ABSTRACT
Current risk assessments for environmental carcinogens rely on animal studies utilizing doses orders of magnitude higher than actual human exposures. Epidemiological studies of people with high exposures (e.g., occupational) are of value, but rely on uncertain exposure data. In addition, exposures are typically not to a single chemical but to mixtures, such as polycyclic aromatic hydrocarbons (PAHs). The extremely high sensitivity of accelerator mass spectrometry (AMS) allows for dosing humans with known carcinogens with de minimus risk. In this study UPLC-AMS was used to assess the toxicokinetics of [14C]-benzo[a]pyrene ([14C]-BaP) when dosed alone or in a binary mixture with phenanthrene (Phe). Plasma was collected for 48 h following a dose of [14C]-BaP (50 ng, 5.4 nCi) or the same dose of [14C]-BaP plus Phe (1250 ng). Following the binary mixture, Cmax of [14C]-BaP significantly decreased (4.4-fold) whereas the volume of distribution (Vd) increased (2-fold). Further, the toxicokinetics of twelve [14C]-BaP metabolites provided evidence of little change in the metabolite profile of [14C]-BaP and the pattern was overall reduction consistent with reduced absorption (decrease in Cmax). Although Phe was shown to be a competitive inhibitor of the major hepatic cytochrome P-450 (CYP) responsible for metabolism of [14C]-BaP, CYP1A2, the high inhibition constant (Ki) and lack of any increase in unmetabolized [14C]-BaP in plasma makes this mechanism unlikely to be responsible. Rather, co-administration of Phe reduces the absorption of [14C]-BaP through a mechanism yet to be determined. This is the first study to provide evidence that, at actual environmental levels of exposure, the toxicokinetics of [14C]-BaP in humans is markedly altered by the presence of a second PAH, Phe, a common component of environmental PAH mixtures.
Subject(s)
Phenanthrenes , Polycyclic Aromatic Hydrocarbons , Animals , Humans , Benzo(a)pyrene/toxicity , Toxicokinetics , Phenanthrenes/toxicity , Phenanthrenes/metabolism , Polycyclic Aromatic Hydrocarbons/metabolism , Mass SpectrometryABSTRACT
Utilizing the atto-zeptomole sensitivity of UPLC-accelerator mass spectrometry (UPLC-AMS), we previously demonstrated significant first-pass metabolism following escalating (25-250 ng) oral micro-dosing in humans of [14C]-benzo[a]pyrene ([14C]-BaP). The present study examines the potential for supplementation with Brussels sprouts (BS) or 3,3'-diindolylmethane (DIM) to alter plasma levels of [14C]-BaP and metabolites over a 48-h period following micro-dosing with 50 ng (5.4 nCi) [14C]-BaP. Volunteers were dosed with [14C]-BaP following fourteen days on a cruciferous vegetable restricted diet, or the same diet supplemented for seven days with 50 g of BS or 300 mg of BR-DIM® prior to dosing. BS or DIM reduced total [14C] recovered from plasma by 56-67% relative to non-intervention. Dietary supplementation with DIM markedly increased Tmax and reduced Cmax for [14C]-BaP indicative of slower absorption. Both dietary treatments significantly reduced Cmax values of four downstream BaP metabolites, consistent with delaying BaP absorption. Dietary treatments also appeared to reduce the T1/2 and the plasma AUC(0,∞) for Unknown Metabolite C, indicating some effect in accelerating clearance of this metabolite. Toxicokinetic constants for other metabolites followed the pattern for [14C]-BaP (metabolite profiles remained relatively consistent) and non-compartmental analysis did not indicate other significant alterations. Significant amounts of metabolites in plasma were at the bay region of [14C]-BaP irrespective of treatment. Although the number of subjects and large interindividual variation are limitations of this study, it represents the first human trial showing dietary intervention altering toxicokinetics of a defined dose of a known human carcinogen.
Subject(s)
Benzo(a)pyrene , Carcinogens , Humans , Dietary Supplements , ToxicokineticsABSTRACT
Benzo[a]pyrene (BaP) is formed by incomplete combustion of organic materials (petroleum, coal, tobacco, etc.). BaP is designated by the International Agency for Research on Cancer as a group 1 known human carcinogen; a classification supported by numerous studies in preclinical models and epidemiology studies of exposed populations. Risk assessment relies on toxicokinetic and cancer studies in rodents at doses 5-6 orders of magnitude greater than average human uptake. Using a dose-response design at environmentally relevant concentrations, this study follows uptake, metabolism, and elimination of [14C]-BaP in human plasma by employing UPLC - accelerator mass spectrometry (UPLC-AMS). Volunteers were administered 25, 50, 100, and 250 ng (2.7-27 nCi) of [14C]-BaP (with interceding minimum 3-week washout periods) with quantification of parent [14C]-BaP and metabolites in plasma measured over 48 h. [14C]-BaP median Tmax was 30 min with Cmax and area under the curve (AUC) approximating dose-dependency. Marked inter-individual variability in plasma pharmacokinetics following a 250 ng dose was seen with 7 volunteers as measured by the Cmax (8.99 ± 7.08 ng × mL-1) and AUC0-48hr (68.6 ± 64.0 fg × hr-1 × mL-1). Approximately 3-6% of the [14C] recovered (AUC0-48 hr) was parent compound, demonstrating extensive metabolism following oral dosing. Metabolite profiles showed that, even at the earliest time-point (30 min), a substantial percentage of [14C] in plasma was polar BaP metabolites. The best fit modeling approach identified non-compartmental apparent volume of distribution of BaP as significantly increasing as a function of dose (p = 0.004). Bay region tetrols and dihydrodiols predominated, suggesting not only was there extensive first pass metabolism but also potentially bioactivation. AMS enables the study of environmental carcinogens in humans with de minimus risk, allowing for important testing and validation of physiologically based pharmacokinetic models derived from animal data, risk assessment, and the interpretation of data from high-risk occupationally exposed populations.
