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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, BaP = 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 46 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 46 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 460 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.

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.

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, BaP=1) are determined from high dose animal data. We employed accelerator mass spectrometry (AMS) to determine pharmacokinetics of [(14)C]-BaP in humans following dosing with 46 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 46 ng of [(14)C]-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 [(14)C]-BaP. Co-administration of smoked salmon, containing a complex mixture of PAHs with an RPF of 460 ng BaP(eq), 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.

Polycyclic aromatic hydrocarbons (PAHs) are a class of over 1500 chemicals formed as incomplete combustion products and released into the environment from both natural (e.g. forest fires) or anthropogenic (e.g. burning of fossil fuels, tobacco, charbroiled meats) sources. Several PAHs, particularly those with more than 4 rings such as benzo[a]pyrene (BaP), dibenzo[def,p]chrysene (DBC), have been designated as Class 1 known or Class 2A probable human carcinogens by the International Agency for Research on Cancer (IARC, 2010). While much of the research on PAH carcinogenicity focuses on individual PAHs and BaP, in particular, most human exposures to PAHs result from chemical mixtures through dietary, inhalation, or dermal routes of exposure. Primary sources of environmental exposure to these PAHs include wood smoke, creosote, and burning of fossil fuels and tobacco (IARC, 2010). Recently, diesel engine exhaust was added to the list of Class 1 known human carcinogens and certain other PAH-containing mixtures, including air pollution, have been designated as probable or possible Class2A/B carcinogens in humans (IARC, 2010, 2014). One of the most difficult challenges for risk assessment is the evaluation of health hazards from exposure to environmental chemical mixtures. Currently, significant data gaps exist for understanding carcinogenicity of PAH mixtures and complex environmental mixtures containing PAHs. Further, little is known about the mechanisms of tumorigenesis for PAH mixtures. Current assessment of cancer risk for PAHs involves testing compounds in the 2-year rodent bioassay, which is not practical for screening large numbers of compounds or mixtures due to expense and time. Therefore, alternative approaches are typically utilized for evaluating the carcinogenic potential of PAHs and PAH-containing mixtures. Currently, the primary method for assessing cancer risk of complex mixtures is the relative potency factor (RPF) approach in which complex mixtures are evaluated based on a subset of individual component PAHs compared with BaP as a surrogate or reference (US EPA, 2010). However, we and others have found this approach inadequate for predicting carcinogenicity of mixtures and certain individual PAHs, particularly those that function through alternate pathways or exhibit greater promotional capacity compared to BaP (Courter et al., 2008; Siddens et al., 2012). Significant challenges have also been identified in utilizing such reference-based approaches for estimating risk from exposure to PAHs in air pollution or waste sites. Complex environmental mixtures subjected to weathering and aging processes can contain many different PAHs, including alkyl-, N-, S-, and O-substituted forms, along with other unknown chemicals; however, only a limited number of unsubstituted PAHs have been characterized for use in RPF calculations. Mixture toxicity for risk assessment is calculated based on select individual components and assumes additivity through a common mechanism of action for PAHs compared to BaP as a standard. Therefore, the RPF approach does not take into consideration mechanistic information about the different pathways, cells, and tissues affected by PAHs during initiation and promotion. This approach is also insufficient for predicting carcinogenicity of complex real-world environmental mixtures of unknown composition. In this study, we propose an innovative model for determining carcinogenic risk of PAH mixtures using mechanistic approaches. We hypothesize that a chemical bioactivity profile measured after short-term exposure to individual and mixture PAHs from global transcriptional profiling can be used to discriminate future carcinogenic potential based on important mechanistic differences among exposures. The bioactivity profile acts as a unique fingerprint for genes and pathways activated by chemicals and mixtures postexposure and can be used for predicting long-term consequences such as cancer outcome. An important aspect of the bioactivity profile is that the gene signatures are linked to chemical mechanism of action and can also provide insight into alternate mechanisms of PAH carcinogenesis and related mechanisms for complex mixtures. Based on preliminary data, we demonstrate that long-term cancer outcome for PAHs and mixtures can be predicted from high-content genomic evaluation of bioactivity after short-term exposure.

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.

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.

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.

