Determination of Cellular Localization of Expression of Flavin ...

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33 Determination of Cellular Localization of Expression of Flavin-Containing Monooxygenase Genes in Mouse Tissues by In Situ Hybridization Azara Janmohamed, Ian R. Phillips, and Elizabeth A. Shephard Summary Methods are described for the cellular localization of expression of flavin-containing monooxygenase (FMO) genes in various mouse tissues by in situ hybridization. These include the production of digoxigenin (DIG)-labeled antisense and sense RNA probes by transcription from FMO cDNA templates, the preparation of paraffin wax-embedded and cryostat tissue sections, the hybridization of RNA probes to tissue sections, and the specific detection of hybridized probes using an antibody to DIG. Key Words: Flavin-containing monooxygenase; antisense RNA probes; in situ hybridization; mouse; digoxigenin-labeled probes; gene expression.

1. Introduction Differences in the patterns of expression of drug-metabolizing enzymes such as the cytochromes P450 (CYPs) and flavin-containing monooxygenases (FMOs) have implications for the ability of organisms to respond to substrates of these enzymes that are present in their environment and diet, or that are used as therapeutic drugs in clinical or veterinary medicine. Members of the FMO family exhibit differential developmental stage- and tissue-specific patterns of expression, which differ among species (1–9). In addition, the expression of FMOs is controlled in a cell-type-specific manner (9–13). In mouse liver, the distribution of FMO1 and FMO5 (9) is similar to that of other phase I enzymes, such as the CYPs, most of which are more highly expressed in hepatocytes of the perivenous region (14). By contrast, FMO2, FMO4, and FMO3 mRNAs are localized to the periportal region (9). The localization of expression of FMO3 to the periportal region of the liver is unusual for a protein whose role is considered to be predominantly one of xenobiotic metabolism. The location of FMO3 within the liver may contribute to increased toxicity of potentially harmful chemicals

From: Methods in Molecular Biology, vol. 320: Cytochrome P450 Protocols: Second Edition Edited by: I. R. Phillips and E. A. Shephard © Humana Press Inc., Totowa, NJ

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activated by FMO3. For example, thiourea, phenylthiourea, and α-naphthylthiourea are toxic to mouse C3H/10T1/2 cells expressing human FMO3, but not to those expressing human FMO1 (15). The antifungal agent ketoconazole is reported to cause hepatotoxicity in humans and rabbits and is a substrate for several FMOs (16,17). Such observations indicate that the differential pattern of FMO expression (and of other drug-metabolizing enzymes) within the liver lobule should be considered when carrying out both in vivo and in vitro xenobiotic metabolism studies on substances that are substrates for these enzymes. In this chapter, we describe the use of nonradioactive, digoxigenin-labeled antisense probes to determine the cellular localization of FMO mRNAs in various mouse tissues including the liver, lung, and brain. For an excellent review on the theory and practice of in situ hybridization (ISH), see ref. 18.

2. Materials 2.1. Preparation of Riboprobe Template 1. 2. 3. 4. 5. 6.

pBluescript (Stratagene Europe, Amsterdam, The Netherlands) plasmid containing the appropriate cDNA fragment (see Note 1). Restriction endonucleases, for linearization of plasmids. 7.5 M Ammonium acetate (filter sterilized). Buffered phenol (pH 6.0–8.0) (Fisher Scientific UK, Loughborough, UK):chloroform (1:1 [v/v]) (see Note 2). Isopropanol. 70% (v/v) Ethanol.

2.2. In Vitro Transcription of Riboprobe From Template 1. DIG RNA labeling kit (SP6/T7) (Roche, Lewes, UK): this kit comes with all the reagents required to carry out the in vitro transcription reaction, as well as a control template DNA encoding the Neo gene. 2. T3 RNA polymerase (Stratagene Europe). 3. 0.5 M Ethylenediamine tetra-acetic acid (EDTA). 4. Fresh 2% (w/v) agarose gel: prepare the gel using equipment that is clean and relatively RNase free.

