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2 Induction in Rat Skeletal Muscle Tissue
Experimental and Molecular Pathology 69, 223–232 (2000) doi:10.1006/exmp.2000.2328, available online at http://www.idealibrary.com on
Ethanol-Mediated CYP1A1/2 Induction in Rat Skeletal Muscle Tissue
Cheryl Smith,* S. Craig Stamm,† Jack E. Riggs,‡ William Stauber,*,§ Veronica Harsh,† Peter M. Gannett,¶ Gerry Hobbs,| and Michael R. Miller†,**,1 *Department of Anatomy, †Department of Biochemistry,‡ Department of Neurology, §Department of Physiology, ¶ School of Pharmacy, and |Department of Community Medicine, West Virginia University Health Sciences Center, Morgantown, West Virginia 26506; and **CDC, NIOSH, Pathology, Physiology Research Branch, Morgantown, West Virginia 26505
Received June 13, 2000
The causes of non-trauma-mediated rhabdomyolysis are not well understood. It has been speculated that ethanol-associated rhabdomyolysis may be attributed to ethanol induction of skeletal muscle cytochrome P450(s), causing drugs such as acetaminophen or cocaine to be metabolized to myotoxic compounds. To examine this possibility, the hypothesis that feeding ethanol induces cytochrome P450 in skeletal muscle was tested. To this end, rats were fed an ethanol-containing diet and skeletal muscle tissue was assessed for induction of CYP2E1 and CYP1A1/2 by immunohistochemical procedures; liver was examined as a positive control tissue. Enzymatic assays and Western blot analyses were also performed on these tissues. In one feeding system, ethanol-containing diets induced CYP1A1/2 in soleus, plantaris, and diaphragm muscles, with immunohistochemical staining predominantly localized to capillaries surrounding myofibers. Antibodies to CYP2E1 did not react with skeletal muscle tissue from animals receiving a control or ethanol-containing diet. However, neither skeletal muscle CYP1A1/2 nor CYP2E1 was induced when ethanol diets were administered by a different feeding system. Ethanol consumption can induce some cytochrome P450 isoforms in skeletal muscle tissue; however, the mechanism of CYP induction is apparently complex and appears to involve factors in addition to ethanol, per se. q 2000 Academic Press Key Words: ethanol; CYP1A1/2 induction; skeletal muscle.
BACKGROUND
Rhabdomyolysis is a disease characterized by skeletal muscle necrosis, causing myoglobin to be released into the plasma, which can precipitate acute renal failure. Rhabdomyolysis can be caused by trauma (i.e., muscle crush injury), muscle ischemia, infections, and abuse of alcohol and drugs (Gabow et al., 1982; Ellinas and Rosner, 1992; Knochel, 1982); however, the mechanisms responsible for nontrauma-induced rhabdomyolysis are not well understood. Although there is an established association between alcohol consumption and rhabdomyolysis (Hed et al., 1955, 1962; Lafair and Myerson, 1968; Kahn and Meyer, 1970; Valaitis et al., 1960), the pathogenesis of alcoholic rhabdomyolysis is unknown. Haller and Drachman (1980) indicated that additional, unidentified factors appeared to potentiate the myotoxic effect of alcohol. In support of this idea, rhabdomyolysis has been reported to be associated with ethanol consumption and concomitant use of acetaminophen (Riggs et al., 1996), cocaine (Roth et al., 1988) or heroin (Richter et al., 1971). This observation led Riggs et al (1996, 1998), to postulate that ethanol might induce skeletal muscle cytochrome P450(s), thereby predisposing individuals to myotoxins, such as acetaminophen or cocaine, which could cause
1
To whom correspondence should be addressed at the Department of Biochemistry, P.O. Box 9142, West Virginia University Health Sciences Center, Morgantown, WV 26506–9142. Fax: (304) 293 6846. E-mail: [email protected].
0014-4800/00 $35.00 Copyright q 2000 by Academic Press All rights of reproduction in any form reserved.
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224 rhabdomyolysis following their metabolism by cytochrome P450 to reactive toxins. The idea that ethanol-induced cytochrome P450s might increase the sensitivity of a tissue to toxic compounds is well established in the liver. In the liver, ethanol consumption is known to predispose animals and humans to the toxic effects of various compounds (Lieber, 1988, 1997; Yang et al., 1992), including acetaminophen (Nelson, 1990), cocaine (Boelsterli and Goldin, 1991), heroin (Jover et al., 1992), vinyl chloride (Yang et al., 1992), dibromoethane (Guengerich, 1994), carbon tetrachloride, and other halogenated hydrocarbons (Stine and Brown, 1996). Increased sensitivity of the liver to many of these compounds is believed to be mediated by ethanol-induction of cytochrome P450 isozymes that metabolize the compounds to toxic intermediates. CYP2E1 is the prominent hepatic cytochrome P450 which is induced by ethanol (Koop et al., 1982; Perrot et al., 1991; Porter and Coon, 1991; Song, 1994). However, ethanol has also been reported to induce hepatic CYP1A in hamster (Ueng et al., 1993) and rat (Roberts et al., 1995); CYP2C7 in rat (Hakkak et al., 1996); and CYP2B1 and 3A1 in rat (DeWaziers et al., 1992; Roberts et al., 1995). Few studies have investigated skeletal muscle cytochrome P450s, and we know of no reports examining ethanol induction of skeletal muscle cytochrome P450 isoforms. Generally, cytochrome P450s are thought to be low in skeletal muscle tissue, relative to liver. Ethoxyresorufin-O-deethylase (EROD) and PROD activities, markers for CYP1A and CYP2B, were undetectable in microsomes from rat type I (slow twitch) and type II (fast twitch) muscles (Crosbie et al., 1997a,b); and cytochrome P450-mediated hexane metabolism in skeletal muscle microsomes was 0.3–2.0% that of hepatic microsomes. Immunocytochemical studies with antibodies to CYP2B1, CYP1A, and CYP2E1 in rat type I and II muscles were interpreted to indicate a low level of these isoforms throughout the muscle fibers, suggesting localization in sarcoplasmic reticulum (Crosbie et al., 1997a,b). Cytochrome P450 aromatase, which converts androgen to estrogen, has been detected in human (Matsumine et al., 1986), mouse (Gray et al., 1995), and monkey (Sholl et al., 1989) skeletal muscle. The goal of the present study was to test the hypothesis that feeding ethanol induces CYP2E1 and/or CYP1A in rat skeletal muscles. METHODS Chemicals. Unless indicated otherwise, all chemicals were purchased from Sigma Chemical Co. (St. Louis, MO) and were the highest quality available.