Multiple drug resistance (MDR) in Mycobacterium tuberculosis (mTB), the causative agent for tuberculosis (TB), has led to increased use of second-line drugs, including ethionamide (ETA). ETA is a prodrug bioactivated by mycobacterial and mammalian flavin-containing monooxygenases (FMOs). FMO2 is the major isoform in the lungs of most mammals, including primates. In humans a polymorphism exists in the expression of FMO2. FMO2.2 (truncated, inactive) protein is produced by the common allele, while the ancestral allele, encoding active FMO2.1, has been documented only in individuals of African and Hispanic origin, at an incidence of up to 50% and 7%, respectively. We hypothesized that FMO2 variability in TB-infected individuals would yield differences in concentrations and ratios of ETA prodrug and metabolites. In this study we assessed the impact of the FMO2 genetic polymorphism on the pharmacokinetics of ETA after administration of a single oral dose of ETA (125 mg/kg) to wild type and triple Fmo1/2/4-null mice, measuring levels of prodrug vs. metabolites in plasma collected from 0 to 3.5 h post-gavage. All mice metabolized ETA to ETA S-oxide (ETASO) and 2-ethyl-4-amidopyridine (ETAA). Wild type mice had higher plasma concentrations of metabolites than of parent compound (p = 0.001). In contrast, Fmo1/2/4-null mice had higher plasma concentrations of parent compound than of metabolites (p = 0.0001). Thus, the human FMO2 genotype could impact the therapeutic efficacy and/or toxicity of ETA.

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

Flavin-containing monooxygenases often are thought not to be inducible but we recently demonstrated aryl hydrocarbon receptor (AHR)-dependent induction of FMO mRNAs in mouse liver by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (Celius et al., Drug Metab Dispos 36:2499, 2008). We now evaluated FMO induction by other AHR ligands and xenobiotic chemicals in vivo and in mouse Hepa1c1c7 hepatoma cells (Hepa-1). In mouse liver, 3-methylcholanthrene (3MC) induced FMO3 mRNA 8-fold. In Hepa-1 cells, 3MC and benzo[a]pyrene (BaP) induced FMO3 mRNA >30-fold. Induction by 3MC and BaP was AHR dependent but, surprisingly, the potent AHR agonist, TCDD, did not induce FMO3 mRNA in Hepa-1 cells nor did chromatin immunoprecipitation assays detect recruitment of AHR or ARNT to Fmo3 regulatory elements after exposure to 3MC in liver or in Hepa-1 cells. However, in Hepa-1, 3MC and BaP (but not TCDD) caused recruitment of p53 protein to a p53 response element in the 5'-flanking region of the Fmo3 gene. We tested the possibility that FMO3 induction in Hepa-1 cells might be mediated by Nrf2/anti-oxidant response pathways, but agents known to activate Nrf2 or to induce oxidative stress did not affect FMO3 mRNA levels. The protein synthesis inhibitor, cycloheximide (which causes "superinduction" of CYP1A1 mRNA in TCDD-treated cells), by itself caused dramatic upregulation (>300-fold) of FMO3 mRNA in Hepa-1 suggesting that cycloheximide prevents synthesis of a labile protein that suppresses FMO3 expression. Although FMO3 mRNA is highly induced by 3MC or TCDD in mouse liver and in Hepa-1 cells, FMO protein levels and FMO catalytic function showed only modest elevation.

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°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 Km 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 kcat 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.

Dibenzo(a,l)pyrene (DBP) is among the most potent carcinogenic polycyclic aromatic hydrocarbons. Previously, we showed that DBP administration to pregnant mice resulted in high mortality of offspring from an aggressive T-cell lymphoma. All mice that survive to 10 months of age exhibit lung tumors with high multiplicity. Recombinant cytochrome P450 (cyp) 1b1 from mice and the homologue 1B1 in humans exhibit high activity toward the metabolic activation of DBP. Targeted disruption of the cyp1b1 gene protects against most DBP-dependent cancers. Mice heterozygous for the disrupted cyp1b1 allele were used to examine the effect of cyp1b1 gene dosage on DBP transplacental carcinogenesis. Dams were treated with 1 or 15 mg/kg of DBP or 50 mg/kg of benzo(a)pyrene. Cyp1b1-null offspring did not develop lymphoma, whereas wild-type and heterozygous siblings, born to dams given the high dose of DBP, exhibited significant mortalities between 10 and 30 weeks of age. At 10 months, all groups had lung adenomas or carcinomas [9.5%, 40.3%, 25.6%, and 100% incidences for controls, benzo(a)pyrene, 1 and 15 mg/kg DBP, respectively]. Cyp1b1 status did not alter benzo(a)pyrene-dependent carcinogenesis. At 1 mg/kg DBP, cyp1b1 status altered the incidence of lung tumors (19.0, 27.8, and 28.6% for nulls, heterozygous, and wild-type, respectively). At 15 mg/kg, tumor multiplicities in cyp1b1 wild-type (9.3) and heterozygous (9.5) offspring were nearly twice that of cyp1b1-null siblings (5.0). These data confirm that cyp1b1 bioactivation of DBP occurs in fetal target tissues, following transplacental exposure, with the thymus and lung as primary and secondary targets, respectively.