2.3. Processing and Sectioning of Tissue 2.3.1. Paraffin Wax-Embedded Sections 1. Phosphate-buffered saline (PBS): prepare a 1X solution by dissolving PBS tablets (SigmaAldrich, Poole, UK) in distilled water per the manufacturer’s instructions. 2. Diethylpyrocarbonate (DEPC)-PBS: add 1 mL of DEPC/L of PBS, incubate at 37°C overnight, and autoclave. 3. Formaldehyde, 37% (v/v) solution (Formalin) (Sigma-Aldrich). 4. 10% (v/v) Formalin: prepare by mixing 1 part 37% formalin with 9 parts DEPC-PBS. 5. DEPC-treated water: add 1mL of DEPC/L of distilled water, incubate at 37°C overnight, and autoclave. 6. 30, 50, 70, 90, and 100% (v/v) ethanol (all made up in DEPC-treated water). 7. Histo-Clear™ II (Flowgen, Ashby de-la Zouch, UK). 8. Histo-Clear II:ethanol (1:1 [v/v]).

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9. Paraplast embedding medium (wax) (Sigma-Aldrich): melt solid wax pellets, in an appropriate container, in a 60°C embedding oven or water bath. 10. Wax:Histo-Clear II (1:1 [v/v]): dilute 1 part melted wax to 1 part Histo-Clear II. 11. Wax:Histo-Clear II (3:1 [v/v]): dilute 3 parts melted wax to 1 part Histo-Clear II. 12. Embedding oven set to 60°C. 13. Hot plate set to 60°C. 14. Plastic molds to embed tissues. 15. Microtome. 16. Superfrost Plus Microscope slides (VWR, Lutterworth, UK).

2.3.2. Cryosections 1. 2. 3. 4. 5.

DEPC-PBS. 10% (v/v) Formalin in DEPC-PBS. Tissue-Tek® OCT Compound (Agar Scientific, Stansted, UK). Cryostat. Superfrost Plus Microscope slides (VWR).

2.4. Preparation of Tissue Sections For Hybridization 1. Glass Coplin jars. 2. 100, 75, 50, and 25% (v/v) Ethanol: dilute ethanol in DEPC-PBS (see Subheading 2.3.1., item 2). 3. 4% (w/v) Paraformaldehyde (Sigma-Aldrich) in DEPC-PBS: in a fume hood, dissolve the appropriate amount of paraformaldehyde in DEPC-PBS. Heat gently and add a couple of pellets of NaOH. Paraformaldehyde is soluble only at an alkaline pH. Once dissolved, cool the solution and filter through No. 1 Whatman filter paper. 4. Proteinase K (Roche): this is supplied as a solution, the concentration of which may vary from batch to batch. 5. Xylene (VWR).

2.5. Hybridization With DIG-Labeled Probes 1. 10X “salts”: 2 M NaCl, 100 mM Tris-HCl, pH 7.5, 50 mM NaH2PO4, and 50 mM Na2HPO4. 2. Baker’s yeast tRNA (Roche): it is important to use high-quality tRNA that dissolves easily to produce a colorless rather than yellow solution. 3. 50% (w/v) Dextran sulfate (Sigma-Aldrich): dissolve the appropriate amount of dextran sulfate in DEPC-treated water by heating to 60°C in a water bath. The stock solution should be kept frozen at –20°C. 4. 50X Denhardt’s solution (Sigma-Aldrich). 5. Hybridization buffer (see Note 3): 1X “salts,” 50% (v/v) deionized formamide (SigmaAldrich), baker’s yeast tRNA (0.1 mg/mL), 10% (w/v) dextran sulfate, and 1X Denhardt’s solution. The volume should be made up with DEPC-treated water. 6. Cover slips: these must be baked or fresh, to ensure that there is no RNase contamination. 7. Bioassay dishes (245 × 245 × 25 mm) (Sigma-Aldrich): these are convenient to clean and hold at least eight slides each. 8. 20X saline sodium citrate (SSC): 175.3 g NaCl and 88.2 g of sodium citrate made up to 1 L with deionized water. The final concentrations of the ingredients are 3 M NaCl, 0.3 M sodium citrate. 9. Whatman 3MM paper wetted in 1X SSC, 50% (v/v) formamide.