Subject(s)
Benzo(a)pyrene , Carcinogens , Animals , Benzo(a)pyrene/pharmacokinetics , Humans , Mass Spectrometry , Risk AssessmentABSTRACT
3,3'-Diindolylmethane (DIM), a major phytochemical derived from ingestion of cruciferous vegetables, is also a dietary supplement. In preclinical models, DIM is an effective cancer chemopreventive agent and has been studied in a number of clinical trials. Previous pharmacokinetic studies in preclinical and clinical models have not reported DIM metabolites in plasma or urine after oral dosing, and the pharmacological actions of DIM on target tissues is assumed to be solely via the parent compound. Seven subjects (6 males and 1 female) ranging from 26-65 years of age, on a cruciferous vegetable-restricted diet prior to and during the study, took 2 BioResponse DIM 150-mg capsules (45.3 mg DIM/capsule) every evening for one week with a final dose the morning of the first blood draw. A complete time course was performed with plasma and urine collected over 48 hours and analyzed by UPLC-MS/MS. In addition to parent DIM, two monohydroxylated metabolites and 1 dihydroxylated metabolite, along with their sulfate and glucuronide conjugates, were present in both plasma and urine. Results reported here are indicative of significant phase 1 and phase 2 metabolism and differ from previous pharmacokinetic studies in rodents and humans, which reported only parent DIM present after oral administration. 3-((1H-indole-3-yl)methyl)indolin-2-one, identified as one of the monohydroxylated products, exhibited greater potency and efficacy as an aryl hydrocarbon receptor agonist when tested in a xenobiotic response element-luciferase reporter assay using Hepa1 cells. In addition to competitive phytochemical-drug adverse reactions, additional metabolites may exhibit pharmacological activity highlighting the importance of further characterization of DIM metabolism in humans. SIGNIFICANCE STATEMENT: 3,3'-Diindolylmethane (DIM), derived from indole-3-carbinol in cruciferous vegetables, is an effective cancer chemopreventive agent in preclinical models and a popular dietary supplement currently in clinical trials. Pharmacokinetic studies to date have found little or no metabolites of DIM in plasma or urine. In marked contrast, we demonstrate rapid appearance of mono- and dihydroxylated metabolites in human plasma and urine as well as their sulfate and glucuronide conjugates. The 3-((1H-indole-3-yl)methyl)indolin-2-one metabolite exhibited significant aryl hydrocarbon receptor agonist activity, emphasizing the need for further characterization of the pharmacological properties of DIM metabolites.
Subject(s)
Indoles , Administration, Oral , Anticarcinogenic Agents/blood , Anticarcinogenic Agents/pharmacokinetics , Anticarcinogenic Agents/urine , Capsules , Dietary Supplements , Drug Development , Drug Elimination Routes , Female , Humans , Inactivation, Metabolic/physiology , Indoles/blood , Indoles/pharmacokinetics , Indoles/urine , Male , Middle Aged , Phytochemicals/blood , Phytochemicals/pharmacokinetics , Phytochemicals/urineABSTRACT
One of the most difficult challenges for risk assessment is evaluation of chemicals that predominately co-occur in mixtures like polycyclic aromatic hydrocarbons (PAHs). We previously developed a classification model in which systems biology data collected from mice short-term after chemical exposure accurately predict tumor outcome. The present study demonstrates translation of this approach into a human in vitro model in which chemical-specific bioactivity profiles from 3D human bronchial epithelial cells (HBEC) classify PAHs by carcinogenic potency. Gene expression profiles were analyzed from HBEC exposed to carcinogenic and non-carcinogenic PAHs and classification accuracies were identified for individual pathway-based gene sets. Posterior probabilities of best performing gene sets were combined via Bayesian integration resulting in a classifier with four gene sets, including aryl hydrocarbon receptor signaling, regulation of epithelial mesenchymal transition, regulation of angiogenesis, and cell cycle G2-M. In addition, transcriptional benchmark dose modeling of benzo[a]pyrene (BAP) showed that the most sensitive gene sets to BAP regulation were largely dissimilar from those that best classified PAH carcinogenicity challenging current assumptions that BAP carcinogenicity (and subsequent mode of action) is reflective of overall PAH carcinogenicity. These results illustrate utility of using systems toxicology approaches to analyze global gene expression towards carcinogenic hazard assessment.
