Xinxin Ding

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

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.

CYP2A13: Variable Expression and Role in Human Lung Microsomal Metabolic Activation of the Tobacco-Specific Carcinogen 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone

CYP2A13 is the most efficient cytochrome P450 enzyme in the metabolic activation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a tobacco-specific lung carcinogen. The aims of this study were to determine the levels of CYP2A13 protein in human lung microsomes and to ascertain whether CYP2A13 plays any role in lung microsomal NNK metabolic activation. The expression of CYP2A6 and CYP2A13 was examined using a high-resolution immunoblotting method, following immunopurification with an anti-CYP2A5 antibody. We found that, of 116 human lung microsomal samples analyzed, ∼90% had detectable CYP2A6, whereas only 12% had detectable CYP2A13 with a detection limit of ∼2 fmol of CYP2A/mg protein. For the majority of microsomal samples analyzed, the level of CYP2A13 was found to be lower than the level of CYP2A6; overall, the highest level of CYP2A13 found (∼20 fmol/mg protein) was ∼10-fold lower than the highest level of CYP2A6 detected. Quantitative RNA-polymerase chain reaction analysis confirmed that the highly variable expression of the CYP2A proteins was consistent with variations in the levels of the corresponding CYP2A mRNAs in the same tissue samples. It is noteworthy that the level of CYP2A13, but not CYP2A6, was correlated with lung microsomal NNK metabolic activation activity. Furthermore, the addition of 8-methoxypsoralen, a CYP2A inhibitor, led to greater inhibition of NNK metabolic activation in microsomes containing relatively high levels of CYP2A13 than in samples containing no detectable CYP2A13. Taken together, these data indicate that human lung microsomal CYP2A13 is active in NNK metabolic activation. Therefore, individuals having relatively high levels of CYP2A13 expression will likely have an increased risk of developing smoking-related lung cancer.

NADPH-cytochrome P450 oxidoreductase: roles in physiology, pharmacology, and toxicology.

This is a report on a symposium sponsored by the American Society for Pharmacology and Experimental Therapeutics and held at the Experimental Biology 2012 meeting in San Diego, California, on April 25, 2012. The symposium speakers summarized and critically evaluated our current understanding of the physiologic, pharmacological, and toxicological roles of NADPH–cytochrome P450 oxidoreductase (POR), a flavoprotein involved in electron transfer to microsomal cytochromes P450 (P450), cytochrome b5, squalene mono-oxygenase, and heme oxygenase. Considerable insight has been derived from the development and characterization of mouse models with conditional Por deletion in particular tissues or partial suppression of POR expression in all tissues. Additional mouse models with global or conditional hepatic deletion of cytochrome b5 are helping to clarify the P450 isoform- and substrate-specific influences of cytochrome b5 on P450 electron transfer and catalytic function. This symposium also considered studies using siRNA to suppress POR expression in a hepatoma cell–culture model to explore the basis of the hepatic lipidosis phenotype observed in mice with conditional deletion of Por in liver. The symposium concluded with a strong translational perspective, relating the basic science of human POR structure and function to the impacts of POR genetic variation on human drug and steroid metabolism.

Generation and Characterization of a Cyp2f2-null Mouse and Studies on the Role of CYP2F2 in Naphthalene-induced Toxicity in the Lung and Nasal Olfactory Mucosa

The CYP2F enzymes, abundantly expressed in the respiratory tract, are active toward many xenobiotic compounds, including naphthalene (NA). However, the precise roles of these enzymes in tissue-selective chemical toxicity have been difficult to resolve. A Cyp2f2-null mouse was generated in this study by disrupting the Cyp2f2 fourth exon. Homozygous Cyp2f2-null mice, which had no CYP2F2 expression and showed no changes in the expression of other P450 genes examined, were viable and fertile and had no in utero lethality or developmental deficits. The loss of CYP2F2 expression led to substantial decreases in the in vitro catalytic efficiency of microsomal NA epoxygenases in lung (up to ∼160-fold), liver (∼3-fold), and nasal olfactory mucosa (OM; up to ∼16-fold), and significant decreases in rates of systemic NA (300 mg/kg i.p.) clearance. The Cyp2f2-null mice were largely resistant to NA-induced cytotoxicity, when examined at 24 h after NA dosing (at 300 mg/kg i.p.), and to NA-induced depletion of total nonprotein sulfhydryl (NPSH), examined at 2 h after dosing, in the lungs. In contrast, the loss of CYP2F2 expression did not alleviate NA-induced NPSH depletion or tissue toxicity in the OM. Mouse CYP2F2 clearly plays an essential role in the bioactivation and toxicity of NA in the lung but not in the OM. The Cyp2f2-null mouse should be valuable for studies on the role of CYP2F2 in the metabolism and toxicity of numerous other xenobiotic compounds and for future production of a CYP2F1-humanized mouse.