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 °C for 10 min 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.

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), but in agreement with a more recent study (Shorey et al., Toxicol. Appl. Pharmacol., 2013), 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.

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 8 h post-initiation. A significant reduction in levels of Cyp 1a1, Nqo1 at 8 h 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 4 h 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 8 h 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.

Arylamine compounds, such as sulfamethoxazole (SMX) and dapsone (DDS), are metabolized in epidermal keratinocytes to arylhydroxylamine metabolites that auto-oxidize to arylnitroso derivatives, which in turn bind to cellular proteins and can act as antigens/immunogens. Previous studies have demonstrated that neither cytochromes P450 nor cyclooxygenases mediate this bioactivation in normal human epidermal keratinocytes (NHEKs). In this investigation, we demonstrated that methimazole (MMZ), a prototypical substrate of the flavin-containing monooxygenases (FMOs), attenuated the protein haptenation observed in NHEKs exposed to SMX or DDS. In addition, recombinant FMO1 and FMO3 were able to bioactivate both SMX and DDS, resulting in covalent adduct formation. Western blot analysis confirmed the presence of FMO3 in NHEKs, whereas FMO1 was not detectable. In addition to MMZ, 4-aminobenzoic acid hydrazide (ABH) also attenuated SMX- and DDS-dependent protein haptenation in NHEKs. ABH did not alter the bioactivation of these drugs by recombinant FMO3, suggesting its inhibitory effect in NHEKs was due to its known ability to inhibit peroxidases. Studies confirmed the presence of peroxidase activity in NHEKs; however, immunoblot analysis and reverse transcription-polymerase chain reaction indicated that myeloperoxidase, lactoperoxidase, and thyroid peroxidase were absent. Thus, our results suggest an important role for FMO3 and yet-to-be identified peroxidases in the bioactivation of sulfonamides in NHEKs.

Flavin-containing monooxygenases (FMO) are membrane-associated enzymes contributing to oxidative metabolism of drugs and other chemicals. There are no known structures similar enough to FMO to provide accurate insights into the structural basis for differences in metabolism observed among FMOs. To develop an FMO amenable to crystallization, we introduced mutations into rabbit FMO2 (rF2) to increase solubility, decrease aggregation, and simplify isolation. Alterations included removal of 26 AA (Delta26) from the carboxyl-terminus, His(6)-fusion to the amino-terminus and a double Ser substitution designed to reduce local hydrophobicity. Only Delta26 FMO variants retained normal activity, increased the yield of cytosolic rF2 and decreased protein aggregation. Delta26 constructs increased rF2 in cytosol in low (from 2 to 13%), and high salt (from 24 to 62%) conditions. His-fusion proteins, while active and useful for purification, did not affect solubility. Delta26 variants should prove useful for identifying conditions suitable for production of an FMO crystal.

Flavin-containing monooxygenase (FMO) oxygenates drugs and xenobiotics containing a “soft-nucleophile”, usually nitrogen or sulfur. FMO, like cytochrome P450 (CYP), is a monooxygenase, utilizing the reducing equivalents of NADPH to reduce 1 atom of molecular oxygen to water, while the other atom is used to oxidize the substrate. FMO and CYP also exhibit similar tissue and cellular location, molecular weight, substrate specificity, and exist as multiple enzymes under developmental control. The human FMO functional gene family is much smaller (5 families each with a single member) than CYP. FMO does not require a reductase to transfer electrons from NADPH and the catalytic cycle of the 2 monooxygenases is strikingly different. Another distinction is the lack of induction of FMOs by xenobiotics. In general, CYP is the major contributor to oxidative xenobiotic metabolism. However, FMO activity may be of significance in a number of cases and should not be overlooked. FMO and CYP have overlapping substrate specificities, but often yield distinct metabolites with potentially significant toxicological/pharmacological consequences. The physiological function(s) of FMO are poorly understood. Three of the 5 expressed human FMO genes, FMO1, FMO2 and FMO3, exhibit genetic polymorphisms. The most studied of these is FMO3 (adult human liver) in which mutant alleles contribute to the disease known as trimethylaminuria. The consequences of these FMO genetic polymorphisms in drug metabolism and human health are areas of research requiring further exploration.

