ARTICLE |
Correspondence to: Jean-Marie Dupret, UMR7000, Faculté de Médecine Pitié-Salpêtrière, 105 bd de l'Hôpital, 75013 Paris, France. E-mail: jmdupret@infobiogen.fr
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The human arylamine N-acetyltransferases (NATs) NAT1 and NAT2 are enzymes responsible for the acetylation of many arylamines and hydrazines, thereby playing an important role in both detoxification and activation of many drugs and carcinogens. Both enzymes show polymorphisms but exhibit key differences in substrate selectivity and tissue expression. In the present study, reverse transcriptase-PCR, Western blotting, and immunohistochemistry were used to investigate the expression of the NATs in human skeletal muscle. Despite the presence of its mRNA, NAT2 enzyme level was below the limit of detection. In contrast, both NAT1 mRNA and enzyme were readily detected in fetal, newborn, and adult muscles. In addition, punctate cytoplasmic and perinuclear NAT1 immunostaining was observed in all tissue sections, the staining being more intense in the fetal tissue. High expression of NAT1 enzyme in fetal muscle was also suggested by Western blotting. Because skeletal muscle accounts for a large proportion of body mass, muscle NAT1 expression may contribute significantly to the total activity in the body. These results further support the involvement of skeletal muscle in the metabolism of xenobiotics.
(J Histochem Cytochem 51:789796, 2003)
Key Words: NATs, xenobiotics, human skeletal muscle, RT-PCR, immunohistochemistry, Western blotting
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
MUSCLES are often subjected to severe conditions provoked by thermal, oxidative, mechanical, or pharmacological stresses. These stresses have been implicated in human skeletal muscle aging (
The XMEs comprise oxidative (phase I) and conjugating (phase II) enzymes involved in biotransformation of many chemical stressors. The acetyl-CoA:arylamine N-acetyltransferases (NATs; EC 2.3.1.5) are phase II enzymes that catalyze the transfer of an acetyl group from Ac-CoA to the nitrogen or oxygen atom of primary arylamines, hydrazines, and their N-hydroxylated metabolites. Therefore, the NATs play an important role in the detoxification and potential metabolic activation of many xenobiotics (
Although the liver is the organ with the primary detoxification functions, skeletal muscle is also involved in the detoxification process. Skeletal muscle cells have been shown to express different types of XMEs, including cytochrome P450 (CYPs) (
These observations prompted us to analyze whether NAT enzymes are expressed in human skeletal muscle. We carried out RT-PCR and immunoblotting on muscle homogenates. Immunohistochemical (IHC) analyses of NAT expression was also carried out on fetal, newborn, and adult human skeletal muscle biopsy specimens.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Antibody Preparation
Polyclonal antibodies that distinguish between human NAT1 and NAT2 were obtained in rabbits using an anti-peptide strategy in which antisera were raised against the divergent C-termini of human NAT1 and NAT2. These antibodies were previously described and used for analysis of NATs expression (
Muscle Biopsies
Biopsies from the quadriceps muscle were obtained during diagnostic or surgical procedures in accordance with French legislation on ethical rules. All samples were derived from individuals with no history of muscle disease. Fetal tissues were obtained immediately after abortion induced by mifepristone (RU486). A postmortem biopsy of a 37-week-old infant and biopsies from a 57- and a 77-year-old were obtained during surgical procedures.
RNA Isolation and Reverse Transcription-polymerase Chain Reaction (RT-PCR)
Total RNA was isolated from skeletal muscle using TRIzol (Gibco BRL; CergyPontoise, France) according to the manufacturer's instructions. For cDNA synthesis, 6 µg of total RNA was used at 70C in a final volume of 20 µl using the C. therm Polymerase RT-PCR kit (Roche). For PCR analysis, 10 µl of cDNA was used as a template in a 50-µl amplification mixture containing 200 µM of each deoxynucleotide triphosphate, 10 mM Tris-HCl, pH 8.3, 1.5 mM MgCl2, 50 mM KCl, 0.3 µM of each primer, and 2.5 U of Expand High Fidelity DNA polymerase (Roche). The NAT primers used in the PCR were as follows: for human NAT1 (specific 387-bp fragment), sense 5'-tagaagacagcaaatacc-3', antisense 5'-agttgataactggtgagc-3'; for human NAT2 (specific 526-bp fragment), sense 5'-tgccaaagaagaaacacc-3', antisense 5'-tgaaaatgtgtccttatg-3'. The PCR conditions were as follows: 35 cycles each consisting of 1-min annealing at 47C, 1-min extension at 72C, and 1-min denaturation at 94C. To assess whether the RNA samples were contaminated by genomic DNA, an additional PCR reaction was carried out under the same conditions with primers for human ß-crystallin (CRYAB); sense 5'-aaggagctgaccagccagct-3', antisense 5'-actggtggggaaactttcttg-3'. According to the intron/exon organization of the human B-crystallin gene, a specific 623-bp fragment is generated from cDNA, whereas a 3055-bp fragment would be amplified from genomic DNA. Absence of genomic DNA contamination was also confirmed using a negative control with no reverse transcriptase. The PCR products were separated by gel electrophoresis in ethidium bromide-stained agarose (1.8%).