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2.6. Posthybridization Washes 1. 2. 3. 4.

Wash buffer: 1X SSC, and 50% (v/v) formamide, 0.1% (v/v) Tween–20 (Sigma-Aldrich). 65°C water bath. 5X MAB: 500 mM maleic acid (Sigma-Aldrich), 750 mM NaCl. MABT: 1X MAB solution containing 0.5% (v/v) Tween-20.

2.7. Blocking and Incubation With Antibody 1. Sheep serum (Chemicon Europe, Chandlers Ford, UK): heat inactivate at 55°C for 30 min. 2. Blocking solution: 1X MABT containing 2% (w/v) blocking reagent (Roche), 20% (v/v) inactivated sheep serum. Dissolve the blocking reagent and MABT by heating to 65°C, and then add the appropriate volume of sheep serum. Once the solution is made it can be stored in aliquots at –20°C. 3. Anti-DIG alkaline phosphatase (AP)-conjugated antibody (Fab fragments) (Roche). 4. Antibody solution: anti-DIG AP-conjugated antibody (Fab fragments) diluted 1:1500 (v/v) in blocking solution. 5. DAKO PAP Pen (DakoCytomation, Ely, UK).

2.8. Postantibody Washes and Color Reaction 1. Prestaining buffer: 100 mM Tris-HCl, pH 9.0, 100 mM NaCl, and 5 mM MgCl2. Prepare prestaining buffer without 5 mM MgCl2 and store at room temperature. Add the MgCl2 just before use. 2. 10% (w/v) Polyvinyl alcohol (PVA) (average mol wt = 70–100 kDa) (Sigma-Aldrich): dissolve the appropriate amount of PVA in prestaining buffer and incubate at 80°C for at least 6 h or overnight. Store at room temperature. 3. Staining buffer: Prestaining buffer, 5% (w/v) PVA, 0.2 mM 5-bromo-4-chloro-3-indolylphosphate (BCIP) (Roche), and 0.2 mM nitroblue tetrazolium salt (NBT) (Roche). Prepare the staining solution just before use by adding equal volumes of a stock solution of 10% (w/v) PVA and prestaining buffer and then adding BCIP and NBT.

2.9. Poststaining Treatment 1. 2. 3. 4.

Graded series of ethanols (30, 60, 80, 95, and 100% [v/v]) made up in deionized water. Xylene. DPX mountant (VWR). Cover slips.

3. Methods The most important factor for success with in situ hybridization of RNA is that all the solutions, glass, and plasticware be RNase free. Solutions for tissue processing, prehybridization, and hybridization must be DEPC treated. All glassware and surgical instruments must be baked at 80°C before use. After hybridization these precautions need not be taken.

3.1. Preparation of Riboprobe Template 1. Digest approx 10 μg of the recombinant plasmid with an appropriate restriction enzyme that cuts the construct once 5' of the cDNA insert. It is important that the antisense probes generated be between 150 and 500 bp (see Note 4). 2. Check the digest by agarose gel electrophoresis, to ensure that >95% of the plasmid is linearized.

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Fig. 1. Integrity of riboprobe. Mouse FMO riboprobes were analysed by electrophoresis through a 2% agarose gel. Lane M, 1-kb+ DNA marker; flanes a and b, FMO1 antisense and sense, respectively; lanes c and d, FMO3, antisense and sense, respectively; lanes e and f, FMO4 antisense and sense, respectively; and lanes g and h, FMO5 antisense and sense, respectively.