Subject(s)
Carcinogens/classification , Carcinogens/toxicity , Epithelial Cells/drug effects , Polycyclic Aromatic Hydrocarbons/classification , Polycyclic Aromatic Hydrocarbons/toxicity , Bronchi/cytology , Cell Survival/drug effects , Cells, Cultured , Epithelial Cells/metabolism , Humans , Reactive Oxygen Species/metabolism , Risk Assessment , Systems Biology , Transcriptome/drug effectsABSTRACT
Current assumption for assessing carcinogenic risk of polycyclic aromatic hydrocarbons (PAHs) is that they function through a common mechanism of action; however, recent studies demonstrate that PAHs can act through unique mechanisms potentially contributing to cancer outcomes in a non-additive manner. Using a primary human 3D bronchial epithelial culture (HBEC) model, we assessed potential differences in mechanism of toxicity for two PAHs, benzo[a]pyrene (BAP) and dibenzo[def,p]chrysene (DBC), compared to a complex PAH mixture based on short-term biosignatures identified from transcriptional profiling. Differentiated bronchial epithelial cells were treated with BAP (100-500⯵g/ml), DBC (10⯵g/ml), and coal tar extract (CTE 500-1500⯵g/ml, SRM1597a) for 48â¯h and gene expression was measured by RNA sequencing or quantitative PCR. Comparison of BAP and DBC gene signatures showed that the majority of genes (~60%) were uniquely regulated by treatment, including signaling pathways for inflammation and DNA damage by DBC and processes for cell cycle, hypoxia and oxidative stress by BAP. Specifically, BAP upregulated targets of AhR, NRF2, and KLF4, while DBC downregulated these same targets, suggesting a chemical-specific pattern in transcriptional regulation involved in antioxidant response, potentially contributing to differences in PAH potency. Other processes were regulated in common by all PAH treatments, BAP, DBC and CTE, including downregulation of genes involved in cell adhesion and reduced functional measurements of barrier integrity. This work supports prior in vivo studies and demonstrates the utility of profiling short-term biosignatures in an organotypic 3D model to identify mechanisms linked to carcinogenic risk of PAHs in humans.
Subject(s)
Benzopyrenes/toxicity , Bronchi/drug effects , Polycyclic Aromatic Hydrocarbons/toxicity , Respiratory Mucosa/drug effects , Benzo(a)pyrene , Bronchi/cytology , Bronchi/metabolism , Cells, Cultured , Dose-Response Relationship, Drug , Gene Expression/drug effects , Humans , Kruppel-Like Factor 4 , L-Lactate Dehydrogenase/metabolism , Respiratory Mucosa/metabolism , Sequence Analysis, RNA , Toxicity Tests/methods , TranscriptomeABSTRACT
Benzo[a]pyrene (BaP), is a known human carcinogen (International Agency for Research on Cancer (IARC) class 1). The remarkable sensitivity (zepto-attomole 14C in biological samples) of accelerator mass spectrometry (AMS) makes possible, with de minimus risk, pharmacokinetic (PK) analysis following [14C]-BaP micro-dosing of humans. A 46â¯ng (5 nCi) dose was given thrice to 5 volunteers with minimum 2â¯weeks between dosing and plasma collected over 72â¯h. [14C]-BaPeq PK analysis gave plasma Tmax and Cmax values of 1.25â¯h and 29-82â¯fg/mL, respectively. PK parameters were assessed by non- compartment and compartment models. Intervals between dosing ranged from 20 to 420â¯days and had little impact on intra-individual variation. DNA, extracted from peripheral blood mononuclear cells (PBMCs) of 4 volunteers, showed measurable levels (LOD ~ 0.5 adducts/1011 nucleotides) in two individuals 2-3â¯h post-dose, approximately three orders of magnitude lower than smokers or occupationally-exposed individuals. Little or no DNA binding was detectable at 48-72â¯h. In volunteers the allelic variants CYP1B1*1/*â1, *1/*3 or *3/*3 and GSTM1*0/0 or *1 had no impact on [14C]-BaPeq PK or DNA adduction with this very limited sample. Plasma metabolites over 72â¯h from two individuals (one CYP1B1*1/*1 and one CYP1B1*3/*3) were analyzed by UPLC-AMS. In both individuals, parent [14C]-BaP was a minor constituent even at the earliest time points and metabolite profiles markedly distinct. AMS, coupled with UPLC, could be used in humans to enhance the accuracy of pharmacokinetics, toxicokinetics and risk assessment of environmental carcinogens.