Objectives Previous work defined two flavin-containing monooxygenase 2 (FMO2) alleles. The major allele, FMO2*2 (g.23,238C>T), encodes truncated inactive protein (p.X472) whereas the minor allele, FMO2*1, present in African- and Hispanic-American populations, encodes active protein (p.Q472). Recently, four common (27 to 51% incidence) FMO2 single nucleotide polymorphisms (SNPs) were detected in African-Americans (N=50); they encode the following protein variants: p.71Ddup, p.V113fs, p.S195L and p.N413 K. Our objectives were to: (1) determine the incidence of these SNPs in 29 Hispanic individuals previously genotyped as g.23,238C (p.Q472) and 124 previously genotyped as homozygous g.23,238 T (p.X472); (2) determine FMO2 haplotypes in this population; and (3) assess the functional impact of SNPs in expressed proteins. Methods SNPs were detected via allele-specific oligonucleotide amplification coupled with real-time or electrophoretic product detection, or single strand conformation polymorphism. Results The g.7,700_7,702dupGAC SNP (p.71Ddup) was absent. The remaining SNPs were present but, except for g.13,732C>T (p.S195L), were less common in the current Hispanic study population versus the previously described African-Americans. Only expressed p.N413 K was as active as p.Q472, as determined by methimazole- and ethylenethiourea-dependent oxidation. Haplotype determination demonstrated that the g.10,951delG (p.V113fs), g.13,732C>T (p.S195L) and g.22,060T>G (p.N413 K) variants segregated with g.23,238C>T (p.X472). Conclusions SNPs would not alter FMO2 activity in individuals possessing at least one FMO2*1 allele. It is likely that these SNPs will segregate similarly in African-American populations. Therefore, estimates that 26% of African-Americans and 2–7% of Hispanic-Americans have at least one FMO2*1 allele should closely reflect the percentages producing active FMO2 protein.

Thioureas are oxygenated by flavin-containing monooxygenases (FMOs), forming reactive sulfenic and/or sulfinic acids. Sulfenic acids can reversibly react with GSH and drive oxidative stress through a redox cycle. For this reason, thiourea S-oxygenation is an example of FMO-dependent bioactivation of a xenobiotic. Functional FMO2 is expressed in the lung of 26% of individuals of African descent and 5% of Hispanics but not in Caucasians or Asians. We have previously demonstrated that human FMO2.1 protein expressed in Sf9 microsomes has high activity toward a series of thioureas that are known or suspected lung toxicants including thiourea, 1-phenylthiourea, and ethylenethiourea. We now show by HPLC and LC-MS that 1-phenylthiourea and alpha-naphthylthiourea are converted to their sulfenic acids. GSH in the incubations at concentrations of 0.5-1.0 mM completely eliminated the sulfenic acid with resultant production of GSSG. These results indicate that individuals with the FMO21 allele may be at enhanced risk of pulmonary damage upon exposure to thioureas.

Flavin-containing monooxygenases (FMOs) comprise a multi-gene family and catalyze the oxygenation of soft nucleophilic sulfur, nitrogen, phosphorus, and selenium in xenobiotics. Previous studies have demonstrated that FMO is regulated developmentally and by the administration of certain steroid hormones. This study examined the expression of FMO form 1 in the livers and kidneys of fetal and neonatal rabbits, from day 25 of gestation through 3 weeks of age, by assaying FMO1 mRNA and protein levels, as well as catalytic activity. FMO1 mRNA and protein expression and FMO catalytic activity were present in fetal livers at the earliest time point measured (day 25 of gestation), although at levels approximately 10% of that found in adult livers. Hepatic FMO1 mRNA levels increased during and after gestation; levels were not significantly different from those measured in adult male livers. FMO1 protein content and activity rose rapidly after birth to reach 70–80% of adult levels by 3 weeks of age. The expression of FMO1 in fetal and neonatal kidneys was markedly lower than in liver. FMO1 mRNA levels never averaged more than 3.4% of adult male liver levels, but did not differ from adult kidney levels at any of the points measured. Protein levels and enzyme activity rose significantly after birth to approximately 30% of the level in adult kidneys by 3 weeks of age. The early developmental appearance of FMO1 suggests a possible role in the metabolism of xenobiotics through transplacental or lactational exposures.