Immunoblot analysis and immunohistochemical characterization of CYP2A expression in human olfactory mucosa

The aim of the present study was to further characterize the expression of the CYP2A genes in human nasal mucosa. Fetal nasal tissues at 12-26 weeks of gestational age and surgical biopsy tissues from various regions of nasal cavity of adult patients were studied to determine whether CYP2A proteins can be detected by immunoblot in adults, whether higher levels of CYP2A proteins are present in adult than in fetal nasal mucosal microsomes, and whether CYP2A13 mRNA is more abundant than CYP2A6 mRNA in fetal nasal mucosa. In adults, immunoblot analysis detected CYP2A proteins in microsomes of the olfactory region from 8 of 10 individuals, but in none of the nasal microsomes of the respiratory region from 47 patients. Quantitative immunoblot analysis confirmed that CYP2A proteins are selectively expressed in the olfactory region in both adult and fetal tissues. Interestingly, the levels of CYP2A proteins in nasal microsomes were generally higher in fetuses than in adults. In the fetus, the level of CYP2A13 mRNA was much higher than that of CYP2A6 mRNA, as has been previously found in adult nasal mucosa. Immunohistochemical studies confirmed that, in the fetus, the CYP2A proteins are expressed in the supporting cells in the olfactory epithelium and in the Bowmans glands in the lamina propria. The prenatal expression of the CYP2A proteins in the olfactory mucosa suggests potential risks of developmental toxicity from maternally derived xenobiotics, since both CYP2A6 and CYP2A13 are known to be efficient in the metabolic activation of tobacco-specific nitrosamines and other respiratory toxicants.

Metabolism of 4-(methyhiitrosamino)-l-(3-pyridyI)-1-butanone (NNK) a tobacco-specific carcinogen, by rabbit nasal microsomes and cytochrome P450s NMa and NMb

Rabbit nasal olfactory and respiratory microsomes were found to catalyze the alpha-hydroxylation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) with specific activities of 262 and 136 pmol/min/mg protein in the formation of keto aldehyde, and of 318 and 190 pmol/min/mg protein in the formation of keto alcohol respectively. The formation of NNK-N-oxide was observed in experiments with rabbit olfactory and respiratory microsomes, but not with rat nasal microsomes. However, the rat nasal microsomes had higher activity in catalyzing the alpha-hydroxylation of NNK. In a reconstituted system, rabbit P450NMa, a major constitutive P450 isozyme in nasal microsomes, displayed high activities in the formation of the keto aldehyde and the keto alcohol with apparent Km values of 15 and 9 microM respectively. In comparison, rabbit olfactory specific P450NMb had a low activity in catalyzing the formation of keto aldehyde (Km = 186 microM) and no activity in the formation of keto alcohol. The P450NMa-catalyzed oxidation of NNK was inhibited by nicotine and diallyl sulfide. Kinetic studies indicated that nicotine is a competitive inhibitor. These results demonstrate that enzymes in rabbit nasal microsomes, especially P450NMa, efficiently catalyze the bioactivation of NNK.

A Mouse Model with Liver-Specific Deletion and Global Suppression of the NADPH-Cytochrome P450 Reductase Gene: Characterization and Utility for in Vivo Studies of Cyclophosphamide Disposition

A mouse model combining liver-specific deletion with global suppression of the NADPH-cytochrome P450 reductase gene (Cpr) has been developed and characterized. These mice (designated “Cpr-low and liver-Cpr-null” or CL-LCN) retain the respective phenotypes of the previously reported Cpr-low (CL) and liver-Cpr-null (LCN) mouse strains, but hepatic deletion of the Cpr gene occurs at an earlier age in the CL-LCN mouse than in the LCN mouse. Residual hepatic microsomal CPR activities are very low in both CL-LCN and LCN mice (at 1.5 and 2.5% of wild-type levels, respectively). The utility of CL-LCN mice for in vivo drug metabolism studies was explored using the cytochrome P450 (P450) prodrug cyclophosphamide (CPA). After i.p. injection of CPA at 100 mg/kg, the t1/2 and the area under the concentration-time curve for plasma CPA were significantly increased in mice deficient in liver CPR compared with wild-type controls, indicating a lower rate of metabolism, with the effects greater in CL-LCN mice than in LCN mice. Correspondingly, substantial decreases in Cmax, and increases in Tmax, and t1/2, of 4-hydroxycyclophosphamide (4-OH-CPA) formation were observed in both LCN and CL-LCN mice relative to wild-type controls. In contrast, CPA and 4-OH-CPA pharmacokinetic parameters were essentially unchanged in CL mice, relative to wild-type controls. The slower elimination of CPA in CL-LCN mice compared with LCN mice suggests a role for extrahepatic P450 in the in vivo metabolism of CPA and demonstrates the utility of the CL-LCN model in determining the role of extrahepatic P450 enzymes in drug metabolism and chemical toxicity.

Role of CYP2A13 in the bioactivation and lung tumorigenicity of the tobacco-specific lung procarcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone: in vivo studies using a CYP2A13-humanized mouse model.