3. Add 0.5 vol of 7.5 M ammonium acetate and extract once with phenol/chloroform. 4. To the aqueous layer add an equal volume of isopropanol and allow the DNA to precipitate at room temperature for 5 min. 5. Centrifuge at 14,000g for 10 min at 4°C. Remove the supernatant and wash the DNA pellet in 70% (v/v) ethanol. Centrifuge as before. Pour off the ethanol. Leave samples to air-dry. 6. Resuspend the DNA in sterile, RNase-free water. Do not use DEPC-treated water, because DEPC interferes with the transcription reaction. 7. Measure the A260 and determine the concentration of the linearized DNA.

3.2. In Vitro Transcription of Riboprobe From Template 1. Add the following components of the DIG RNA labeling kit to a 1.5-mL tube on ice: 1 μg of linearized template (see Subheading 3.1.), 2 μL of 10X NTP labeling mixture, 2 μL of 10X transcription buffer, 1 μL of RNase inhibitor, and 2 μL of RNA polymerase (SP6, T3, or T7) (20 U/μL) (see Note 5). Make up to 20 μL with sterile water. Mix the contents gently and incubate at 37°C for 2 h. 2. Add 2 μL of 0.5 M EDTA to stop the reaction. Make the volume of the reaction up to 100 μL with DEPC-treated water. 3. Electrophorese a 5-μL aliquot of the probe on a fresh 2% (w/v) agarose gel. Although the size of the transcript cannot be determined using such a gel, it provides a quick way of determining the integrity of the riboprobe (Fig. 1).

3.3. Processing and Sectioning of Tissue All solutions, glassware, and surgical instruments must be RNase free. In our laboratory, we use paraffin wax-embedded sections, because retention of tissue morphology is much better in these than in cryosections. The only disadvantage of using paraffin-embedded tissues is the processing time required. The procedures detailed here for processing tissues have been optimized for ISH and give reproducible results without loss of signal and with no variability among different tissues.

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3.3.1. Paraffin Embedding and Sectioning of Tissues 1. Anesthetize a mouse using standard techniques (see Note 6). 2. Perfuse the mouse (through the left ventricle of the heart) with fresh 10% formalin (prewarmed to 37°C). 3. Remove all the required tissues and fix in 10% formalin overnight. 4. The following day, wash the tissues twice for 20 min each time in DEPC-PBS. 5. Replace the PBS sequentially with 30, 50, and then 70% ethanol. Allow 30 min for each incubation (with shaking). If using the brain, increase incubation times to 1 h. 6. Remove the final ethanol wash and cover the tissue with fresh 70% ethanol. The tissues can then be stored in 70% ethanol at –20°C. 7. To continue the processing, remove the 70% ethanol and incubate the tissues in 90% ethanol for 30 min (allow 1 h for the brain). Wash twice in 100% ethanol for 15 min each time (increase incubation time to 30 min for the brain). 8. Transfer the tissues to Histo-Clear II:ethanol (1:1) for 10 min, followed by 100% HistoClear II for 5 min. Do not incubate the tissues for more than 5 min in Histo-Clear II, because this makes them brittle when sectioning is carried out. 9. Place the tissues in a 1:1 mixture of wax:Histo-Clear II at 60°C for 10 min. 10. Transfer to a 3:1 mixture of wax:Histo-Clear II at 60°C for 10 min and finally to wax (at 60°C) for about 30 min. Then place the tissues in fresh wax for a further 30 min. When working with brain tissue, increase the wax treatment to at least 3 to 4 h. 11. Using plastic molds placed on a hot plate, embed the tissues in fresh wax. Ensure that there are no bubbles and that the wax is liquid when embedding the tissues; otherwise, the tissue blocks will give uneven sections. 12. Using a microtome, cut 10-μm sections and remove creases in the ribbons of sections by floating on top of water in a water bath at 37°C. To place sections on slides, immerse Superfrost slides in the water bath (close to a ribbon of sections) and use the slides to lift the sections out. The sections will stick to the surface of the slides. Allow the slides to dry overnight at 37°C.