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Animals. Female Sprague–Dawley rats (Charles River, Wilmington, MA) were housed individually in polycarbonate cages containing aspen chip bedding, in the WVU Animal Quarters on a 12-h light/dark diurnal cycle. The WVU animal quarters is under the supervision of a licensed DVM; animals were humanely treated at all times and their general health was monitored daily. For the first week, animals received certified rodent chow blocks (Purina, Richmond, IN) and water ad libitum. Animals were then acclimated to Lieber/DeCarli control liquid diet (BioServe, Frenchtown, NJ) for 1 week. Control animals remained on the control liquid diet for 2 weeks, while ethanol-treated animals received an isocaloric liquid diet containing 2% ethanol for 2 days, 4% ethanol for the next 2 days, and then 6% ethanol for 10 additional days. In initial studies, liquid diets were supplied via glass water bottles containing a neoprene, black rubber stopper with inserted stainless steel feeding tube (Feeding System 1); later studies utilized all glass feeding tubes (Feeding System 2) supplied by BioServe. Daily food consumption for each animal was monitored; there was no significant difference between the amount of food consumed in the control or ethanol groups using Feeding System 1 or 2. Liver and plantaris, soleus, and diaphragm muscles were dissected from rats under deep anesthesia after exsanguination by cardiac puncture. Ethanol purification. After initial studies, ethanol was purified by distillation, to ensure that alteration of cytochrome P450 enzymes was not attributed to contaminants in the commercial ethanol. Magnesium chips (5 g) were added to ethanol [50 ml, 200 proof (Warner-Graham, Co., Cockeysville, MD)] in a round-bottom flask fitted with a reflux condenser. Iodine (0.1 g) was then added and the resulting mixture refluxed until the color (reddish brown) had been discharged. The balance of the ethanol (950 ml) was added and the mixture then heated at reflux for 1 h. The ethanol was distilled through a 20-cm column containing glass helices. A forerun was collected (100 ml) and discarded, and the next 700 ml was then collected and used in the feeding studies. Microsome preparation and enzyme assays. Microsomes were prepared from liver and skeletal muscle tissue as described (Lipscomb et al., 1998), with the following minor modifications. Tissues were diced into small pieces using a scalpel prior to homogenization in a glass tube/ Teflon pestle (Kontes, Vineland, NJ); each tissue received 15 homogenization strokes; only the surface of microsomal pellets was washed (33) prior to resuspension in the storage buffer (Lipscomb et al., 1998). Microsomal “beads” (,25 ml each) were prepared by resuspending pellets in 1.5 vol of microsomal storage buffer and adding them dropwise to
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liquid nitrogen. These beads were stable, stored in a 2808C freezer, and subsequently used for protein, enzyme, and Western blot assays. Microsomal protein concentration was determined with a Bio-Rad (Hercules, CA) protein assay using bovine serum albumin as a standard (Bradford, 1976). Total microsomal cytochrome P450 content was measured by the carbon monoxide difference spectra (Omura and Sato, 1964). CYP2E1 activity was measured using a p-nitrophenol (PNP) colorimetric assay (Wu and Cederbaum, 1996); results are expressed as pmol nitrocatechol/min/mg protein. CYP1A1/2 activity was measured by an EROD fluorometric assay (Burke et al., 1985; Rutten et al., 1992); results are expressed as pmol resorufin/min/mg protein. Western blot analysis. Rat liver and skeletal muscle microsomes, as well as molecular weight standards (Bio-Rad), were electrophoresed on 10% sodium dodecyl sulfate/polyacrylamide (SDS/PAGE) gels using a Hoeffer SE250 unit (San Francisco, CA) at 20 mA of current per gel. Proteins were electrophoretically transferred onto PVDF membranes (Bio-Rad). After the membranes were blocked with PBS containing 3% bovine serum albumin and 0.1% Triton X100, monoclonal primary mouse anti-rat antibodies directed against CYP4501A1/2 or against CYP4502E1 (PM16 or PM25, respectively, at a 1:1000 dilution, Oxford Biomedical, Rochester Hills, MI) were incubated with the membranes overnight on a rocker/shaker at 58C. After the PVDF membranes were washed three times with 0.1% Triton X-100/ PBS, they were incubated for 1 h at room temperature with goat anti-mouse IgGs conjugated with horse radish peroxidase (DC 02L at a 1:1000 dilution, Calbiochem, Cambridge, MA). The membranes were then washed three times again and developed using a TMB membrane peroxidase substrate system (KPL Laboratories, Gaithersburg, MD). Development of blots was terminated before reactivity reached a “saturating” level. The relative optical density of protein bands corresponding to CYP1A1/2 or CYP2E1 was determined by analyzing blots on an Optimas system in the WVU Image Analysis Facility. Reported relative optical density values take into account the average “gray” value for a specific band and the area of the band. Microsomes were serially diluted to ensure that relative optical densities determined on Western blots were in the linear range. Statistics. Enzyme assays were analyzed by Student’s t test to determine if differences between control and ethanol groups were statistically significant. Probability, P values, used in t tests is indicated in table legends. Western blots were analyzed using the ANOVA method on logarithmically transformed data.
Immunohistochemical analysis. Immediately after removing liver and skeletal muscle tissue from rats, the tissues were cut, mounted on corks with OCT compound, and frozen in isopentane cooled by liquid nitrogen. The frozen tissues were oriented and sectioned at 2208C. The frozen sections (8–12 mm) were collected on glass slides, air dried, and placed in slide boxes at 2808C until use. The same primary monoclonal antibodies directed against CYP1A1/2 and CYP2E1 used in Western blotting (described above) were used for immunohistochemical analysis. Slides with tissues were rinsed with PBS, then incubated with a 1:40–1:60 dilution of primary antibodies for 30 min at room temperature, washed 5 min in PBS, and incubated with a 1:20 dilution of fluorescein-labeled sheep anti-mouse IgG (Sigma Chemical ) for 30 min at room temperature. After a final wash in PBS, glass coverslips were applied over the tissue sections with 50% glycerine–50% PBS and they were viewed on a Leitz DAS fluorescence microscope.
RESULTS
Induction of hepatic CYP2E1 and CYP1A1/2. As positive controls, experiments were conducted to establish that feeding ethanol-containing diets resulted in induction of hepatic CYP2E1 and CYP1A1/2. Table 1 demonstrates that when rats were administered ethanol for 2 weeks via Feeding System 1, total CYP450 was elevated ,1.5-fold above control levels. PNP activity was elevated ,2.6-fold by the ethanol diet, as expected (Koop et al., 1982; Perrot et al., 1989), and EROD activity was elevated ,1.8-fold following ethanol consumption, consistent with a previous report (Roberts et al., 1995). Ethanol induction of PNP and EROD activity is reported to be due to increased protein levels of CYP2E1 and CYP1A, respectively (Koop and Tierney, 1990; Koop et al., 1982; Perrot et al., 1989; Roberts et al., 1995; Ueng et al., 1993). Western blot analysis indicated that consuming ethanol induced immunoreactive CYP2E1 (Fig. 1A) and CYP1A1/2 (Fig. 1B). Furthermore, the Western blots (Fig. 1) establish that the antibodies for CYP2E1 or CYP1A1/2 react as expected, with proteins of molecular weight ,52 or ,53–58 kDal, respectively. Under the SDS/page condition used, the CYP1A1 and CYP1A2 isoforms were not well resolved. These results also demonstrate that the antibodies are highly specific for their specific antigens and can be used with confidence in immunohistochemical studies. Liver samples from the same rats used to derive data in Table 1 and Fig. 1 were used for immunohistochemical
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TABLE 1 Effect of Feeding Ethanol via System I on Hepatic Total CYP, PNP, and EROD Sample
Total CYP450
PNP
EROD
C1 C2 C3 C4 Mean
0.44 0.56 0.47 0.43 0.48 6 0.06
0.58 0.60 0.73 0.53 0.61 6 0.08
211 169 413 129 230 6 126
E1 E2 E3 E4 E5 Mean
0.62 0.62 0.76 0.85 0.75 0.72 6 0.10a
2.17 1.58 1.24 1.43 1.45 1.58 6 0.35a
272 435 475 559 333 415 6 114b
Note. Four rats were fed a liquid diet that did not contain ethanol (C1–4), and five rats were fed an isocaloric liquid diet that contained 6% ethanol (E1–5), as described, using Feeding System 1. Liver microsomes were then prepared and assayed for total cytochrome P450 (nmol/mg protein), PNP activity (pmol nitrocatechol/min/mg protein), and EROD activity (pmol resorufin/min/mg protein), as described under Methods. Measurements for individual rats are shown above; the mean values 6 standard deviation for the control and ethanol groups are presented. a Significantly different from control at P , 0.05. b Not significantly different from control at P , 0.05, but different from control at P , 0.06.