Protein Sample Preparation, SDS-PAGE, and Immunoblot Analysis
Muscle proteins were extracted by crushing muscle biopsy specimens (2030 mg) in 500 µl Tris-HCl 25 mM, pH 7.5, 1 mM DTT, 1 mM EDTA, 0.2% SDS, 0.5% Triton X-100, and protease inhibitors. To selectively separate proteins in the muscle extracts from contaminating species, such as nucleic acids and lipids, that would otherwise interfere with electrophoresis, trichloroacetic acid (TCA) precipitation was used. In addition, TCA precipitation allows more proteins to be loaded in acrylamide gels. Briefly, cold TCA was added (to a final concentration of 25%) to 250 µl of muscle extracts (protein concentration adjusted to 3 mg/ml) and the proteins were allowed to precipitate on ice for 30 min. The mixture was then centrifuged for 30 min at 15,000 x g and the protein pellet washed with cold acetone to remove residual TCA. After centrifugation (30 min, 15,000 x g), proteins were resuspended in a small volume of 4 x SDS sample buffer (3040 µl) and heated at 100C before SDS-PAGE (10% acrylamide) under reducing conditions. After electrophoresis, proteins were transferred to a nitrocellulose membrane. Immunodetection of NAT1 or NAT2 isoform was carried out using the polyclonal antibodies described above at a dilution of 1:1500. Visualization of immunoreactivity was performed using a secondary goat anti-rabbit IgG conjugated with horseradish peroxidase. Recombinant human GST-NAT1 and GST-NAT2 proteins were used as controls.
Immunohistochemical Analysis
All experiments were performed on 8- µm frozen transverse or longitudinal cryostat sections. Double immunofluorescence was performed using the purified polyclonal anti-NAT1 antibody (1:100) with either a monoclonal antibody against the slow myosin heavy chain (1:5) (NCL-MHCs; Novacastra, Newcastle upon Tyne, UK) or a monoclonal antibody against the intermediate filament protein, desmin (1:200) (D33; Dako, Trappes, France). Briefly, on unfixed sections, primary antibodies were simultaneously incubated overnight at 4C. The anti-NAT1 antibody was visualized with an AlexaFluor 488 (Molecular Probes; Montluçon, France) directly coupled to an anti-rabbit secondary antibody, and the anti-slow and anti-desmin antibodies were visualized with an AlexaFluor 594 directly coupled to an anti-mouse secondary antibody. Secondary antibodies in the absence of the primary antibodies were used as negative controls. Finally, to visualize nuclei, the sections were mounted in medium (Mowiol; CalbiochemNovabiochem, San Diego, CA) containing bis-benzimide (0.0001% w/v, Hoechst no. 33258; Sigma, St Louis, MO). All images were digitalized using the MetaView image analysis system.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
RT-PCR Analysis
We initially investigated the expression of NAT1 and NAT2 in human skeletal muscle using RT-PCR. The primer pairs were designed to yield PCR products of different sizes (387 bp for NAT1 and 526 bp for NAT2). Both NAT1 and NAT2 PCR-derived bands were detected in two adult muscle samples (Fig 1, Lanes 1 and 4 for NAT1; Lanes 2 and 5 for NAT2). Identical results were obtained from mRNA extracted from a third adult muscle biopsy specimen (data not shown). These results strongly suggest that NAT1 and NAT2 mRNA are present in adult skeletal muscle. The co-amplification of B-crystallin (CRYAB) mRNA revealed no genomic DNA contamination (Fig 1, Lanes 3 and 6). Absence of genomic DNA contamination was also confirmed using a negative control with no reverse transcriptase in the RT experiment (Fig 1, Lane 7; data not shown for other subjects).