Subject(s)
Benzo(a)pyrene/pharmacokinetics , Carcinogens/pharmacokinetics , Chromatography, Liquid/methods , Mass Spectrometry , Administration, Oral , Adult , Aged , Benzo(a)pyrene/administration & dosage , Benzo(a)pyrene/adverse effects , Carcinogens/administration & dosage , Carcinogens/toxicity , Cytochrome P-450 CYP1B1/genetics , Cytochrome P-450 CYP1B1/metabolism , DNA Adducts/metabolism , Female , Glutathione Transferase/genetics , Glutathione Transferase/metabolism , Humans , Male , Middle Aged , Models, Biological , Pharmacogenomic Variants , Risk Assessment , Young AdultABSTRACT
Benzo[a]pyrene (BaP), a polycyclic aromatic hydrocarbon (PAH), is a known human carcinogen. In non-smoking adults greater than 95% of BaP exposure is through diet. The carcinogenicity of BaP is utilized by the U.S. EPA to assess relative potency of complex PAH mixtures. PAH relative potency factors (RPFs, BaPâ¯=â¯1) are determined from high dose animal data. We employed accelerator mass spectrometry (AMS) to determine pharmacokinetics of [14C]-BaP in humans following dosing with 46â¯ng (an order of magnitude lower than human dietary daily exposure and million-fold lower than animal cancer models). To assess the impact of co-administration of food with a complex PAH mixture, humans were dosed with 46â¯ng of [14C]-BaP with or without smoked salmon. Subjects were asked to avoid high BaP-containing diets and a 3-day dietary questionnaire given to assess dietary exposure prior to dosing and three days post-dosing with [14C]-BaP. Co-administration of smoked salmon, containing a complex mixture of PAHs with an RPF of 460â¯ng BaPeq, reduced and delayed absorption. Administration of canned commercial salmon, containing very low amounts of PAHs, showed the impacts on pharmacokinetics were not due to high amounts of PAHs but rather a food matrix effect.
Subject(s)
Benzo(a)pyrene/pharmacokinetics , Carcinogens/pharmacokinetics , Fish Products/analysis , Salmon/metabolism , Adult , Aged , Animals , Benzo(a)pyrene/metabolism , Carbon Radioisotopes/analysis , Carcinogens/metabolism , Cooking , Female , Fish Products/adverse effects , Food Safety , Humans , Male , Middle Aged , Polycyclic Aromatic Hydrocarbons/metabolism , Polycyclic Aromatic Hydrocarbons/pharmacokinetics , Young AdultABSTRACT
The cytochrome P450 (CYP) 1 family is active toward numerous environmental pollutants, including polycyclic aromatic hydrocarbons (PAHs). Utilizing a mouse model, null for Cyp1b1 and expressing human CYP1B1, we tested the hypothesis that hCYP1B1 is important for dibenzo[def,p]chrysene (DBC) transplacental carcinogenesis. Wild-type mCyp1b1, transgenic hCYP1B1 (mCyp1b1 null background), and mCyp1b1 null mice were assessed. Each litter had an equal number of siblings with Ahrb-1/d and Ahrd/d alleles. Pregnant mice were dosed (gavage) on gestation day 17 with 6.5 or 12 mg/kg of DBC or corn oil. At 10 months of age, mortality, general health, lymphoid disease and lung tumor incidence, and multiplicity were assessed. hCYP1B1 genotype did not impact lung tumor multiplicity, but tended to enhance incidence compared to Cyp1b1 wild-type mice (P = 0.07). As with Cyp1b1 in wild-type mice, constitutive hCYP1B1 protein is non-detectable in liver but was induced with 2,3,7,8-tetrachlorodibenzo-p-dioxin. Wild-type mice were 59% more likely to succumb to T-cell Acute Lymphoblastic Leukemia (T-ALL). Unlike an earlier examination of the Ahr genotype in this model (Yu et al., Cancer Res, 2006;66:755-762), but in agreement with a more recent study (Shorey et al., Toxicol Appl Pharmacol, 2013;270:60-69), this genotype was not associated with lung tumor incidence, multiplicity, or mortality. Sex was not significant with respect to lung tumor incidence or mortality but males exhibited significantly greater multiplicity. Lung tumor incidence was greater in mCyp1b1 nulls compared to wild-type mice. To our knowledge, this is the first application of a humanized mouse model in transplacental carcinogenesis. © 2016 Wiley Periodicals, Inc.
Subject(s)
Carcinogenesis/genetics , Cytochrome P-450 CYP1B1/genetics , Lung Neoplasms/genetics , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Pregnancy Complications, Neoplastic/genetics , Animals , Carcinogenesis/chemically induced , Carcinogenesis/pathology , Carcinogens , Chrysenes , Female , Gene Expression Regulation, Neoplastic , Humans , Lung/pathology , Lung Neoplasms/chemically induced , Lung Neoplasms/pathology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Placenta/pathology , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/chemically induced , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/pathology , Pregnancy , Pregnancy Complications, Neoplastic/chemically induced , Pregnancy Complications, Neoplastic/pathologyABSTRACT
FVB/N mice wild-type, heterozygous or null for Cyp 1b1 were used in a two-stage skin tumor study comparing PAH, benzo[a]pyrene (BaP), dibenzo[def,p]chrysene (DBC), and coal tar extract (CTE, SRM 1597a). Following 20 weeks of promotion with TPA the Cyp 1b1 null mice, initiated with DBC, exhibited reductions in incidence, multiplicity, and progression. None of these effects were observed with BaP or CTE. The mechanism of Cyp 1b1-dependent alteration of DBC skin carcinogenesis was further investigated by determining expression of select genes in skin from DBC-treated mice 2, 4 and 8h post-initiation. A significant reduction in levels of Cyp 1a1, Nqo1 at 8h and Akr 1c14 mRNA was observed in Cyp 1b1 null (but not wt or het) mice, whereas no impact was observed in Gst a1, Nqo 1 at 2 and 4h or Akr 1c19 at any time point. Cyp 1b1 mRNA was not elevated by DBC. The major covalent DNA adducts, dibenzo[def,p]chrysene-(±)-11,12-dihydrodiol-cis and trans-13,14-epoxide-deoxyadenosine (DBCDE-dA) were quantified by UHPLC-MS/MS 8h post-initiation. Loss of Cyp1 b1 expression reduced DBCDE-dA adducts in the skin but not to a statistically significant degree. The ratio of cis- to trans-DBCDE-dA adducts was higher in the skin than other target tissues such as the spleen, lung and liver (oral dosing). These results document that Cyp 1b1 plays a significant role in bioactivation and carcinogenesis of DBC in a two-stage mouse skin tumor model and that loss of Cyp 1b1 has little impact on tumor response with BaP or CTE as initiators.