We describe the isolation and characterization of cDNAs for FMO2 from the laboratory rat. In contrast to FMO2 in other animals, each of which contain 535 amino acid residues, analysis of the sequence of the cDNAs and of a section of the corresponding gene revealed that the ORF of the laboratory rat FMO2 encodes a polypeptide of only 432 residues. This truncated protein is due to the presence of a double deletion corresponding to 1263 and 1264 nucleotides of the orthologous FMO2 cDNAs. This double deletion provokes a frame-shift, with the appearance of a premature stop codon in position 1297-1299. By Northern blotting, the probe for FMO2 hybridized a 2.5-kb transcript in lung and kidney samples only. Heterologous expression of the cDNA revealed that the truncated protein was catalytically inactive. By Western blotting, FMO2 was faintly detected at approximately 50 kDa in laboratory rat lung.

Full-length human (hFMO2.1) and monkey (mFMO2) flavin-containing monooxygenase proteins, which share 97% sequence identity, were produced by baculovirus-mediated expression in insect cells and assayed for S-oxygenation under conditions known to affect FMO activity. Both enzymes demonstrated maximal activity at pH 9.5; but hFMO2.1 retained significantly more activity than mFMO2 did at pH 9.0 and higher. hFMO2.1 also retained significantly more activity than mFMO2 did in the presence of magnesium and all detergents tested. Although hFMO2.1 had more residual activity after heating at 45 degrees C than mFMO2, under some conditions, both had less than 10% of control activity, whereas expressed rabbit FMO2 retained over 50% activity. Screening for NADPH-oxygenation by hFMO2.1, indicated that substituted thioureas with a small cross-sectional area (2.4-4.3 A) are good substrates, whereas 1,3-diphenylthiourea (11.2 A) was not oxygenated. We confirmed the presence of hFMO2.1 in lung tissue from a heterozygous individual (hFMO2*1/hFMO2*2A) by Western analysis and confirmed activity by S-oxygenation. These microsomes also demonstrated a heat-associated loss of activity similar to expressed hFMO2.1. The heat sensitivity of hFMO2.1 may partially explain why activity in post mortem human lung samples has previously been unreported. Individuals that have the FMO2*1 allele-encoding full-length hFMO2.1 may exhibit altered drug metabolism in the lung.

Mammalian flavin-containing monooxygenase functions in the oxygenation of numerous xenobiotics containing a soft nucleophile, usually a nitrogen or sulfur. A total of five distinct flavin monooxygenase (FMO) isoforms are expressed in mammals. Individual isoforms are expressed in a sex-, age-, and tissue-specific fashion. In this study, we document the early developmental appearance of the major isoform in rabbit lung, FMO2. FMO2 catalytic activity as well as protein and mRNA are not only present in fetal and neonatal lung but, in some instances, approach levels found in the adult. The expression pattern of FMO2 is similar to that of the two major constitutive cytochromes P450 found in rabbit lung, 2B4 and 4B1. The early developmental appearance of these monooxygenases indicate an important role in the protection of the fetus and neonate against toxic insult from foreign chemicals.

Pulmonary microsomes from Rhesus macaque express a flavin-containing monooxygenase (FMO) resembling the FM02 ortholog from rabbit with respect to immunochemical cross-reactivity and expression in lung, but not liver. A full-length cDNA was cloned following screening of a Rhesus macaque lung cDNA library. The nucleotide sequence contained an open reading frame encoding 535 amino acids with 85 and 84% identity to FMO2 from rabbit and guinea pig, respectively, and an identical location of the putative FAD- and NADP-binding sites. Northern blots of monkey lung mRNA revealed multiple size FMO2 transcripts. These mRNA transcripts are expressed in lung, but not in liver or kidney.