3.3.2. Cryosection Processing 1. Excise the tissue from the perfused animal (see Subheading 3.3.1., item 2). 2. Wash the tissue in DEPC-PBS; then fix in 10% formalin overnight. 3. Place OCT medium in a plastic mold and embed the tissues in the OCT medium. Place the mold inside a cryostat to solidify. 4. Mount the tissue block onto the cryostat and cut 10-μm sections. 5. Remove the creases in the ribbon of sections by gently teasing them out with a soft brush. Place a slide on the sections; they will attach immediately to the slide. 6. Air-dry the sections and store at –20°C.

3.4. Preparation of Tissue Sections For Hybridization All procedures are carried out in baked, glass Coplin jars.

3.4.1. Paraffin Sections 1. To dewax the sections, place slides in xylene for 10 min. Repeat this step. 2. Transfer the slides to 100% ethanol for 3 min. Discard the ethanol. Repeat this step twice. 3. Hydrate the sections by incubating the slides sequentially in 75, 50, and then 25% ethanol for 3 min each time. Wash the slides twice in DEPC-PBS for 5 min each time.

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4. Place the slides in 4% paraformaldehyde for 20 min. Wash three times in PBS for 5 min each time. 5. Dilute proteinase K to a final concentration of 10 μg/mL in PBS. A solution of 50 mL is sufficient to immerse slides in a Coplin jar (see Note 7). Incubate the slides for 10–15 min at 37°C. 6. Return the slides to 4% paraformaldehyde solution (the same solution as that used in step 4 can be used) and incubate for 20 min (at room temperature). 7. Wash the slides three times for 5 min each time with DEPC-PBS. Dehydrate in the graded alcohol series used in step 3 but in the reverse order (25, 50, 75, and then 100%) for 1 min each time. 8. Air-dry the slides. They are now ready for hybridization.

3.4.2. Cryosections 1. Before hybridization thaw the sections and allow them to come to room temperature. 2. Wash the sections three times in DEPC-PBS for 5 min each time. The sections are now ready for hybridization.

3.5. Hybridization With DIG-Labeled Probes 1. Dilute DIG-labeled riboprobes in hybridization buffer. We usually use a dilution of about 1:1000 (v/v). Depending on the efficiency of the transcription reaction, the dilution may need to be varied; determine this empirically. 2. Denature the mixture of probe and hybridization buffer for 10 min at 70°C. 3. Vortex to mix and centrifuge briefly. 4. Place the slides flat (sections side upward) inside a Perspex box containing two sheets of Whatman 3MM paper wetted in 1X SSC, 50% formamide (see Note 8). Add 100–150 μL of probe to each slide. Gently lower cover slips onto the slides. Ensure that all the sections on the slides are covered with the hybridization mixture. Hybridize overnight at 55–65°C.

3.6. Posthybridization Washes 1. Fill a Coplin jar with wash buffer and warm to 65°C. 2. Transfer the slides (do not remove the cover slips) to the Coplin jar, and incubate for 15 min or until the cover slips have fallen off, or slide off easily. 3. Wash the slides a further two times for 30 min each time in wash buffer at 65°C. 4. Wash the slides twice for 30 min each time in 1X MABT at room temperature.

3.7. Blocking and Incubation With Antibody 1. Dry off the slides using tissue paper, being careful not to touch the sections. 2. Circle the sections using a DAKO PAP Pen. 3. Transfer the slides to a humidified chamber (see Note 9). Place the slides with the sections facing upward. Cover the sections with blocking solution (approx 300 μL/slide). Incubate the sections for at least 1 h at room temperature. 4. Remove the blocking solution by gently tipping it off the slides. Cover the sections with anti-DIG AP-conjugated antibody (Fab fragments) diluted 1:1500 (v/v) in blocking solution. Incubate the slides in this solution overnight at 4°C.

3.8. Postantibody Washes and Color Reaction 1. Transfer the slides to Coplin jars containing MABT and wash five times for 30 min each time at room temperature.