analysis. Ethanol feeding via Feeding System 1 induced additional immunohistochemical reactivity of CYP2E1 (Figs. 2C and 2D) and CYP1A1/2 (Figs. 2A and 2B) in liver, consistent with enzymatic (Table 1) and Western blot (Fig. 1) results. Induction of skeletal muscle CYP2E1 and CYP1A1/2. Various skeletal muscle tissues were assessed for the presence of CYP2E1 and for CYP1A1/2 using immunohistochemistry. CYP2E1 was not detected in soleus (Fig. 3D), plantaris, or diaphragm (not shown) of animals fed the control liquid diet or the diet containing ethanol. CYP1A1/2 was barely detectable in soleus muscles from animals on the control diet (Fig. 3C). However, marked CYP1A1/2 immunoreactivity was consistently apparent in soleus (Fig. 3A), plantaris (Fig. 3B), and diaphragm (not shown) of animals fed the diet containing ethanol. In all skeletal muscle tissues examined, the CYP1A1/2 immunoreactivity appeared to be localized to capillaries surrounding myofibers. Purified ethanol. Feeding commercial ethanol induced both CYP2E1 and CYP1A1/2 in liver (Table 1 and Figs. 1 and 2), confirming other reports (Koop and Tierney, 1990; Koop et al., 1982; Perrot et al., 1989; Roberts et al., 1995; Ueng et al., 1993); however, the finding that feeding ethanol
induced CYP1A1/2, but not CYP2E1, in skeletal muscle capillary cells was unexpected. To ensure that CYP1A1/2 induction in skeletal muscle was not due to a contaminant in the commercial ethanol added to the liquid diet, ethanol was highly purified immediately before use by distillation, as described, and used in all subsequent experiments. Feeding highly purified ethanol to rats using Feeding System 1 resulted in the same immunological staining pattern in the liver, soleus, plantaris, and diaphragm (not shown) as obtained using commercial ethanol that was not distilled (Figs. 2 and 3 and described above). In addition, using highly purified ethanol also resulted in induction of hepatic PNP and EROD activities (not shown) similar to that shown in Table 1. Microsomes prepared from skeletal muscle (soleus, plantaris, diaphragm) of rats fed liquid diets (control or containing highly purified ethanol) via Feeding System 1 were assayed for total cytochrome P450 and EROD activity. Total CYP450 was undetectable in microsomes from all tissues examined, and EROD activity was only detectable (#46 pmol resorufin/min/mg protein) in microsomes from diaphragm of animals fed diets containing ethanol. Detectable EROD activity in diaphragm microsomes from animals fed ethanol is consistent with localization of induced CYP1A1/ 2 in the capillaries of skeletal muscle (Fig. 3) and with the higher vascular content of this tissue, relative to soleus or plantaris. Ethanol induction of CYP1A1/2 is dependent on the feeding system. Although the results indicating that ethanol induces CYP1A1/2 and CYP2E1 in liver (Table 1 and Fig. 1) are consistent with those of other reports (Koop et al., 1982; Perrot et al., 1989; Roberts et al., 1995; Ueng et al., 1993), it was important to demonstrate that CYP1A1/2 induction by ethanol in skeletal muscle (in the absence of CYP2E1 induction) was not due to components of Feeding System 1 (rubber stopper, stainless steel tubing, glass bottle). To this end, all-glass feeding systems (Feeding System 2) were used to deliver liquid diets to rats for 2 weeks. Immunohistochemical analysis of skeletal muscle consistently failed to show CYP1A1/2 or CYP2E1 reactivity in rats receiving either control or ethanol-containing liquid diets (not shown). Microsomes were prepared from livers of these animals and subjected to EROD and Western blotting analysis. Table 2 shows that feeding ethanol to rats by Feeding System 2 did not induce hepatic EROD activity. Additional studies confirmed that administering ethanol in Feeding System 2 did not induce EROD activity in rat liver or immunoreactive CYP1A1/2 in the soleus, plantaris, or diaphragm muscles (not shown). A direct comparison between administering
CYP1A1/2 IN RAT SKELETAL MUSCLE
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FIG. 1. The same liver microsomes described in Table 1, which were prepared from rats fed control (C1–4) or ethanol-containing (E1–5) diets, were subjected to SDS–polyacrylamide gel electrophoresis (100 mg each) and Western blot analysis with primary antibodies directed to CYP2E1 (A) or CYP1A1/2 (B). Lane 1, E1 (16.5); lane 2, C1, (4.2); lane 3, E2 (42.7); lane 4, C2 (2.7); lane 5, E3 (18.6); lane 6, C3 (29.4); lane 7, E4 (67.7); lane 8, C4 (2.5); lane 9, E5 (6.6). Numbers in parentheses represent relative optical densities determined with an Optimas (Bothell, WA) image analysis system for blots depicted in Fig. 1B.
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FIG. 2. Immunohistochemical analysis of CYP1A1/2 and CYP2E1 in rat liver. Rats received a control or ethanol-containing liquid diet as described. Liver tissue was processed and reacted with primary antibodies directed to CYP1A1/2 or to CYP2E1, followed by FITC-conjugated secondary antibodies. Samples were observed under a fluorescent microscope; (A) control diet, anti-CYP1A1/2; (B) ethanol diet, anti-CYP1A1/ 2; (C) control diet, anti-CYP2E1; (D) ethanol diet, anti-CYP2E1. Photographs were taken using an autoexposure setting; this setting provides optimum visualization of FITC staining, but minimizes differences between the fluorescent intensities of different samples. Note that in the samples from rats on an ethanol diet, staining of CYP1A1/2 (B) and CYP2E1 (D) covers more of the liver area than is observed when rats received the control liquid diet (A and C, respectively). Bar in D, 100 mm.
ethanol to rats in Feeding Systems 1 and 2 was then undertaken to assess the effects on induction of hepatic CYP1A1/ 2 and CYP2E1. Western blot analysis of hepatic microsomes from rats fed liquid diets by Feeding System 1 or 2 were probed with CYP2E1-specific antibodies and with CYP1A1/ 2-specific antibodies; the relative immunoreactivity of each of these CYP450 isoforms was determined by image analysis as described. Table 3 demonstrates that CYP2E1 was induced ,two-fold when ethanol was administered by both Feeding Systems 1 and 2. In contrast, CYP1A1/2 was only induced in ethanol-fed animals when ethanol was administered by Feeding System 1.
DISCUSSION
The objective of this study was to test the hypothesis that ethanol consumption induced skeletal muscle tissue CYP2E1 and/or CYP1A1/2. This study focused on CYP1A1/ 2 and CYP2E1 because Riggs et al. (1996) postulated that ethanol consumption may have induced skeletal muscle cytochrome P450s, which metabolized acetaminophen to a myotoxin, precipitating rhabdomyolysis. CYP2E1 and CYP1A1 are reported to be ethanol-inducible and to metabolize acetaminophen to the toxic quinone imine (Haravisonn and Guengerich, 1988; Patten et al., 1993; Raucy et al.,
CYP1A1/2 IN RAT SKELETAL MUSCLE
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FIG. 3. Immunohistochemical analysis of CYP1A1/2 and CYP2E1 in rat skeletal muscle. Rats received control or ethanol-containing liquid diets as described. Soleus and plantaris muscles were processed and reacted with primary antibodies to CYP1A1/2 or to CYP2E1, as described; (A) ethanol diet, soleus reacted with anti-CYP1A1/2; (B) ethanol diet, plantaris reacted with anti-CYP1A1/2; (C) control diet, soleus reacted with anti-CYP1A1/2; (D) ethanol diet, soleus reacted with anti-CYP2E1. Note that very low levels of CYP1A1/2 staining were observed in some capillaries surrounding muscle bundles in tissue from animals on the control diet (C); intense CYP1A1/2 staining was observed in capillaries surrounding muscle tissue from animals receiving the ethanol-containing diet (A and B); CYP2E1 staining was not observed in any muscle tissue from animals receiving the control or ethanol-containing (D) liquid diet. Bar in D, 100 mm.