Persistent viruses occur intracellularly in brown algae, specifically the Ectocarpales, and as reported here in the genus Feldmannia. Feldmannia species are small (1 mm–several cm), filamentous forms with single-celled meiotic sporangia that normally produce haploid zoospores. In the isolate reported here, spores were not observed in the sporangia but rather numerous (∼10 6 per cell) polyhedral viruses are formed in their place. Two dsDNA genome classes of 158 and 178 kbp, with two restriction site variants of each, are described. The individual abundance of each genome in viral preparations is affected by culture temperature. A cosmid library was used to generate circular restriction enzyme ( Bam HI, Not I, and Pst I) site maps.

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.

Dietary modulation of cancer & cancer biomarkers. Dietary item or component studied: indole-3-carbinol (I3C)Outcome studied: lung tumor multiplicity. Study type: mice resulting from reciprocal crosses between B6129 SF1/J [AHR(b-1/d)] and 129Sv/ImJ [AHR(d/d)] mice. Tissue/biological material/sample size: heart, thymus, lung, spleen, liver, kidney, abnormal lymph node, testes or ovaries, colon and skinMode of exposure: dibenzo[a,l]pyrene (DBP) was administered to pregnant mice (15 mg/kg, gavage) on gestation day 17, and 2000 p.p.m. indole-3-carbinol (I3C) was fed to half of the mice from gestation day 9 until weaning. Impact on outcome (including dose-response): Offspring born to dams fed I3C exhibited markedly fewer mortalities (P < 0.0001). Maternal dietary exposure to I3C also significantly lowered lung tumor multiplicity (P = 0.035) in offspring surviving to 10 months of age. The I3C chemoprotection was independent of either maternal or fetal AHR genotype. The bioavailability of DBP to fetal target tissue was demonstrated by assessing DNA covalent adduction with a (33)P-post-labeling assay. The bioavailability of I3C was determined by dosing a subset of pregnant mice with [(14)C]-I3C. KEYWORD CLASSIFICATION: administration & dosage;analysis;Animals;antagonists & inhibitors;Anticarcinogenic Agents;Benzopyrenes;Biological Availability;chemically induced;Diet;dietary modulation of cancer & cancer biomarkers;DNA Adducts;Female;Fetal Diseases;humans;Indoles;Liver Neoplasms,Experimental;Lung;Lung Neoplasms;Lymphoma;Male;Maternal-Fetal Exchange;metabolism;Mice;mortality;NF-kappa B;nursing;pharmacokinetics;Pregnancy;prevention & control;Research;toxicity;Toxicology; The fetus and neonate are sensitive targets for chemically induced carcinogenesis. Few studies have examined the risk/benefit of chemoprotective phytochemicals, given in the maternal diet, against transplacental carcinogenesis. In this study, B6129 SF1/J (AHR(b-1/d)) and 129Sv/ImJ (AHR(d/d)) mice were cross-bred. The polycyclic aromatic hydrocarbon, dibenzo[a,l]pyrene (DBP), was administered to pregnant mice (15 mg/kg, gavage) on gestation day 17, and 2000 p.p.m. indole-3-carbinol (I3C), a chemoprotective phytochemical from cruciferous vegetables, was fed to half of the mice from gestation day 9 until weaning. Offspring born to dams fed I3C exhibited markedly fewer mortalities (P < 0.0001). Maternal dietary exposure to I3C also significantly lowered lung tumor multiplicity (P = 0.035) in offspring surviving to 10 months of age. The I3C chemoprotection was independent of either maternal or fetal AHR genotype. The bioavailability of DBP to fetal target tissue was demonstrated by assessing DNA covalent adduction with a (33)P-post-labeling assay. The bioavailability of I3C was determined by dosing a subset of pregnant mice with [(14)C]-I3C. Addition of chemoprotective agents to the maternal diet during pregnancy and nursing may be an effective new approach in reducing the incidence of cancers in children and young adults.