Subject(s)
Carcinogens/toxicity , Coal Tar/toxicity , Cytochrome P-450 CYP1B1/metabolism , Polycyclic Aromatic Hydrocarbons/toxicity , Skin Neoplasms/chemically induced , Animals , Benzopyrenes , Cytochrome P-450 CYP1B1/genetics , DNA Adducts/metabolism , Female , Gene Expression , Mice , Mice, Knockout , RNA, Messenger/biosynthesis , Tandem Mass Spectrometry , Time FactorsABSTRACT
We have previously shown that relative potency factors and DNA adduct measurements are inadequate for predicting carcinogenicity of certain polycyclic aromatic hydrocarbons (PAHs) and PAH mixtures, particularly those that function through alternate pathways or exhibit greater promotional activity compared to benzo[a]pyrene (BaP). Therefore, we developed a pathway-based approach for classification of tumor outcome after dermal exposure to PAH/mixtures. FVB/N mice were exposed to dibenzo[def,p]chrysene (DBC), BaP, or environmental PAH mixtures (Mix 1-3) following a 2-stage initiation/promotion skin tumor protocol. Resulting tumor incidence could be categorized by carcinogenic potency as DBC >> BaP = Mix2 = Mix3 > Mix1 = Control, based on statistical significance. Gene expression profiles measured in skin of mice collected 12 h post-initiation were compared with tumor outcome for identification of short-term bioactivity profiles. A Bayesian integration model was utilized to identify biological pathways predictive of PAH carcinogenic potential during initiation. Integration of probability matrices from four enriched pathways (P < .05) for DNA damage, apoptosis, response to chemical stimulus, and interferon gamma signaling resulted in the highest classification accuracy with leave-one-out cross validation. This pathway-driven approach was successfully utilized to distinguish early regulatory events during initiation prognostic for tumor outcome and provides proof-of-concept for using short-term initiation studies to classify carcinogenic potential of environmental PAH mixtures. These data further provide a 'source-to-outcome' model that could be used to predict PAH interactions during tumorigenesis and provide an example of how mode-of-action-based risk assessment could be employed for environmental PAH mixtures.
Subject(s)
Carcinogens/classification , Carcinogens/toxicity , Polycyclic Aromatic Hydrocarbons/classification , Polycyclic Aromatic Hydrocarbons/toxicity , Animals , MiceABSTRACT
Mammalian flavin-containing monooxygenase (FMO) is active towards many drugs with a heteroatom having the properties of a soft nucleophile. Thiocarbamides and thiones are S-oxygenated to the sulfenic acid which can either react with glutathione and initiate a redox-cycle or be oxygenated a second time to the unstable sulfinic acid. In this study, we utilized LC-MS/MS to demonstrate that the oxygenation by hFMO of the thioureas under test terminated at the sulfenic acid. With thiones, hFMO catalyzed the second reaction and the sulfinic acid rapidly lost sulfite to form the corresponding imidazole. Thioureas are often pulmonary toxicants in mammals and, as previously reported by our laboratory, are excellent substrates for hFMO2. This isoform is expressed at high levels in the lung of most mammals, including non-human primates. Genotyping to date indicates that individuals of African (up to 49%) or Hispanic (2-7%) ancestry have at least one allele for functional hFMO2 in lung, but not Caucasians nor Asians. In this study the major metabolite formed by hFMO2 with thioureas from Allergan, Inc. was the sulfenic acid that reacted with glutathione. The majority of thiones were poor substrates for hFMO3, the major form in adult human liver. However, hFMO1, the major isoform expressed in infant and neonatal liver and adult kidney and intestine, readily S-oxygenated thiones under test, with Kms ranging from 7 to 160 µM and turnover numbers of 30-40 min(-1). The product formed was identified by LC-MS/MS as the imidazole. The activities of the mouse and human FMO1 and FMO3 orthologs were in good agreement with the exception of some thiones for which activity was much greater with hFMO1 than mFMO1.