1989). Our studies demonstrate for the first time that consuming a 6% ethanol diet may mediate induction of CYP1A1/ 2 in rat skeletal muscle tissue. Furthermore, the induced CYP1A1/2 is predominantly localized in capillaries surrounding type I and II myofibers (Fig. 3). However, the ethanol-mediated induction of CYP1A1/2 in skeletal muscle and in liver is complex; purified ethanol administered by the all-glass Feeding System 2 did not result in CYP1A1/2 induction (Tables 2 and 3). Induction of CYP1A1/2 in skeletal muscle by the ethanol-containing diet administered via Feeding System 1 may be due to ethanol solubilizing a compound from rubber stoppers or from the stainless steel tubing in this feeding system. Alternatively, because rats chew the rubber stoppers, it is possible that a CYP1A1/2 inducer is produced in the stomach when ethanol
is present; or the combination of ethanol, a component in the liquid diet, and rubber stopper/stainless steel tubing may generate a CYP1A1/2-inducing compound. The stoppers in Feeding System 1 were composed of : poly 2-chlorobutadiene 1, 3 (Neoprene WRT) (62.50%); N-phenyl-a-naphthylamine (Neozone A) (1.25%); distilled stearic acid (Wecoline 300) (0.31%); magnesium oxide (Maglite Y) (1.25%); 2mercaptomidazoline (NA-22) (0.31%); zinc oxide surface treated with propionic acid (Protox-166) (3.13%); and Intermediate superabrasion oil furnace black (ISAF or Carbon Black) (31.25%). The Carbon Black contains adsorbed polyaromatic hydrocarbons (,0.1% of the carbon black), potent inducers of CYP1A1/2. However, if rubber stoppers are the source of a CYP1A1/2-inducing compound, apparently ethanol is required to render the compound “biologically
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used to “reseal” opened alcoholic beverages (wine, beer, liquor) contain rubber components; and some ethanol-containing pharmaceuticals are placed in vials containing rubber stoppers or are delivered through syringes which use black rubber plungers. Data in this study also demonstrate that purified ethanol administered by an all-glass feeding system (Feeding System 2) did not significantly induce hepatic EROD activity (Tables 2 and 3). This contradicts two reports indicating that ethanol administered via a Lieber/DeCarli liquid diet induces CYP1A1 in rats (Roberts et al., 1995) and hamsters (Ueng et al., 1993). Potential explanations for these discrepancies include the facts that rats and hamsters were fed an ethanol diet for 3 weeks (Roberts et al., 1995; Ueng et al., 1993), in contrast to the 2-week period used in this study; highly purified ethanol was not used in the two previous reports (Roberts et al., 1995; Ueng et al., 1993); the feeding system used to administer ethanol in a liquid diet to hamsters was not described (Ueng et al., 1993) and may have contained an ethanol-extractable CYP1A1/2-inducer, like Feeding System 1, and a contaminant may have been responsible for inducing CYP1A1; and studies herein utilized female rats, while the gender of rats in a previous study (Roberts et al., 1995) was not specified, and male hamsters were used (Ueng et al., 1993). Gender differences in ethanol stimulation of
FIG. 4. Means of logarithmically transformed data from Western blot analysis of CYP1A1/2 (A) and CYP2E1 (B) by diet (control or ethanol) and Feeding System 1(m) and Feeding System 2 (v); means 6 standard error.
available"; rats consuming the control diet chew and ingest rubber stoppers but do not exhibit induced CYP1A1/2. In addition, preliminary studies exposed rubber stoppers in Feeding System 1 to water or water containing 6% ethanol; the solutions were then analyzed by GC/MS and by HPLC/ UV. Polyaromatic hydrocarbons were not detected in either 6% ethanol or water solutions. The results of this study are consistent with the conclusion that additional factors may potentiate the myotoxic effects of ethanol (Haller and Drachman, 1980), and additional studies are in progress to identify the mechanism by which ethanol in Feeding System 1 induces CYP1A1/2. In addition, it will be important to determine if ethanolmediated CYP1A1/2 induction, exhibited by Feeding System 1, can occur in humans using ethanol-containing beverages or pharmaceutical drugs. For instance, some devices
TABLE 2 Effect of Feeding Ethanol via System 2 on Hepatic EROD Activity
Sample
EROD (pmol resorufin/min/mg)
C1 C2 C3 C4 Mean
180 138 142 151 153 6 19
E1 E2 E3 E4 ES E6 Mean
105 169 109 138 165 99 118 6 28a
Note. Four rats were fed a liquid diet that did not contain ethanol (C1–4), and six rats were fed an isocaloric liquid diet that contained ethanol (E1–6) for 2 weeks, using Feeding System 2. Liver microsomes were then prepared and assayed for total EROD activity, as described under Methods. Measurements for individual rats are shown above; the mean values 6 standard deviation for the control and ethanol groups are presented. a Not statistically different from control at P , 0.l0.
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CYP1A1/2 IN RAT SKELETAL MUSCLE TABLE 3 Western Blot Analysis of Hepatic Microsomes from Rats Fed Liquid Diets with Feeding System I or 2 Relative immunoreactivity Feeding System System 2 C1 C2 E1 E2 E3 E4 System 1 C3 E5 E6
CYP1A1/2
CYP2E1
2.6 2.8 2.0 3.2 2.2 2.9
16.1 16.9 31.7 31.4 28.1 36.7
4.2 43.3 32.3
20.0 38.5 38.1
Note. Rats received a control liquid diet (C1–3) or a liquid diet containing ethanol (E1–6) for 2 weeks via Feeding System 1 or 2 (described under Methods). Liver microsomes were then prepared and 100 mg of each sample was subjected to Western blot analysis and probed with primary antibodies specific for CYP1A1/2 or CYP2E1, as described. Relative optical densities of protein bands corresponding to CYP1A1/2 or CYP2E1 were then analyzed by Optimas image analysis, which is depicted above. For CYP1A1/ 2, analysis demonstrated a significant interaction between Feeding System and ethanol containing diet (P , 0.0006) (see Fig. 4). For CYP2E1 there was a small but significant (P 0.331) effect due to Feeding System and a larger (P 0.0002) effect due to ethanol-containing diet, and there was no evidence of an interaction between them (see Fig. 4B).
hepatic benzo[a]pyrene hydroxylase activity have been noted (Lieber et al., 1979); in female rats, feeding ethanol stimulated microsomal benzo[a]pyrene hydroxylase activity, but in male rats feeding ethanol did not stimulate microsomal benzo[a]pyrene hydroxylase. Additional experiments will be required to clarify why an ethanol-containing diet induced hepatic CYP1A1 in several studies, but highly purified ethanol in all-glass feeders did not induce hepatic CYP1A1/2 in this study.
ACKNOWLEDGMENTS
This work was supported in part by a West Virginia University School of Medicine Research Development grant, by NIH/NCI (CA45131-09 to M.R.M.), by ACS (RPG-57-066-01-VM to M.R.M.), and by NIOSH/CDC (R01 OHAR02918 to W.T.S.).