Tamoxifen is utilized in breast cancer therapy and in chemoprevention. Tamoxifen may enhance risk for other neoplasias, especially endometrial cancer. The risk:benefit depends on the rate of metabolic activation versus detoxication. Cytochrome P450-dependent alpha-hydroxylation, followed by sulfonation, represents a metabolic activation pathway, producing products capable of covalent DNA adduction. In contrast, tamoxifen N-oxygenation represents a detoxication pathway, with the caveat that N-oxides can be reduced back to the parent amines. The N-oxygenation pathway will be the focus for this review. Dr. David Kupfer pioneered studies on cytochrome P450 and flavin-containing monooxygenase (FMO) tamoxifen metabolism. We collaborated with Dr. Kupfer's laboratory and recently determined that the low level of tamoxifen N-oxide production in human liver microsomes may be explained by the kinetics of FMO1 versus FMO3.

Lymphoma and leukemia are the most common cancers in children and young adults; in utero carcinogen exposure may contribute to the etiology of these cancers. A polycyclic aromatic hydrocarbon (PAH), dibenzo[a,l]pyrene (DBP), was given to pregnant mice (15 mg/kg body weight, gavage) on gestation day 17. Significant mortalities in offspring, beginning at 12 weeks of age, were observed due to an aggressive T-cell lymphoblastic lymphoma. Lymphocytes invaded numerous tissues. All mice surviving 10 months, exposed in utero to DBP, exhibited lung tumors; some mice also had liver tumors. To assess the role of the aryl hydrocarbon receptor (AHR) in DBP transplacental cancer, B6129SF1/J (AHRb-1/d, responsive) mice were crossed with strain 129S1/SvIm (AHRd/d, nonresponsive) to determine the effect of maternal and fetal AHR status on carcinogenesis. Offspring born to nonresponsive mothers had greater susceptibility to lymphoma, irrespective of offspring phenotype. However, when the mother was responsive, an AHR-responsive phenotype in offspring increased mortality by 2-fold. In DBP-induced lymphomas, no evidence was found for TP53, β-catenin, or Ki-ras mutations but lung adenomas of mice surviving to 10 months of age had mutations in Ki-ras codons 12 and 13. Lung adenomas exhibited a 50% decrease and a 35-fold increase in expression of Rb and p19/ARF mRNA, respectively. This is the first demonstration that transplacental exposure to an environmental PAH can induce a highly aggressive lymphoma in mice and raises the possibility that PAH exposures to pregnant women could contribute to similar cancers in children and young adults. (Cancer Res 2006; 66(2): 755-62)

A polymorphism for the phase I drug-metabolizing enzyme, flavin-containing monooxygenase isoform 2 (FMO2), encoding either truncated inactive protein, FMO2X472 (FMO2.2A), or full-length active enzyme, FMO2Q472 (FMO2.1), is known and exhibits significant interethnic differences in allelic frequency. FMO2 is the major or sole FMO isoform expressed in the lung of most mammals, including nonhuman primates. To date, FMO2.1 has been found only in African-American and Hispanic populations, rendering individuals with this allele subject to drug metabolism that is potentially different from that of the general population. Approximately 26% of African-Americans (n = 180) possess the FMO2*1 allele. In preliminary studies, we initially estimated that 5% of Hispanics (n = 40) have the FMO2*1 allele, but access to large cohorts of individuals of defined national origin has allowed us to determine the occurrence among Mexican-American and Puerto Rican-American groups. We used allele-specific genotyping to detect FMO2*1 from 632 Hispanic individuals, including 280 individuals of Mexican origin and 327 individuals of Puerto Rican origin. Statistical analysis indicated that results from Mexican (five sample sources) and Puerto Rican (three sample sources) samples were consistent with the hypothesis of homogeneity within each group from different sources. Data were subsequently pooled across sources to test for evidence of a difference in occurrence of FMO2*1 between ethnic groups. There was strong evidence (p = 0.0066) that FMO2*1 is more common among Puerto Ricans (7%) than among individuals of Mexican descent (2%). The overall occurrence of FMO2*1 among Hispanics of all origins is estimated to be between 2 and 7%.