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2. Wash the slides twice for 10 min each time in prestaining buffer. 3. Incubate the slides in staining buffer at 37°C in the dark. 4. Check for staining every few hours until satisfactory. This can take from 3 to 48 h (see Note 10). Staining is observed as a purple color and can be clearly distinguished under a light microscope (Fig. 2). 5. To stop the reaction, remove the slides from the staining buffer and rinse several times in distilled water.

3.9. Poststaining Treatment 1. Dehydrate the sections sequentially through 30, 60, 80, 95, and then 100% ethanol for at least 1 min each. 2. Wash twice in xylene for at least 1 min each time. However, an overnight incubation in xylene gives the best results. 3. Place a cover slip on each slide using DPX mountant and leave for 24 h to set fully. Store the slides in the dark to prevent the color from fading.

4. Notes 1. Any plasmid containing promoters such as T3, T7, or SP6 can be used for the in vitro transcription reaction. However, we have experienced problems when carrying out in vitro transcription from cDNAs cloned into the TOPO-TA vector (Invitrogen, Paisley, UK). 2. The appropriate precautions should be taken when using phenol:chloroform. All extractions should be done in a fume hood and gloves should be worn. 3. This is the most crucial component of the ISH procedure and must be made with great care, ensuring no RNase contamination. The highest-quality reagents and water must be used. 4. Probes differ in their tissue permeability, depending on their length. We have observed problems with permeability of tissues when probes are longer than 500 bp. Sometimes probes derived from certain regions of a cDNA will not hybridize to a tissue. If staining is expected but not observed, try using a probe transcribed from a different region of the same cDNA. 5. When using T3 or T7 RNA polymerase from Stratagene, the enzyme must be diluted to 20 U/μL. If this is not done, the efficiency of transcription will be very low and the probe yield will decrease significantly. 6. All animal procedures must be carried out according to local regulations. 7. The concentration of proteinase K used may need to be varied from tissue to tissue. We have found that a concentration of 10 μg/mL works very well for all soft tissues, such as the liver and brain. Other tissues, such as muscle, may require higher concentrations of the enzyme. This must be determined empirically. 8. The mix of 1X SSC, 50% formamide, instead of water, is used to wet the Whatman paper, because this ensures that the atmosphere in which the slides are incubated is saturated with formamide vapors, which prevents the slides from drying out. 9. Any Tupperware or Perspex box can be used for this. The slides should not touch the bottom of the box. To achieve this, disposable pipets can be placed horizontally inside the box and the slides placed on top of the pipets. 10. The inclusion of 5% PVA in the staining buffer reduces background and intensifies the signal, even after a long period of incubation. In our experience, incubation at 37°C for 24 h is sufficient to detect rare mRNA molecules.

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Fig. 2. Localization of FMO mRNAs in mouse liver, lung and brain. (A) Female mouse liver was hybridized with antisense probes for FMO3, and glutamine synthetase, which served as a control for perivenous expression. Black arrowheads indicate the central veins and the white arrowhead the portal vein. Bar = 200 μm. (B) Lung was hybridized with an antisense probe for FMO3 mRNA. The arrowhead indicates the terminal bronchiole. Staining is observed specifically in the epithelial cells lining the alveoli. Bar = 100 μm. (C) An antisense probe for FMO1 mRNA was hybridized with a section of the cerebrum of mouse brain. Arrowheads indicate specific staining observed in the neurons. Bar = 200 μm.