REFERENCES
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232 Khan, L. B., and Myer, J. S. (1970) Acute myopathy in chronic alcoholism: a study of 22 autopsy cases, with ultrastructural observations. Am. J. Clin. Pathol. 53, 516–530. Knochel, J. P. (1982) Rhabdomyolysis and myoglobinuria. Annu. Rev. Med. 33, 435–443. Koop, D. R., and Tierney, D. J. (1990). Multiple mechanisms in the regulation of ethanol-inducible cytochrome P-450IIE1. Bioessays 12, 429–435. Koop, D. R., Morgan, E. T., Tarr, G. E., and Coon, M. J. (1982) Purification and characterization of a unique isozyme of cytochrome P-450 from liver microsomes of ethanol-treated rabbits. J. Biol. Chem. 257, 8472–8480. Lafair, J. S., and Myerson, R. M. (1968) Alcoholic myopathy, with special reference to the significance of creatine phosphokinase. Arch. Intern. Med. 122, 417–422. Lieber, C. S. (1988) Biochemical and molecular basis of alcoholinduced injury to liver and other tissues. N. Engl. J. Med. 319, 1639– 1650. Lieber, C. S. (1997) Cytochrome P-4502E1: Its physiological and pathological role. Physiol. Rev. 77, 517–544. Lieber, C. S., Seitz, H. K., Garro, A. J., and Worner, T. M. (1979) Alcohol-related diseases and carcinogenesis. Cancer Res. 39, 2863– 2886. Lipscomb, J. C., Confer, P. E., Miller, M. R., Stamm, S. C., Snawder, J. E., and Bandiera, S. T. (1998) Metabolism of trichloroethylene and chloral hydrate by the japanese medaka (Oryzias latipes) in vitro. Environ. Toxicol. Chem. 17, 325–332. Matsumine, H., Hirato, K., Yanaihara, T., Tamada, T., and Yoshida, M. (1986) Aromatization by skeletal muscle. J. Clin. Endocrinol. Metab. 63, 717–720. Nelson, S. D. (1990) Molecular mechanisms of the hepatotoxicity caused by acetaminophen. Semin. Liver Dis. 10, 267–278. Omura, I., and Sato, R. (1964) The carbon monoxide-binding pigment of liver microsomes. I. Evidence for its hemoprotein nature. J. Biol. Chem. 239, 2370–2378. Patten, C. J., Thomas, P. E., Guy, R. L., Lee, J., Gonzalez, H., Guengerich, F. P., and Yang, C. S. (1993). Cytochrome P450 enzymes involved in acetaminophen activation by rat and human liver microsomes and their kinetics. Chem. Res. Toxicol. 6, 511–518. Perrot, N., Chesne, C., DeWaziers, I., Conner, J., Beaune, P. H., and Guillouzo, A. (1991). Effects of ethanol and clofibrate on expression of cytochrome P450 enzymes and epoxide hydrolase in cultures and cocultures of rat hepatocytes. Eur. J. Biochem. 200, 255–261. Perrot, N., Nalpas, B., Yang, C. S., and Beaune, P. (1989) Modulation of cytochrome P450 isozymes in human liver, by ethanol and drug intake. Eur. J. Clin. 19, 549–555. Porter, T. D., and Coon, M. J. (1991) Cytochrome P-450. Multiplicity
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of isoforms, substrates, and catalytic and regulatory mechanisms. J. Biol. Chem. 266, 13469–13472. Raucy, J. L., Lasker, J. M., Lieber, C. S., and Black, M. (1989) Acetaminophen activation by human liver cytochromes P450 2E1 and P450 1A2. Arch. Biochem. Biophys. 271, 270–283. Richer, R. W., Challenor, Y. B., Pearson, J., Kagen, L. J., Hamilton, L. L., and Ramsey, W. H. (1971) Acute myoglobinuria associated with heroin addiction. J. Am. Med. Assoc. 216, 1172–1176. Riggs, J. E. (1998) Alcohol-associated rhabdomyolysis: Ethanol induction of cytochrome P450 may potentiate myotoxicity. Clin. Neuropharmacol. 21, 363–364. Riggs, J. E., Schochet, S. S., and Parmar, J. P. (1996) Rhabdomyolysis with acute renal failure and disseminated intravascular coagulation: Association with acetaminophen and ethanol. Military Med. 161, 708–709. Roberts, B. J., Shoaf, S. E., and Song, B. J. (1995) Rapid changes in cytochrome P4502E1 (CYP2E1) activity and other P450 isozymes following ethanol withdrawal in rats. Biochem. Pharmacol. 49, 1665–1673. Roth, D., Alarcon, F. J., Fernandez, J. A., Preston, R. A., and Bourgoignie, J.J. (1988). Acute rahbdomyolysis associated with cocaine intoxication. N. Engl. J. Med. 319, 673–677. Rutten, A. A. J. H. L., Falke, H. E., Catsburg, J. F., Wortelboer, H. M., Blaauboer, B. J., Doom, L., vanLeeuwen, F. X. R., Theelen, R., and Rietjens, I. M. C. M. (1992) Interlaboratory comparison of microsomal ethoxyresorufin and pentoxyresorufin O-dealkylation determinations: Standardization of assay conditions. Arch. Toxicol. 66, 237–244. Sholl, S. A., Goy, R. W., and Kim, K. L. (1989) 5 alpha-reductase, aromatase, and androgen receptor levels in the monkey brain during fetal development. Endocrinology. 124, 627–634. Song, B. J. (1994) Gene structure and multiple regulations of the ethanol-inducible cytochrome P-4502E1 (CYP2EI) subfamily. In: “Alcohol and Hormones” (R. R. Watson, Ed.) pp. 177–192. Humana Press, Totowa. Stine, K. E., and Brown, T. M. (1996) “Principles of Toxicology,” pp. 149–157. Lewis, CRC Press, Boca Raton. Ueng, I-H., Ueng, Y-F., Tsai, J-N., Chao, I-C., Chen, T-L., Park, S. S., Iwasaki, M., and Guengerich, F. P. (1993). Induction and inhibition of cytochrome P-450-dependent monooxygenases in hamster tissues by ethanol. Toxicology 81, 145–154. Valaitis, J., Pilz, C. G., Oliner, H., and Chomet, B. (1960) Myoglobinuria, myoglobinuric nephrosis and alcoholism. Arch. Pathol. 70, 195–202. Wu, D., and Cederbaum, A. L. (1996) Ethanol cytotoxicity to a transfected HepG2 cell line expressing human cytochrome P4502E1. J. Biol. Chem. 271, 23914–23919. Yang, C. S., Brady, J. F., and Hong, J-Y. (1992) Dietary effects on cytochmomes P450, xenobiotic metabolism, and toxicity. FASEB. J. 6, 737–744.