|
Detection of NAT Expression by Western Blot Analysis
To study NAT expression at the protein level, we used specific NAT antibodies. We first investigated whether NAT1 and NAT2 proteins were present in human skeletal muscle extracts. To do this, fetal (n=2) and adult (n=2) muscles were used to prepare total protein extracts at a concentration of 3 mg/ml. Equal amounts of extracts were then concentrated by TCA precipitation to selectively separate the proteins from the contaminating species (e.g., nucleic acids, lipids) and to increase the protein fraction of the samples. Western blot analysis with the specific antibody against human NAT1 revealed the presence of NAT1 protein in all of the skeletal muscle protein extracts tested (Fig 2). In addition, the results suggested that NAT1 protein was expressed at a high level in fetal tissue (Fig 2, Lanes 1 and 3). The expression of NAT2 protein in these samples was also studied by Western blot analysis. However, the NAT2 protein was not detectable at the level of sensitivity attained in these experiments (data not shown).
|
Immunohistochemistry
To further analyze the expression of NAT1 protein in human skeletal muscle, IHC experiments were performed on fetal, newborn, and adult muscle sections. In transverse sections, the most intense NAT1 immunostaining was observed in fetal muscle (Fig 3A) whereas weaker immunostaining was obtained in newborn and adult muscles (Fig 3B and Fig 3C, respectively). To determine whether the localization of NAT1 was fiber type-specific, double immunofluorescence staining with the slow myosin heavy chain antibody was carried out (Fig 3D3F). The results obtained demonstrate that NAT1 is evenly distributed within slow and fast fibers, with no preferential localization in either type of muscle fibers. The localization of NAT1 immunostaining in fetal muscle was similar to that observed in newborn and adult muscles, with punctate cytoplasmic staining but also with a punctate perinuclear arrangement (Fig 4A4F). When immunostaining was performed in the absence of the primary antibody, no staining of the muscle sections was visible (data not shown). These results were consistent with the Western blot data that suggest a strong expression of NAT1 protein in fetal tissues (Fig 2). To further characterize the immunostaining of NAT1, we used an antibody against desmin, a Z-line-associated intermediate filament that appears as collars surrounding Z-discs of the myofibril (
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We have demonstrated by RT-PCR, Western blotting, and IHC, that human skeletal muscle synthesizes the arylamine N-acetyltransferase NAT1. Although RT-PCR revealed that the NAT2 mRNA is also present in human skeletal muscles, its expression product was not detected by immunoblotting. Such a difference between the expression profiles of NAT1 and NAT2 has been reported in other cell types. Both NAT1 and NAT2 mRNA have been detected in liver, gastrointestinal tract tissues, ureter, bladder, lung (
NATs are polymorphic enzymes and the differences in immunoreactive recombinant protein variants have been reported in yeast expression systems. Thus, NAT1 variants encoded by NAT1*14B, NAT1*15, NAT1*17, NAT1*19, and NAT1*22 have been shown to exhibit NAT1 protein expression levels below the limit of detection as measured by Western blotting (
Our results suggest that NAT1 is expressed at a high level in fetal compared to newborn and adult skeletal muscle (Fig 2 and Fig 3). In contrast, the level of expression of several metabolic enzymes is developmentally regulated, with increased levels being present in the adult (
Skeletal muscle enzymes, such as ß-enolase (
The early expression of NAT1 and its persistence in adult skeletal muscle have potential pharmacological consequences. Because skeletal muscle constitutes a large proportion of total body mass, it is likely that the total activity of NAT1 in muscle contributes significantly to the total activity in the body, as suggested for other xenobiotic-metabolizing enzymes (
It has been proposed that human NAT1 plays a role in folate catabolism through the acetylation of the folate para-aminobenzoylglutamate (
Variations in muscle cell activity related to aging (
In conclusion, the expression of NAT1 in muscle cells may be an important factor in the detoxification/activation processes because of the potential involvement of the muscle in the pharmacokinetics of many xenobiotics (
![]() |
Acknowledgments |
---|
Supported by grants from the Association Française contre les Myopathies, Alliance program, and the Association pour la Recherche contre le Cancer.
Received for publication November 14, 2002; accepted January 22, 2003.