Subject(s)
Oxygenases/metabolism , Thiones/metabolism , Thiourea/metabolism , Animals , Cell Line , Humans , Insecta , Mice , Oxidation-Reduction , Oxygenases/chemistry , Oxygenases/genetics , Thiones/chemistry , Thiourea/chemistryABSTRACT
Flavin-containing monooxygenase (FMO) oxygenates drugs/xenobiotics containing a soft nucleophile through a C4a hydroperoxy-FAD intermediate. Human FMOs 1, 2 and 3, expressed in Sf9 insect microsomes, released 30-50% of O2 consumed as H2O2 upon addition of NADPH. Addition of substrate had little effect on H2O2 production. Two common FMO2 (the major isoform in the lung) genetic polymorphisms, S195L and N413K, were examined for generation of H2O2. FMO2 S195L exhibited higher "leakage", producing much greater amounts of H2O2, than ancestral FMO2 (FMO2.1) or the N413K variant. S195L was distinct in that H2O2 generation was much higher in the absence of substrate. Addition of superoxide dismutase did not impact H2O2 release. Catalase did not reduce levels of H2O2 with either FMO2.1 or FMO3 but inhibited H2O2 generated by FMO2 allelic variants N413K and S195L. These data are consistent with FMO molecular models. S195L resides in the GxGxSG/A NADP(+) binding motif, in which serine is highly conserved (76/89 known FMOs). We hypothesize that FMO, especially allelic variants such as FMO2 S195L, may enhance the toxicity of xenobiotics such as thioureas/thiocarbamides both by generation of sulfenic and sulfinic acid metabolites and enhanced release of reactive oxygen species (ROS) in the form of H2O2.
Subject(s)
Hydrogen Peroxide/metabolism , Oxygenases/metabolism , Animals , Humans , Sf9 CellsABSTRACT
Polycyclic aromatic hydrocarbons (PAHs) are present in the environment as complex mixtures with components that have diverse carcinogenic potencies and mostly unknown interactive effects. Non-additive PAH interactions have been observed in regulation of cytochrome P450 (CYP) gene expression in the CYP1 family. To better understand and predict biological effects of complex mixtures, such as environmental PAHs, an 11 gene input-1 gene output fuzzy neural network (FNN) was developed for predicting PAH-mediated perturbations of dermal Cyp1b1 transcription in mice. Input values were generalized using fuzzy logic into low, medium, and high fuzzy subsets, and sorted using k-means clustering to create Mamdani logic functions for predicting Cyp1b1 mRNA expression. Model testing was performed with data from microarray analysis of skin samples from FVB/N mice treated with toluene (vehicle control), dibenzo[def,p]chrysene (DBC), benzo[a]pyrene (BaP), or 1 of 3 combinations of diesel particulate extract (DPE), coal tar extract (CTE) and cigarette smoke condensate (CSC) using leave-one-out cross-validation. Predictions were within 1 log(2) fold change unit of microarray data, with the exception of the DBC treatment group, where the unexpected down-regulation of Cyp1b1 expression was predicted but did not reach statistical significance on the microarrays. Adding CTE to DPE was predicted to increase Cyp1b1 expression, whereas adding CSC to CTE and DPE was predicted to have no effect, in agreement with microarray results. The aryl hydrocarbon receptor repressor (Ahrr) was determined to be the most significant input variable for model predictions using back-propagation and normalization of FNN weights.
Subject(s)
Aryl Hydrocarbon Hydroxylases/genetics , Fuzzy Logic , Gene Regulatory Networks/drug effects , Neural Networks, Computer , Polycyclic Aromatic Hydrocarbons/toxicity , Skin/drug effects , Animals , Cytochrome P-450 CYP1B1 , Female , Mice , Risk AssessmentABSTRACT
The polycyclic aromatic hydrocarbon (PAH), benzo[a]pyrene (BaP), was compared to dibenzo[def,p]chrysene (DBC) and combinations of three environmental PAH mixtures (coal tar, diesel particulate and cigarette smoke condensate) using a two stage, FVB/N mouse skin tumor model. DBC (4nmol) was most potent, reaching 100% tumor incidence with a shorter latency to tumor formation, less than 20 weeks of 12-O-tetradecanoylphorbol-13-acetate (TPA) promotion compared to all other treatments. Multiplicity was 4 times greater than BaP (400 nmol). Both PAHs produced primarily papillomas followed by squamous cell carcinoma and carcinoma in situ. Diesel particulate extract (1 mg SRM 1650b; mix 1) did not differ from toluene controls and failed to elicit a carcinogenic response. Addition of coal tar extract (1 mg SRM 1597a; mix 2) produced a response similar to BaP. Further addition of 2 mg of cigarette smoke condensate (mix 3) did not alter the response with mix 2. PAH-DNA adducts measured in epidermis 12 h post initiation and analyzed by ³²P post-labeling, did not correlate with tumor incidence. PAH-dependent alteration in transcriptome of skin 12 h post initiation was assessed by microarray. Principal component analysis (sum of all treatments) of the 922 significantly altered genes (p<0.05), showed DBC and BaP to cluster distinct from PAH mixtures and each other. BaP and mixtures up-regulated phase 1 and phase 2 metabolizing enzymes while DBC did not. The carcinogenicity with DBC and two of the mixtures was much greater than would be predicted based on published Relative Potency Factors (RPFs).
