]fluoranthene alone. Arsenite is stable for at least 48 h in the HepG2 cell medium with respect to its ability to diminish CYP1A1 benzo[k]fluoranthene induction. Arsenite did not affect benzo[k]fluoranthene induction directly through XRE sites, nor did it affect the stability of CYP1A1 mRNA. Thus, arsenite affects the transcriptional regulation of the benzo[k]fluoranthene-mediated induction of CYP1A1 and could diminish PAH carcinogenicity by decreasing bioactivation by CYP1A1.

Exfoliated cytologic specimens from mouth (buccal) epithelium may contain viable cells, permitting assay of gene expression for direct and noninvasive measurement of gene-environment interactions, such as for inhalation (e.g., tobacco smoke) exposures. We determined specific mRNA levels in exfoliated buccal cells collected by cytologic brush, using a recently developed RNA-specific real-time quantitative reverse transcription-PCR strategy. In a pilot study, metabolic activity of exfoliated buccal cells was verified by 3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium assay in vitro. Transcriptional activity was observed, after timed in vivo exposure to mainstream tobacco smoke resulted in induction of CYP1B1 in serially collected buccal samples from the one subject examined. For a set of 11 subjects, mRNA expression of nine genes encoding carcinogen- and oxidant-metabolizing enzymes qualitatively detected in buccal cells was then shown to correlate with that in laser-microdissected lung from the same individuals (Chi2 = 52.91, P < 0.001). Finally, quantitative real-time reverse transcription-PCR assays for seven target gene (AhR, CYP1A1, CYP1B1, GSTM1, GSTM3, GSTP1, and GSTT1) and three reference gene [glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-actin, and 36B4] transcripts were performed on buccal specimens from 42 subjects. In multivariate analyses, gender, tobacco smoke exposure, and other factors were associated with the level of expression of CYP1B1, GSTP1, and other transcripts on a gene-specific basis, but substantial interindividual variability in mRNA expression remained unexplained. Within the power limits of this pilot study, gene expression signature was not clearly predictive of lung cancer case or control status. This noninvasive and quantitative method may be incorporated into high-throughput human applications for probing gene-environment interactions associated with cancer.

The expression of biotransformation enzymes in mouse small intestine is poorly characterized, which limits the utility of transgenic or knockout mouse models for first-pass drug metabolism studies. In response, we have systematically examined the composition and inducibility of cytochrome P450 (P450) protein and mRNA in mouse small intestinal epithelial cells (enterocytes). RNA-PCR was conducted to confirm the expression and identity of CYP1A1, 1B1, 2B10, 2B19, 2B20, 2C29, 2C38, 2C40, 2E1, 3A11, 3A13, 3A16, 3A25, and 3A44 in the enterocytes of untreated mice, but CYP1A2, 2A4/5, 2A12, 2C37, 2C39, and 2F2 were not detected. The inducibility of CYP2B, 2C, and 3A subfamily forms was determined by real-time quantitative RNA-PCR. All five CYP3A forms were induced, in a range from 1.7- to 4.5-fold, by dexamethasone (DEX). Phenobarbital (PB) induced CYP2B9, CYP2B10, and CYP2B20 mRNAs and suppressed CYP2B19 mRNA levels. PB also induced CYP2C29 and CYP2C40, but not CYP2C38 mRNA. At the protein level, CYP1A1, CYP1B1, CYP2B, CYP2C, CYP2E1, and CYP3A were detected in enterocytes from untreated mice by immunoblot analysis. CYP1A1 was inducible by beta-naphthoflavone (BNF), CYP2B and CYP2C by PB, and CYP3A by DEX. CYP2B, 2C, and 3A proteins were all expressed at high levels proximally, and decreased distally. The inducibility of CYP1A1 followed a similar pattern. Intestinal P450 expression was compared between C57BL/6 (B6) and 129/sv (129) mice, strains commonly used in the preparation of transgenic and knockout mouse models. There was no significant strain difference in constitutive levels or induction patterns for CYP2B, 2C, and 3A protein. However, CYP1A1 was induced to a high level by BNF in B6 mice, but was not induced in the 129 mice.

The mammalian small intestine serves principally as the site for absorption of nutrients, water, and both beneficial and potentially harmful xenobiotics. However, it has become apparent over the past 20 years, and most notably during the past 10 years, that an array of metabolic machinery is also

PURPOSE:The regulation of carcinogen metabolism machinery may involve proximate tobacco smoke exposure, hormonal and other endogenous coregulatory factors, and an individual's underlying genetic responsiveness. The mRNA and protein expression patterns of known carcinogen metabolism genes encoding the aromatic hydrocarbon receptor Ahr; the cytochromes P450 CYP1A1 and CYP1B1; glutathione S-transferases GSTM1, GSTM3, GSTP1, and GSTT1; and NADPH quinone oxidoreductase NQO1 were examined.EXPERIMENTAL DESIGN:Paired tumor and nontumor lung tissue from 45 subjects was subject to a recently devised RNA-specific qualitative reverse transcription-PCR strategy, as well as Western immunoblotting. Tobacco exposure measured by plasma biomarkers nicotine and cotinine, plasma estradiol levels, alpha and beta estrogen receptor (ER) expression in the lung, gender, age, and histological diagnosis were then analyzed using multivariate regression models.RESULTS:In nontumor lung tissue, multivariate models identified several correlates of mRNA expression: (a) CYP1B1 in females (positively: smoke status, P=0.024; ER-beta expression, P=0.024); (b) GSTT1 in females (positively: cotinine, P=0.007; negatively: age, P=0.001; ER-beta expression, P=0.005) and in males (positively: plasma estradiol, P=0.015; ER-beta expression, P=0.025); and (c) NQO1 in females (positively: smoke status, P=0.002) and in males (positively: ER-beta expression, P=0.001). CYP1A1 (mRNA, 9.1%) and GSTM1 (mRNA, 17.5%) are uncommonly expressed in human lung. Confirmation by Western immunoassayed protein is described. The results in nontumor tissue differed from that in tumor tissue.CONCLUSIONS:Regulation of carcinogen metabolism genes expressed in human lung seems impacted by hormonal and gender factors, as well as ongoing tobacco exposure. Expression differences between tumor and nontumor tissue in this pathway have both susceptibility and therapeutic implications.