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Acknowledgment This work was supported by a grant from the Wellcome Trust (no. 053590). References 1. Tynes, R. E. and Philpot, R. M. (1987) Tissue- and species-dependent expression of multiple forms of mammalian microsomal flavin-containing monooxygenase. Mol. Pharmacol. 31, 569–574. 2. Lawton, M. P., Gasser, R., Tynes, R. E., Hodgson, E., and Philpot, R. M. (1990) The flavin-containing monooxygenase enzymes expressed in rabbit liver and lung are products of related but distinctly different genes. J. Biol. Chem. 265, 5855–561. 3. Shehin-Johnson, S. E., Williams, D. E., Larsen Su, S., Stresser, D. M., and Hines, R. N. (1995) Tissue-specific expression of flavin-containing monooxygenase (FMO) forms 1 and 2 in the rabbit. J. Pharmacol. Exp. Ther. 272, 1293–1299. 4. Phillips, I. R., Dolphin, C. T., Clair, P., et al. (1995) The molecular biology of the flavincontaining monooxygenases of man. Chem. Biol. Interact. 96,17–32. 5. Dolphin, C. T., Cullingford, T. E., Shephard, E. A., Smith, R. L., and Phillips, I. R. (1996) Differential developmental and tissue-specific regulation of expression of the genes encoding three members of the flavin-containing monooxygenase family of man, FMO1, FMO3 and FMO4. Eur. J. Biochem. 235, 683–689. 6. Cherrington, N. J., Cao, Y., Cherrington, J. W., Rose, R. L., and Hodgson, E. (1998) Physiological factors affecting protein expression of flavin-containing monooxygenases 1, 3 and 5. Xenobiotica 28, 673–682. 7. Ripp, S. L., Itagaki, K., Philpot, R. M., and Elfarra, A. A. (1999) Species and sex differences in expression of flavin-containing monooxygenase form 3 in liver and kidney microsomes. Drug Metab. Dispos. 27, 46–52. 8. Lattard, V., Buronfosse, T., Lachuer, J., Longin-Sauvageon, C., Moulin, C., and Benoit, E. (2001) Cloning, sequencing, tissue distribution, and heterologous expression of rat flavin-containing monooxygenase 3. Arch. Biochem. Biophys. 391, 30–40. 9. Janmohamed, A., Hernandez, D., Phillips, I. R., and Shephard, E. A. (2004) Cell-, tissue-, sex- and developmental stage-specific expression of mouse flavin-containing monooxygenases (Fmos). Biochem. Pharmacol. 68, 73–83. 10. Devereux, T. R., Diliberto, J. J., and Fouts, J. R. (1985) Cytochrome P-450 monooxygenase, epoxide hydrolase and flavin monooxygenase activities in Clara cells and alveolar type II cells isolated from rabbit. Cell Biol. Toxicol. 1, 57–65. 11. Bhamre, S., Shankar, S. K., Bhagwat, S. V., and Ravindranath, V. (1993) Catalytic activity and immunohistochemical localization of flavin-containing monooxygenase in rat kidney. Life Sci. 52, 1601–1607. 12. Overby, L., Nishio, S. J., Lawton, M. P., Plopper, C. G., and Philpot, R. M. (1992) Cellular localization of flavin-containing monooxygenase in rabbit lung. Exp. Lung Res. 18, 131–144. 13. Janmohamed, A., Dolphin, C. T., Phillips, I. R., and Shephard, E. A. (2001) Quantification and cellular localization of expression in human skin of genes encoding flavincontaining monooxygenases and cytochromes P450. Biochem. Pharmacol. 62, 777–786. 14. Lindros, K. O. (1997) Zonation of cytochrome P450 expression, drug metabolism and toxicity in liver. Gen. Pharmacol. 28, 191–196. 15. Smith, P. B. and Crespi, C. (2002) Thiourea toxicity in mouse C3H/10T1/2 cells expressing human flavin-dependent monooxygenase 3. Biochem. Pharmacol. 63, 1941–1948.

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16. Rodriguez, R. J. and Acosta, D. Jr. (1997) Metabolism of ketoconazole and deacetylated ketoconazole by rat hepatic microsomes and flavin-containing monooxygenases. Drug Metab, Dispos, 25, 772–777. 17. Rodriguez, R. J. and Miranda, C. L. (2000) Isoform specificity of N-deacetyl ketoconazole by human and rabbit flavin-containing monooxygenases. Drug Metab. Dispos. 28, 1083–1086. 18. Wilkinson, D. G. (1992) The Theory and Practice of In Situ Hybridization, IRL, Oxford, UK, pp. 1–13.