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Atalay M, Seene T, Hanninen O, Sen CK (1996) Skeletal muscle and heart antioxidant defences in response to sprint training. Acta Physiol Scand 158:129-134[Medline]
Blum M, Grant DM, McBride W, Heim M, Meyer UA (1990) Human arylamine N-acetyltransferase genes: isolation, chromosomal localization, and functional expression. DNA Cell Biol 9:193-203[Medline]
Butcher NJ, Ilett KF, Minchin RF (2000a) Inactivation of human arylamine N-acetyltransferase 1 by the hydroxylamine of p-aminobenzoic acid. Biochem Pharmacol 60:1829-1836[Medline]
Butcher NJ, Ilett KF, Minchin RF (2000b) Substrate-dependent regulation of human arylamine N-acetyltransferase-1 in cultured cells. Mol Pharmacol 57:468-473
Chowrashi P, Mittal B, Sanger JM, Sanger JW (2002) Amorphin is phosphorylase: phosphorylase is an alpha-actinin-binding protein. Cell Motil Cytoskel 53:125-135[Medline]
Crosbie SJ, Blain PG, Williams FM (1997) An investigation into the role of rat skeletal muscle as a site for xenobiotic metabolism using microsomes and isolated cells. Hum Exp Toxicol 16:138-145[Medline]
Davidson M, Collins M, Byrne J, Vora S (1983) Alterations in phosphofructokinase isoenzymes during early human development. Establishment of adult organ-specific patterns. Biochem J 214:703-710[Medline]
Derewlany LO, Knie B, Koren G (1994) Arylamine N-acetyltransferase activity of the human placenta. J Pharmacol Exp Ther 269:756-760[Abstract]
Evans DAP, White TA (1964) Human acetylation polymorphism. J Lab Clin Med 63:394-403
Fougerousse F, EdomVovard F, Merkulova T, Ott MO, Durand M, ButlerBrowne G, Keller A (2002) The muscle-specific enolase is an early marker of human myogenesis. J Muscle Res Cell Motil 22:535-544
Fretland AJ, Doll MA, Leff MA, Hein DW (2001a) Functional characterization of nucleotide polymorphisms in the coding region of N-acetyltransferase 1. Pharmacogenetics 11:511-520[Medline]
Fretland AJ, Leff MA, Doll MA, Hein DW (2001b) Functional characterization of human N-acetyltransferase 2 (NAT2) single nucleotide polymorphisms. Pharmacogenetics 11:207-215[Medline]
Guengerich FP (1992) Metabolic activation of carcinogens. Pharmacol Ther 54:17-61[Medline]
Hein D (2002) Molecular genetics and function of NAT1 and NAT2: role in aromatic amine metabolism and carcinogenesis. Mutat Res 65:506-507
Hein DW, Doll MA, Fretland AJ, Leff MA, Webb SJ, Xiao GH, Devanaboyina US et al. (2000) Molecular genetics and epidemiology of the NAT1 and NAT2 acetylation polymorphisms. Cancer Epidemiol Biomarkers Prev 9:29-42
Hickman D, Pope J, Patil SD, Fakis G, Smelt V, Stanley LA, Payton M et al. (1998) Expression of arylamine N-acetyltransferase in human intestine. Gut 42:402-409
Hightower LE (1998) The promise of molecular biomarkers for environmental monitoring. Biol Chem 379:1213-1215[Medline]
Hughes NC, Janezic SA, McQueen KL, Jewett MA, Castranio T, Bell DA, Grant DM (1998) Identification and characterization of variant alleles of human acetyltransferase NAT1 with defective function using p-aminosalicylate as an in-vivo and in-vitro probe. Pharmacogenetics 8:55-66[Medline]
Hussey AJ, Kerr LA, Cronshaw AD, Harrison DJ, Hayes JD (1991) Variation in the expression of Mu-class glutathione S-transferase isoenzymes from human skeletal muscle. Evidence for the existence of heterodimers. Biochem J 273:323-332[Medline]
Ilett KF, Ingram DM, Carpenter DS, Teitel CH, Lang NP, Kadlubar FF, Minchin RF (1994) Expression of monomorphic and polymorphic N-acetyltransferases in human colon. Biochem Pharmacol 47:914-917[Medline]
Ip W (1999) Guidebook to the Cytoskeletal and Motor Proteins. Oxford, Oxford University Press
Irshaid YM, al-Hadidi HF, Abuirjeie MA, Rawashdeh NM, Gharaibeh NS (1993) Acetylation of dapsone by human whole blood. Int J Clin Pharmacol Ther Toxicol 31:18-22[Medline]
Kawakubo Y, Yamazoe Y, Kato R, Nishikawa T (1990) High capacity of human skin for N-acetylation of arylamines. Skin Pharmacol 3:180-185[Medline]
Khazaeinia T, Ramsey AA, Tam YK (2000) The effects of exercise on the pharmacokinetics of drugs. J Pharm Pharm Sci 3:292-302[Medline]