Ethanol-Mediated CYP1A1/2 Induction in Rat Skeletal Muscle Tissue
Cheryl Smith,* S. Craig Stamm,† Jack E. Riggs,‡ William Stauber,*,§ Veronica Harsh,† Peter M. Gannett,¶ Gerry Hobbs,| and Michael R. Miller†,**,1 *Department of Anatomy, †Department of Biochemistry,‡ Department of Neurology, §Department of Physiology, ¶ School of Pharmacy, and |Department of Community Medicine, West Virginia University Health Sciences Center, Morgantown, West Virginia 26506; and **CDC, NIOSH, Pathology, Physiology Research Branch, Morgantown, West Virginia 26505
Received June 13, 2000
The causes of non-trauma-mediated rhabdomyolysis are not well understood. It has been speculated that ethanol-associated rhabdomyolysis may be attributed to ethanol induction of skeletal muscle cytochrome P450(s), causing drugs such as acetaminophen or cocaine to be metabolized to myotoxic compounds. To examine this possibility, the hypothesis that feeding ethanol induces cytochrome P450 in skeletal muscle was tested. To this end, rats were fed an ethanol-containing diet and skeletal muscle tissue was assessed for induction of CYP2E1 and CYP1A1/2 by immunohistochemical procedures; liver was examined as a positive control tissue. Enzymatic assays and Western blot analyses were also performed on these tissues. In one feeding system, ethanol-containing diets induced CYP1A1/2 in soleus, plantaris, and diaphragm muscles, with immunohistochemical staining predominantly localized to capillaries surrounding myofibers. Antibodies to CYP2E1 did not react with skeletal muscle tissue from animals receiving a control or ethanol-containing diet. However, neither skeletal muscle CYP1A1/2 nor CYP2E1 was induced when ethanol diets were administered by a different feeding system. Ethanol consumption can induce some cytochrome P450 isoforms in skeletal muscle tissue; however, the mechanism of CYP induction is apparently complex and appears to involve factors in addition to ethanol, per se. q 2000 Academic Press Key Words: ethanol; CYP1A1/2 induction; skeletal muscle.
BACKGROUND
Rhabdomyolysis is a disease characterized by skeletal muscle necrosis, causing myoglobin to be released into the plasma, which can precipitate acute renal failure. Rhabdomyolysis can be caused by trauma (i.e., muscle crush injury), muscle ischemia, infections, and abuse of alcohol and drugs (Gabow et al., 1982; Ellinas and Rosner, 1992; Knochel, 1982); however, the mechanisms responsible for nontrauma-induced rhabdomyolysis are not well understood. Although there is an established association between alcohol consumption and rhabdomyolysis (Hed et al., 1955, 1962; Lafair and Myerson, 1968; Kahn and Meyer, 1970; Valaitis et al., 1960), the pathogenesis of alcoholic rhabdomyolysis is unknown. Haller and Drachman (1980) indicated that additional, unidentified factors appeared to potentiate the myotoxic effect of alcohol. In support of this idea, rhabdomyolysis has been reported to be associated with ethanol consumption and concomitant use of acetaminophen (Riggs et al., 1996), cocaine (Roth et al., 1988) or heroin (Richter et al., 1971). This observation led Riggs et al (1996, 1998), to postulate that ethanol might induce skeletal muscle cytochrome P450(s), thereby predisposing individuals to myotoxins, such as acetaminophen or cocaine, which could cause
1
To whom correspondence should be addressed at the Department of Biochemistry, P.O. Box 9142, West Virginia University Health Sciences Center, Morgantown, WV 26506–9142. Fax: (304) 293 6846. E-mail: [email protected].
0014-4800/00 $35.00 Copyright q 2000 by Academic Press All rights of reproduction in any form reserved.
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224 rhabdomyolysis following their metabolism by cytochrome P450 to reactive toxins. The idea that ethanol-induced cytochrome P450s might increase the sensitivity of a tissue to toxic compounds is well established in the liver. In the liver, ethanol consumption is known to predispose animals and humans to the toxic effects of various compounds (Lieber, 1988, 1997; Yang et al., 1992), including acetaminophen (Nelson, 1990), cocaine (Boelsterli and Goldin, 1991), heroin (Jover et al., 1992), vinyl chloride (Yang et al., 1992), dibromoethane (Guengerich, 1994), carbon tetrachloride, and other halogenated hydrocarbons (Stine and Brown, 1996). Increased sensitivity of the liver to many of these compounds is believed to be mediated by ethanol-induction of cytochrome P450 isozymes that metabolize the compounds to toxic intermediates. CYP2E1 is the prominent hepatic cytochrome P450 which is induced by ethanol (Koop et al., 1982; Perrot et al., 1991; Porter and Coon, 1991; Song, 1994). However, ethanol has also been reported to induce hepatic CYP1A in hamster (Ueng et al., 1993) and rat (Roberts et al., 1995); CYP2C7 in rat (Hakkak et al., 1996); and CYP2B1 and 3A1 in rat (DeWaziers et al., 1992; Roberts et al., 1995). Few studies have investigated skeletal muscle cytochrome P450s, and we know of no reports examining ethanol induction of skeletal muscle cytochrome P450 isoforms. Generally, cytochrome P450s are thought to be low in skeletal muscle tissue, relative to liver. Ethoxyresorufin-O-deethylase (EROD) and PROD activities, markers for CYP1A and CYP2B, were undetectable in microsomes from rat type I (slow twitch) and type II (fast twitch) muscles (Crosbie et al., 1997a,b); and cytochrome P450-mediated hexane metabolism in skeletal muscle microsomes was 0.3–2.0% that of hepatic microsomes. Immunocytochemical studies with antibodies to CYP2B1, CYP1A, and CYP2E1 in rat type I and II muscles were interpreted to indicate a low level of these isoforms throughout the muscle fibers, suggesting localization in sarcoplasmic reticulum (Crosbie et al., 1997a,b). Cytochrome P450 aromatase, which converts androgen to estrogen, has been detected in human (Matsumine et al., 1986), mouse (Gray et al., 1995), and monkey (Sholl et al., 1989) skeletal muscle. The goal of the present study was to test the hypothesis that feeding ethanol induces CYP2E1 and/or CYP1A in rat skeletal muscles. METHODS Chemicals. Unless indicated otherwise, all chemicals were purchased from Sigma Chemical Co. (St. Louis, MO) and were the highest quality available.