Subject(s)
Benzo(a)pyrene/toxicity , Benzopyrenes/toxicity , Carcinogens, Environmental/toxicity , Skin Neoplasms/chemically induced , Animals , Benzo(a)pyrene/metabolism , Benzopyrenes/metabolism , Carcinogens, Environmental/metabolism , Gene Expression Regulation, Neoplastic/drug effects , Mice , Mice, Inbred Strains , Molecular Structure , Principal Component Analysis , Protein Array Analysis , Skin Neoplasms/metabolism , TranscriptomeABSTRACT
Dibenzo[def,p]chrysene (DBC) is a transplacental carcinogen in mice (15mg/kg; gestation day (GD) 17). To mimic residual exposure throughout pregnancy, dams received four smaller doses of DBC (3.75mg/kg) on GD 5, 9, 13 and 17. This regimen alleviated the previously established carcinogenic responses in the thymus, lung, and liver. However, there was a marked increase in ovarian tumors (females) and hyperplastic testes (males). [(14)C]-DBC (GD 17) dosing revealed transplacental distribution to fetal tissues at 10-fold lower concentrations than in paired maternal tissue and residual [(14)C] 3weeks post-dose. This study highlights the importance of developmental stage in susceptibility to environmental carcinogens.
Subject(s)
Benzopyrenes/toxicity , Carcinogens/toxicity , Maternal Exposure , Maternal-Fetal Exchange , Neoplasms, Experimental/chemically induced , Placental Circulation , Prenatal Exposure Delayed Effects , Animals , Aryl Hydrocarbon Hydroxylases/genetics , Aryl Hydrocarbon Hydroxylases/metabolism , Benzopyrenes/administration & dosage , Benzopyrenes/pharmacokinetics , Carcinogens/administration & dosage , Carcinogens/pharmacokinetics , Cytochrome P-450 CYP1B1 , Female , Fetus/drug effects , Fetus/metabolism , Gene Expression Regulation, Developmental , Gene Expression Regulation, Enzymologic , Gestational Age , Male , Mice , Mice, 129 Strain , Neoplasms, Experimental/pathology , Pregnancy , Time Factors , Tissue DistributionABSTRACT
Catalytically active human flavin-containing monooxygenase isoform 2 (FMO2.1) is encoded by an allele detected only in individuals of African or Hispanic origin. Genotyping and haplotyping studies indicate that S195L and N413K occasionally occur secondary to the functional FMO2*1 allele encoding reference protein Gln472. Sulfoxygenation under a range of conditions reveals the role these alterations may play in individuals expressing active FMO2 and provides insight into FMO structure. Expressed S195L lost rather than gained activity as pH was increased or when cholate was present. The activity of S195L was mostly eliminated after heating at 45 degrees C for 5 min in the absence of NADPH, but activity was preserved if NADPH was present. By contrast, Gln472 was less sensitive to heat, a response not affected by NADPH. A major consequence of the S195L mutation was a mean 12-fold increase in K(m) for NADPH compared with Gln472. Modeling an S213L substitution, the equivalent site, in the structural model of FMO from the Methylophaga bacterium leads to disruption of interactions with NADP(+). N413K had the same pattern of activity as Gln472 in response to pH, cholate, and magnesium, but product formation was always elevated by comparison. N413K also lost more activity when heated than Gln472; however, NADPH attenuated this loss. The major effects of N413K were increases in velocity and k(cat) compared with Gln472. Although these allelic variants are expected to occur infrequently as mutations to the FMO2*1 allele, they contribute to our overall understanding of mammalian FMO structure and function.
Subject(s)
Oxygenases/metabolism , Polymorphism, Single Nucleotide , Sulfoxides/metabolism , Cholates/metabolism , Databases, Protein , Enzyme Stability , Genotype , Hot Temperature , Humans , Hydrogen-Ion Concentration , Isoenzymes , Kinetics , Magnesium/metabolism , Models, Molecular , NADP/metabolism , Oxidation-Reduction , Oxygenases/chemistry , Oxygenases/genetics , Phenotype , Protein Conformation , Protein Denaturation , Recombinant Proteins/metabolism , Structure-Activity RelationshipABSTRACT
Tuberculosis (TB) results from infection with Mycobacterium tuberculosis and remains endemic throughout the world with one-third of the world's population infected. The prevalence of multi-drug resistant strains necessitates the use of more toxic second-line drugs such as ethionamide (ETA), a pro-drug requiring bioactivation to exert toxicity. M. tuberculosis possesses a flavin monooxygenase (EtaA) that oxygenates ETA first to the sulfoxide and then to 2-ethyl-4-amidopyridine, presumably through a second oxygenation involving sulfinic acid. ETA is also a substrate for mammalian flavin-containing monooxygenases (FMOs). We examined activity of expressed human and mouse FMOs toward ETA, as well as liver and lung microsomes. All FMOs converted ETA to the S-oxide (ETASO), the first step in bioactivation. Compared to M. tuberculosis, the second S-oxygenation to the sulfinic acid is slow. Mouse liver and lung microsomes, as well as human lung microsomes from an individual expressing active FMO, oxygenated ETA in the same manner as expressed FMOs, confirming this reaction functions in the major target organs for therapeutics (lung) and toxicity (liver). Inhibition by thiourea, and lack of inhibition by SKF-525A, confirm ETASO formation is primarily via FMO, particularly in lung. ETASO production was attenuated in a concentration-dependent manner by glutathione. FMO3 in human liver may contribute to the toxicity and/or affect efficacy of ETA administration. Additionally, there may be therapeutic implications of efficacy and toxicity in human lung based on the FMO2 genetic polymorphism, though further studies are needed to confirm that suggestion.