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), which is abundant in tobacco smoke, is a potent lung procarcinogen. The present study was aimed to prove that transgenic expression of human cytochrome P450 2A13 (CYP2A13), known to be selectively expressed in the respiratory tract and be the most efficient enzyme for NNK bioactivation in vitro, will enhance NNK bioactivation and NNK-induced tumorigenesis in the mouse lung. Kinetic parameters of NNK bioactivation in vitro and incidence of NNK-induced lung tumors in vivo were determined for wild-type, Cyp2a5-null and CYP2A13-humanized (CYP2A13-transgenic/Cyp2a5-null) mice. As expected, in both liver and lung microsomes, the loss of CYP2A5 resulted in significant increases in Michaelis constant (K m) values for the formation of 4-oxo-4-(3-pyridyl)-butanal, representing the reactive intermediate that can lead to the formation of O(6)-methylguanine (O(6)-mG) DNA adducts; however, the gain of CYP2A13 at a fraction of the level of mouse lung CYP2A5 led to recovery of the activity in the lung, but not in the liver. The levels of O(6)-mG, the DNA adduct highly correlated with lung tumorigenesis, were significantly higher in the lungs of CYP2A13-humanized mice than in Cyp2a5-null mice. Moreover, incidences of lung tumorigenesis were significantly greater in CYP2A13-humanized mice than in Cyp2a5-null mice, and the magnitude of the differences in incidence was greater at low (30mg/kg) than at high (200mg/kg) NNK doses. These results indicate that CYP2A13 is a low K m enzyme in catalyzing NNK bioactivation in vivo and support the notion that genetic polymorphisms of CYP2A13 can influence the risks of tobacco-induced lung tumorigenesis in humans.

Mechanisms of olfactory toxicity of the herbicide 2,6-dichlorobenzonitrile: Essential roles of CYP2A5 and target-tissue metabolic activation

The herbicide 2,6-dichlorobenzonitril (DCBN) is a potent and tissue-specific toxicant to the olfactory mucosa (OM). The toxicity of DCBN is mediated by cytochrome P450 (P450)-catalyzed bioactivation; however, it is not known whether target-tissue metabolic activation is essential for toxicity. CYP2A5, expressed abundantly in both liver and OM, was previously found to be one of the P450 enzymes active in DCBN bioactivation in vitro. The aims of this study were to determine the role of CYP2A5 in DCBN toxicity in vivo, by comparing the extents of DCBN toxicity between Cyp2a5-null and wild-type (WT) mice, and to determine whether hepatic microsomal P450 enzymes (including CYP2A5) are essential for the DCBN toxicity, by comparing the extents of DCBN toxicity between liver-Cpr-null (LCN) mice, which have little P450 activity in hepatocytes, and WT mice. We show that the loss of CYP2A5 expression did not alter systemic clearance of DCBN (at 25 mg/kg); but it did inhibit DCBN-induced non-protein thiol depletion and cytotoxicity in the OM. Thus, CYP2A5 plays an essential role in mediating DCBN toxicity in the OM. In contrast to the results seen in the Cyp2a5-null mice, the rates of systemic DCBN clearance were substantially reduced, while the extents of DCBN-induced nasal toxicity were increased, rather than decreased, in the LCN mice, compared to WT mice. Therefore, hepatic P450 enzymes, although essential for DCBN clearance, are not necessary for DCBN-induced OM toxicity. Our findings form the basis for a mechanism-based approach to assessing the potential risks of DCBN nasal toxicity in humans.

Generation and Characterization of a Novel Cyp2a(4/5)bgs-null Mouse Model

Knockout mouse models targeting various cytochrome P450 (P450 or CYP) genes are valuable for determining P450’s biologic functions, including roles in drug metabolism and chemical toxicity. In this study, a novel Cyp2a(4/5)bgs-null mouse model was generated, in which a 1.2-megabase pair genomic fragment containing nine Cyp genes in mouse chromosome 7 (including, sequentially, Cyp2a5, 2g1, 2b19, 2b23, 2a4, 2b9, 2b13, 2b10, and 2s1) are deleted, through Cre-mediated recombination in vivo. The resultant mouse strain was viable and fertile, without any developmental deficits or morphologic abnormalities. Deletion of the constitutive genes in the cluster was confirmed by polymerase chain reaction analysis of the genes and the mRNAs in tissues known to express each gene. The loss of this gene cluster led to significant decreases in microsomal activities toward testosterone hydroxylation in various tissues examined, including olfactory mucosa (OM), lung, liver, and brain. In addition, systemic clearance of pentobarbital was decreased in Cyp2a(4/5)bgs-null mice, as indicated by >60% increases in pentobarbital-induced sleeping time, compared with wild-type (WT) mice. This novel Cyp2a(4/5)bgs-null mouse model will be valuable for in vivo studies of drug metabolism and chemical toxicities in various tissues, including the liver, lung, brain, intestine, kidney, skin, and OM, where one or more of the targeted Cyp genes are known to be expressed in WT mice. The model will also be valuable for preparation of humanized mice that express human CYP2A6, CYP2A13, CYP2B6, or CYP2S1, and as a knockout mouse model for five non-P450 genes (Vmn1r184, Nalp9c, Nalp4a, Nalp9a, and Vmn1r185) that were also deleted.