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Animals. Female Sprague–Dawley rats (Charles River, Wilmington, MA) were housed individually in polycarbonate cages containing aspen chip bedding, in the WVU Animal Quarters on a 12-h light/dark diurnal cycle. The WVU animal quarters is under the supervision of a licensed DVM; animals were humanely treated at all times and their general health was monitored daily. For the first week, animals received certified rodent chow blocks (Purina, Richmond, IN) and water ad libitum. Animals were then acclimated to Lieber/DeCarli control liquid diet (BioServe, Frenchtown, NJ) for 1 week. Control animals remained on the control liquid diet for 2 weeks, while ethanol-treated animals received an isocaloric liquid diet containing 2% ethanol for 2 days, 4% ethanol for the next 2 days, and then 6% ethanol for 10 additional days. In initial studies, liquid diets were supplied via glass water bottles containing a neoprene, black rubber stopper with inserted stainless steel feeding tube (Feeding System 1); later studies utilized all glass feeding tubes (Feeding System 2) supplied by BioServe. Daily food consumption for each animal was monitored; there was no significant difference between the amount of food consumed in the control or ethanol groups using Feeding System 1 or 2. Liver and plantaris, soleus, and diaphragm muscles were dissected from rats under deep anesthesia after exsanguination by cardiac puncture. Ethanol purification. After initial studies, ethanol was purified by distillation, to ensure that alteration of cytochrome P450 enzymes was not attributed to contaminants in the commercial ethanol. Magnesium chips (5 g) were added to ethanol [50 ml, 200 proof (Warner-Graham, Co., Cockeysville, MD)] in a round-bottom flask fitted with a reflux condenser. Iodine (0.1 g) was then added and the resulting mixture refluxed until the color (reddish brown) had been discharged. The balance of the ethanol (950 ml) was added and the mixture then heated at reflux for 1 h. The ethanol was distilled through a 20-cm column containing glass helices. A forerun was collected (100 ml) and discarded, and the next 700 ml was then collected and used in the feeding studies. Microsome preparation and enzyme assays. Microsomes were prepared from liver and skeletal muscle tissue as described (Lipscomb et al., 1998), with the following minor modifications. Tissues were diced into small pieces using a scalpel prior to homogenization in a glass tube/ Teflon pestle (Kontes, Vineland, NJ); each tissue received 15 homogenization strokes; only the surface of microsomal pellets was washed (33) prior to resuspension in the storage buffer (Lipscomb et al., 1998). Microsomal “beads” (,25 ml each) were prepared by resuspending pellets in 1.5 vol of microsomal storage buffer and adding them dropwise to
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liquid nitrogen. These beads were stable, stored in a 2808C freezer, and subsequently used for protein, enzyme, and Western blot assays. Microsomal protein concentration was determined with a Bio-Rad (Hercules, CA) protein assay using bovine serum albumin as a standard (Bradford, 1976). Total microsomal cytochrome P450 content was measured by the carbon monoxide difference spectra (Omura and Sato, 1964). CYP2E1 activity was measured using a p-nitrophenol (PNP) colorimetric assay (Wu and Cederbaum, 1996); results are expressed as pmol nitrocatechol/min/mg protein. CYP1A1/2 activity was measured by an EROD fluorometric assay (Burke et al., 1985; Rutten et al., 1992); results are expressed as pmol resorufin/min/mg protein. Western blot analysis. Rat liver and skeletal muscle microsomes, as well as molecular weight standards (Bio-Rad), were electrophoresed on 10% sodium dodecyl sulfate/polyacrylamide (SDS/PAGE) gels using a Hoeffer SE250 unit (San Francisco, CA) at 20 mA of current per gel. Proteins were electrophoretically transferred onto PVDF membranes (Bio-Rad). After the membranes were blocked with PBS containing 3% bovine serum albumin and 0.1% Triton X100, monoclonal primary mouse anti-rat antibodies directed against CYP4501A1/2 or against CYP4502E1 (PM16 or PM25, respectively, at a 1:1000 dilution, Oxford Biomedical, Rochester Hills, MI) were incubated with the membranes overnight on a rocker/shaker at 58C. After the PVDF membranes were washed three times with 0.1% Triton X-100/ PBS, they were incubated for 1 h at room temperature with goat anti-mouse IgGs conjugated with horse radish peroxidase (DC 02L at a 1:1000 dilution, Calbiochem, Cambridge, MA). The membranes were then washed three times again and developed using a TMB membrane peroxidase substrate system (KPL Laboratories, Gaithersburg, MD). Development of blots was terminated before reactivity reached a “saturating” level. The relative optical density of protein bands corresponding to CYP1A1/2 or CYP2E1 was determined by analyzing blots on an Optimas system in the WVU Image Analysis Facility. Reported relative optical density values take into account the average “gray” value for a specific band and the area of the band. Microsomes were serially diluted to ensure that relative optical densities determined on Western blots were in the linear range. Statistics. Enzyme assays were analyzed by Student’s t test to determine if differences between control and ethanol groups were statistically significant. Probability, P values, used in t tests is indicated in table legends. Western blots were analyzed using the ANOVA method on logarithmically transformed data.
Immunohistochemical analysis. Immediately after removing liver and skeletal muscle tissue from rats, the tissues were cut, mounted on corks with OCT compound, and frozen in isopentane cooled by liquid nitrogen. The frozen tissues were oriented and sectioned at 2208C. The frozen sections (8–12 mm) were collected on glass slides, air dried, and placed in slide boxes at 2808C until use. The same primary monoclonal antibodies directed against CYP1A1/2 and CYP2E1 used in Western blotting (described above) were used for immunohistochemical analysis. Slides with tissues were rinsed with PBS, then incubated with a 1:40–1:60 dilution of primary antibodies for 30 min at room temperature, washed 5 min in PBS, and incubated with a 1:20 dilution of fluorescein-labeled sheep anti-mouse IgG (Sigma Chemical ) for 30 min at room temperature. After a final wash in PBS, glass coverslips were applied over the tissue sections with 50% glycerine–50% PBS and they were viewed on a Leitz DAS fluorescence microscope.
RESULTS
Induction of hepatic CYP2E1 and CYP1A1/2. As positive controls, experiments were conducted to establish that feeding ethanol-containing diets resulted in induction of hepatic CYP2E1 and CYP1A1/2. Table 1 demonstrates that when rats were administered ethanol for 2 weeks via Feeding System 1, total CYP450 was elevated ,1.5-fold above control levels. PNP activity was elevated ,2.6-fold by the ethanol diet, as expected (Koop et al., 1982; Perrot et al., 1989), and EROD activity was elevated ,1.8-fold following ethanol consumption, consistent with a previous report (Roberts et al., 1995). Ethanol induction of PNP and EROD activity is reported to be due to increased protein levels of CYP2E1 and CYP1A, respectively (Koop and Tierney, 1990; Koop et al., 1982; Perrot et al., 1989; Roberts et al., 1995; Ueng et al., 1993). Western blot analysis indicated that consuming ethanol induced immunoreactive CYP2E1 (Fig. 1A) and CYP1A1/2 (Fig. 1B). Furthermore, the Western blots (Fig. 1) establish that the antibodies for CYP2E1 or CYP1A1/2 react as expected, with proteins of molecular weight ,52 or ,53–58 kDal, respectively. Under the SDS/page condition used, the CYP1A1 and CYP1A2 isoforms were not well resolved. These results also demonstrate that the antibodies are highly specific for their specific antigens and can be used with confidence in immunohistochemical studies. Liver samples from the same rats used to derive data in Table 1 and Fig. 1 were used for immunohistochemical
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TABLE 1 Effect of Feeding Ethanol via System I on Hepatic Total CYP, PNP, and EROD Sample
Total CYP450
PNP
EROD
C1 C2 C3 C4 Mean
0.44 0.56 0.47 0.43 0.48 6 0.06
0.58 0.60 0.73 0.53 0.61 6 0.08
211 169 413 129 230 6 126
E1 E2 E3 E4 E5 Mean
0.62 0.62 0.76 0.85 0.75 0.72 6 0.10a
2.17 1.58 1.24 1.43 1.45 1.58 6 0.35a
272 435 475 559 333 415 6 114b
Note. Four rats were fed a liquid diet that did not contain ethanol (C1–4), and five rats were fed an isocaloric liquid diet that contained 6% ethanol (E1–5), as described, using Feeding System 1. Liver microsomes were then prepared and assayed for total cytochrome P450 (nmol/mg protein), PNP activity (pmol nitrocatechol/min/mg protein), and EROD activity (pmol resorufin/min/mg protein), as described under Methods. Measurements for individual rats are shown above; the mean values 6 standard deviation for the control and ethanol groups are presented. a Significantly different from control at P , 0.05. b Not significantly different from control at P , 0.05, but different from control at P , 0.06.