Subject(s)
Antitubercular Agents/metabolism , Ethionamide/metabolism , Lung/metabolism , Microsomes, Liver/metabolism , Oxygenases/metabolism , Animals , Antitubercular Agents/pharmacokinetics , Chromatography, High Pressure Liquid , Ethionamide/pharmacokinetics , Glutathione/metabolism , Humans , In Vitro Techniques , Lung/cytology , Lung/drug effects , Lung/enzymology , Mass Spectrometry , Mice , Microsomes/drug effects , Microsomes/enzymology , Microsomes/metabolism , Microsomes, Liver/drug effects , Microsomes, Liver/enzymologyABSTRACT
The significance of active versus inactive flavin-containing monooxygenase 2 (FMO2) for human drug and xenobiotic metabolism and sensitivity is unknown, but the underlying ethnic polymorphism is well documented. We used quantitative real-time PCR to measure message levels of Fmo1, Fmo2, Fmo3 and Fmo5 in lung and liver from eight strains of 8 week old female mice to determine if a strain could be identified that predominately expressed Fmo2 in lung, recapitulating the human FMO expression profile and being the ideal strain for Fmo2 knockout studies. We also characterized enzyme activity of baculovirus expressed mouse Fmo1, Fmo2 and Fmo3 to identify a substrate or incubation conditions capable of discriminating Fmo2 from Fmo mixtures. Fmo transcript expression patterns were similar for all strains. In lung, 59% of total FMO message was Fmo2, but Fmo1 levels were also high, averaging 34%, whereas Fmo3 and Fmo5 levels were 2 and 5%, respectively. In liver, Fmo1, Fmo2, Fmo3 and Fmo5 contributed 16, 1, 7 and 76% respectively, of detected message. Peak activity varied by isoform and was pH- and substrate-dependent. Fmo3 oxidation of methyl p-tolyl sulfide was negligible at pH 9.5, but Fmo3 oxidation of methimazole was comparable to Fmo1 and Fmo2. Heating microsomes at 50 degrees C for 10min eliminated most Fmo1 and Fmo3 activity, while 94% of Fmo2 activity remained. Measurement of activity in heated and unheated lung and liver microsomes verified relative transcript abundance. Our results show that dual Fmo1/2 knockouts will be required to model the human lung FMO profile.
Subject(s)
Isoenzymes/genetics , Liver/enzymology , Lung/enzymology , Oxygenases/genetics , RNA, Messenger/analysis , Animals , Female , Kinetics , Mice , Mice, Inbred Strains , Oxygenases/metabolismABSTRACT
Flavin-containing monooxygenases (FMOs) are important for the disposition of many therapeutics, environmental toxicants, and nutrients. FMO3, the major adult hepatic FMO enzyme, exhibits significant interindividual variation. Eighteen FMO3 single-nucleotide polymorphism (SNP) frequencies were determined in 202 Hispanics (Mexican descent), 201 African Americans, and 200 non-Latino whites. Using expressed recombinant enzyme with methimazole, trimethylamine, sulindac, and ethylenethiourea, the novel structural variants FMO3 E24D and K416N were shown to cause modest changes in catalytic efficiency, whereas a third novel variant, FMO3 N61K, was essentially devoid of activity. The latter variant was present at an allelic frequency of 5.2% in non-Latino whites and 3.5% in African Americans, but it was absent in Hispanics. Inferring haplotypes using PHASE, version 2.1, the greatest haplotype diversity was observed in African Americans followed by non-Latino whites and Hispanics. Haplotype 2A and 2B, consisting of a hypermorphic promoter SNP cluster (-2650C>G, -2543T>A, and -2177G>C) in linkage with synonymous structural variants was inferred at a frequency of 27% in the Hispanic population, but only 5% in non-Latino whites and African Americans. This same promoter SNP cluster in linkage with one or more hypomorphic structural variant also was inferred in multiple haplotypes at a total frequency of 5.6% in the African-American study group but less than 1% in the other two groups. The sum frequencies of the hypomorphic haplotypes H3 [15,167G>A (E158K)], H5B [-2650C>G, 15,167G>A (E158K), 21,375C>T (N285N), 21,443A>G (E308G)], and H6 [15,167G>A (E158K), 21,375C>T (N285N)] was 28% in Hispanics, 23% in non-Latino whites, and 24% in African Americans.