Although all forms of vitamin E are absorbed, the liver preferentially secretes alpha-, but not gamma-tocopherol, into plasma. Liver alpha-tocopherol secretion is under the control of the alpha-tocopherol transfer protein (TTP). Therefore, to assess gamma-tocopherol bioactivities Ttpa-/-, +/- and +/+ mice were fed for 5 weeks diets containing gamma-tocopherol 550 (gamma-T550), gamma-tocopherol 60 (gamma-T60) mg/kg that also contained trace amounts of alpha-tocopherol, a vitamin E-deficient diet, or a control diet. Plasma and tissues from mice fed gamma-T550 diets were found to contain similar gamma- and alpha-tocopherol concentrations despite the high dietary gamma-tocopherol content; nervous tissues contained almost no gamma-tocopherol. Liver vitamin E metabolites (carboxyethyl hydroxychromans, CEHCs) were also measured. In mice with widely ranging liver alpha- (from 0.7 to 16 nmol/g) and gamma-tocopherol concentrations (0 to 13 nmol/g), hepatic alpha-CEHC was undetectable, but gamma-CEHC concentrations (0.1 to 0.8 nmol/g) were correlated with both alpha- and gamma-tocopherol concentrations (P0.004). Hepatic cytochrome P450s (CYPs) involved in vitamin E metabolism, Cyp4f and Cyp3a, were also measured. There were no variations in Cyp4f protein expression as related to diet or mouse genotype. However, Cyp3a was correlated (P0.0001) with liver alpha-, but not gamma-tocopherol concentrations. These data support the hypothesis that alpha-tocopherol modulates xenobiotic metabolism by increasing Cyp3a expression, gamma-CEHC formation, and the excretion of both gamma-tocopherol and gamma-CEHC.

Phorate and disulfoton are organophosphate insecticides containing three oxidizable sulfurs, including a thioether. Previous studies have shown that only the thioether is oxygenated by flavin-containing monooxygenase (FMO) and the sole product is the sulfoxide with no oxygenation to the sulfone. The major FMO in lung of most mammals, including non-human primates, is FMO2. The FMO2*2 allele, found in all Caucasians and Asians genotyped to date, codes for a truncated, non-functional, protein (FMO2.2A). Twenty-six percent of individuals of African descent and 5% of Hispanics have the FMO2*1 allele, coding for full-length, functional protein (FMO2.1). We have here demonstrated that the thioether-containing organophosphate insecticides, phorate and disulfoton, are substrates for expressed human FMO2.1 with Km of 57 and 32 microM, respectively. LC/MS confirmed the addition of oxygen and formation of a single polar metabolite for each chemical. MS/MS analysis confirmed the metabolites to be the respective sulfoxides. Co-incubations with glutathione did not reduce yield, suggesting they are not highly electrophilic. As the sulfoxide of phorate is a markedly less effective acetylcholinesterase inhibitor than the cytochrome P450 metabolites (oxon, oxon sulfoxide or oxon sulfone), humans possessing the FMO2*1 allele may be more resistant to organophosphate-mediated toxicity when pulmonary metabolism is an important route of exposure or disposition.

Mammalian flavin-containing monooxygenase (FMO) exists as six gene families and metabolizes a plethora of drugs and xenobiotics. The major FMO in adult human liver, FMO3, is responsible for trimethylamine (TMA) N-oxygenation. A number of FMO3 mutant alleles have been described and associated with a disease termed trimethylaminuria (TMAU). The TMAU patient excretes large amounts of TMA in urine and sweat. A more recent ethnically related polymorphism in expression of the major FMO in lung, FMO2, has been described. All Caucasians and Asians genotyped to date are homozygous for a CAG --> TAG amber mutation resulting in a premature stop codon and a nonfunctional protein truncated at AA 472 (wildtype FMO2 is 535 AA). This allele has been designated hFMO2*2A. Twenty-six percent of individuals of African descent and 5% of Hispanics genotyped to date carry at least one allele coding for full-length FMO2 (hFMO2*1 allele). Preliminary evidence indicates that FMO2.1 is very active toward the S-oxygenation of low MW thioureas, including the lung toxicant ethylene thiourea. Polymorphic expression of functional FMO2 in the individuals of African and Hispanic descent may markedly influence drug metabolism and/or xenobiotic toxicity in the lung.

The brown filamentous algaFeldmanniasp. contains a large icosahedral dsDNA virus, FsV, of which there are multiple variants. A 4.5-kbSstI–HindIII fragment (SH4.5) that is conserved among all genome variants was sequenced. Three open reading frames (ORF-1, -2, and -3, containing 555, 2022, and 411 bp, respectively) were shown to be transcriptionally active by ribonuclease protection assay. A “RING” zinc finger motif and a nucleotide binding site motif were identified in ORF-2.