Kondo H, Nakagaki I, Sasaki S, Hori S, Itokawa Y (1993) Mechanism of oxidative stress in skeletal muscle atrophied by immobilization. Am J Physiol 265:E839-844[Medline]
Liu Y, Steinacker JM (2001) Changes in skeletal muscle heat shock proteins: pathological significance. Front Biosci 6:D12-25[Medline]
Matas N, Thygesen P, Stacey M, Risch A, Sim E (1997) Mapping AAC1, AAC2 and AACP, the genes for arylamine N-acetyltransferases, carcinogen metabolising enzymes on human chromosome 8p22, a region frequently deleted in tumours. Cytogenet Cell Genet 77:290-295[Medline]
McArdle A, Vasilaki A, Jackson M (2002) Exercise and skeletal muscle ageing: cellular and molecular mechanisms. Ageing Res Rev 1:79-93[Medline]
Meisel P (2002) Arylamine N-acetyltransferases and drug response. Pharmacogenomics 3:349-366[Medline]
Meisel P, Giebel J, Peters M, Foerster K, Cascorbi I, Wulff K, Fanghaenel J, Kocher T (2002) Expression of N-acetyltransferases in periodontal granulation tissue. J Dent Res 81:349-353
Meyer DJ, Ketterer B (1995) Purification of soluble human glutathione S-transferases. Methods Enzymol 252:53-65[Medline]
Minchin RF (1995) Acetylation of p-aminobenzoylglutamate, a folic acid catabolite, by recombinant human arylamine N-acetyltransferase and U937 cells. Biochem J 307:1-3[Medline]
Ohsako S, Deguchi T (1990) Cloning and expression of cDNAs for polymorphic and monomorphic arylamine N-acetyltransferases from human liver. J Biol Chem 265:4630-4634
Pacifici GM, Bencini C, Rane A (1986) Acetyltransferase in humans: development and tissue distribution. Pharmacology 32:283-291[Medline]
Pansarasa O, Bertorelli L, Vecchiet J, Felzani G, Marzatico F (1999) Age-dependent changes of antioxidant activities and markers of free radical damage in human skeletal muscle. Free Radic Biol Med 27:617-622[Medline]
Ricart E, Taylor WR, Loftus EV, O'Kane D, Weinshilboum RM, Tremaine WJ, Harmsen WS et al. (2002) N-acetyltransferase 1 and 2 genotypes do not predict response or toxicity to treatment with mesalamine and sulfasalazine in patients with ulcerative colitis. Am J Gastroenterol 97:1763-1768[Medline]
Riggs JE (1998) Alcohol-associated rhabdomyolysis: ethanol induction of cytochrome P450 may potentiate myotoxicity. Clin Neuropharmacol 21:363-364[Medline]
Sadrieh N, Davis CD, Snyderwine EG (1996) N-acetyltransferase expression and metabolic activation of the food-derived heterocyclic amines in the human mammary gland. Cancer Res 56:2683-2687[Abstract]
Sim E, Payton M, Noble M, Minchin R (2000) An update on genetic, structural and functional studies of arylamine N-acetyltransferases in eucaryotes and procaryotes. Hum Mol Genet 9:2435-2441
Smelt VA, Upton A, Adjaye J, Payton MA, Boukouvala S, Johnson N, Mardon HJ et al. (2000) Expression of arylamine N-acetyltransferases in pre-term placentas and in human pre-implantation embryos. Hum Mol Genet 9:1101-1107
Smith C, Stamm SC, Riggs JE, Stauber W, Harsh V, Gannett PM, Hobbs G et al. (2000) Ethanol-mediated CYP1A1/2 induction in rat skeletal muscle tissue. Exp Mol Pathol 69:223-232[Medline]
Sonawane BR, Lucier GW (1975) Hepatic and extrahepatic N-acetyltransferase. Perinatal development using a new radioassay. Biochim Biophys Acta 411:97-105[Medline]
Stanley LA, Coroneos E, Cuff R, Hickman D, Ward A, Sim E (1996) Immunochemical detection of arylamine N-acetyltransferase in normal and neoplastic bladder. J Histochem Cytochem 44:1059-1067
Tomarev SI, Piatigorsky J (1996) Lens crystallins of invertebratesdiversity and recruitment from detoxification enzymes and novel proteins. Eur J Biochem 235:449-465[Abstract]
Upton A, Johnson N, Sandy J, Sim E (2001) Arylamine N-acetyltransferasesof mice, men and microorganisms. Trends Pharmacol Sci 22:140-146[Medline]
Wang AM, Doyle MV, Mark DF (1989) Quantitation of mRNA by the polymerase chain reaction. Proc Natl Acad Sci USA 86:9717-9721[Abstract]
Windmill KF, Gaedigk A, Hall PM, Samaratunga H, Grant DM, McManus ME (2000) Localization of N-acetyltransferases NAT1 and NAT2 in human tissues. Toxicol Sci 54:19-29