analysis. Ethanol feeding via Feeding System 1 induced additional immunohistochemical reactivity of CYP2E1 (Figs. 2C and 2D) and CYP1A1/2 (Figs. 2A and 2B) in liver, consistent with enzymatic (Table 1) and Western blot (Fig. 1) results. Induction of skeletal muscle CYP2E1 and CYP1A1/2. Various skeletal muscle tissues were assessed for the presence of CYP2E1 and for CYP1A1/2 using immunohistochemistry. CYP2E1 was not detected in soleus (Fig. 3D), plantaris, or diaphragm (not shown) of animals fed the control liquid diet or the diet containing ethanol. CYP1A1/2 was barely detectable in soleus muscles from animals on the control diet (Fig. 3C). However, marked CYP1A1/2 immunoreactivity was consistently apparent in soleus (Fig. 3A), plantaris (Fig. 3B), and diaphragm (not shown) of animals fed the diet containing ethanol. In all skeletal muscle tissues examined, the CYP1A1/2 immunoreactivity appeared to be localized to capillaries surrounding myofibers. Purified ethanol. Feeding commercial ethanol induced both CYP2E1 and CYP1A1/2 in liver (Table 1 and Figs. 1 and 2), confirming other reports (Koop and Tierney, 1990; Koop et al., 1982; Perrot et al., 1989; Roberts et al., 1995; Ueng et al., 1993); however, the finding that feeding ethanol
induced CYP1A1/2, but not CYP2E1, in skeletal muscle capillary cells was unexpected. To ensure that CYP1A1/2 induction in skeletal muscle was not due to a contaminant in the commercial ethanol added to the liquid diet, ethanol was highly purified immediately before use by distillation, as described, and used in all subsequent experiments. Feeding highly purified ethanol to rats using Feeding System 1 resulted in the same immunological staining pattern in the liver, soleus, plantaris, and diaphragm (not shown) as obtained using commercial ethanol that was not distilled (Figs. 2 and 3 and described above). In addition, using highly purified ethanol also resulted in induction of hepatic PNP and EROD activities (not shown) similar to that shown in Table 1. Microsomes prepared from skeletal muscle (soleus, plantaris, diaphragm) of rats fed liquid diets (control or containing highly purified ethanol) via Feeding System 1 were assayed for total cytochrome P450 and EROD activity. Total CYP450 was undetectable in microsomes from all tissues examined, and EROD activity was only detectable (#46 pmol resorufin/min/mg protein) in microsomes from diaphragm of animals fed diets containing ethanol. Detectable EROD activity in diaphragm microsomes from animals fed ethanol is consistent with localization of induced CYP1A1/ 2 in the capillaries of skeletal muscle (Fig. 3) and with the higher vascular content of this tissue, relative to soleus or plantaris. Ethanol induction of CYP1A1/2 is dependent on the feeding system. Although the results indicating that ethanol induces CYP1A1/2 and CYP2E1 in liver (Table 1 and Fig. 1) are consistent with those of other reports (Koop et al., 1982; Perrot et al., 1989; Roberts et al., 1995; Ueng et al., 1993), it was important to demonstrate that CYP1A1/2 induction by ethanol in skeletal muscle (in the absence of CYP2E1 induction) was not due to components of Feeding System 1 (rubber stopper, stainless steel tubing, glass bottle). To this end, all-glass feeding systems (Feeding System 2) were used to deliver liquid diets to rats for 2 weeks. Immunohistochemical analysis of skeletal muscle consistently failed to show CYP1A1/2 or CYP2E1 reactivity in rats receiving either control or ethanol-containing liquid diets (not shown). Microsomes were prepared from livers of these animals and subjected to EROD and Western blotting analysis. Table 2 shows that feeding ethanol to rats by Feeding System 2 did not induce hepatic EROD activity. Additional studies confirmed that administering ethanol in Feeding System 2 did not induce EROD activity in rat liver or immunoreactive CYP1A1/2 in the soleus, plantaris, or diaphragm muscles (not shown). A direct comparison between administering
CYP1A1/2 IN RAT SKELETAL MUSCLE
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FIG. 1. The same liver microsomes described in Table 1, which were prepared from rats fed control (C1–4) or ethanol-containing (E1–5) diets, were subjected to SDS–polyacrylamide gel electrophoresis (100 mg each) and Western blot analysis with primary antibodies directed to CYP2E1 (A) or CYP1A1/2 (B). Lane 1, E1 (16.5); lane 2, C1, (4.2); lane 3, E2 (42.7); lane 4, C2 (2.7); lane 5, E3 (18.6); lane 6, C3 (29.4); lane 7, E4 (67.7); lane 8, C4 (2.5); lane 9, E5 (6.6). Numbers in parentheses represent relative optical densities determined with an Optimas (Bothell, WA) image analysis system for blots depicted in Fig. 1B.
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FIG. 2. Immunohistochemical analysis of CYP1A1/2 and CYP2E1 in rat liver. Rats received a control or ethanol-containing liquid diet as described. Liver tissue was processed and reacted with primary antibodies directed to CYP1A1/2 or to CYP2E1, followed by FITC-conjugated secondary antibodies. Samples were observed under a fluorescent microscope; (A) control diet, anti-CYP1A1/2; (B) ethanol diet, anti-CYP1A1/ 2; (C) control diet, anti-CYP2E1; (D) ethanol diet, anti-CYP2E1. Photographs were taken using an autoexposure setting; this setting provides optimum visualization of FITC staining, but minimizes differences between the fluorescent intensities of different samples. Note that in the samples from rats on an ethanol diet, staining of CYP1A1/2 (B) and CYP2E1 (D) covers more of the liver area than is observed when rats received the control liquid diet (A and C, respectively). Bar in D, 100 mm.
ethanol to rats in Feeding Systems 1 and 2 was then undertaken to assess the effects on induction of hepatic CYP1A1/ 2 and CYP2E1. Western blot analysis of hepatic microsomes from rats fed liquid diets by Feeding System 1 or 2 were probed with CYP2E1-specific antibodies and with CYP1A1/ 2-specific antibodies; the relative immunoreactivity of each of these CYP450 isoforms was determined by image analysis as described. Table 3 demonstrates that CYP2E1 was induced ,two-fold when ethanol was administered by both Feeding Systems 1 and 2. In contrast, CYP1A1/2 was only induced in ethanol-fed animals when ethanol was administered by Feeding System 1.
DISCUSSION
The objective of this study was to test the hypothesis that ethanol consumption induced skeletal muscle tissue CYP2E1 and/or CYP1A1/2. This study focused on CYP1A1/ 2 and CYP2E1 because Riggs et al. (1996) postulated that ethanol consumption may have induced skeletal muscle cytochrome P450s, which metabolized acetaminophen to a myotoxin, precipitating rhabdomyolysis. CYP2E1 and CYP1A1 are reported to be ethanol-inducible and to metabolize acetaminophen to the toxic quinone imine (Haravisonn and Guengerich, 1988; Patten et al., 1993; Raucy et al.,
CYP1A1/2 IN RAT SKELETAL MUSCLE
229
FIG. 3. Immunohistochemical analysis of CYP1A1/2 and CYP2E1 in rat skeletal muscle. Rats received control or ethanol-containing liquid diets as described. Soleus and plantaris muscles were processed and reacted with primary antibodies to CYP1A1/2 or to CYP2E1, as described; (A) ethanol diet, soleus reacted with anti-CYP1A1/2; (B) ethanol diet, plantaris reacted with anti-CYP1A1/2; (C) control diet, soleus reacted with anti-CYP1A1/2; (D) ethanol diet, soleus reacted with anti-CYP2E1. Note that very low levels of CYP1A1/2 staining were observed in some capillaries surrounding muscle bundles in tissue from animals on the control diet (C); intense CYP1A1/2 staining was observed in capillaries surrounding muscle tissue from animals receiving the ethanol-containing diet (A and B); CYP2E1 staining was not observed in any muscle tissue from animals receiving the control or ethanol-containing (D) liquid diet. Bar in D, 100 mm.
1989). Our studies demonstrate for the first time that consuming a 6% ethanol diet may mediate induction of CYP1A1/ 2 in rat skeletal muscle tissue. Furthermore, the induced CYP1A1/2 is predominantly localized in capillaries surrounding type I and II myofibers (Fig. 3). However, the ethanol-mediated induction of CYP1A1/2 in skeletal muscle and in liver is complex; purified ethanol administered by the all-glass Feeding System 2 did not result in CYP1A1/2 induction (Tables 2 and 3). Induction of CYP1A1/2 in skeletal muscle by the ethanol-containing diet administered via Feeding System 1 may be due to ethanol solubilizing a compound from rubber stoppers or from the stainless steel tubing in this feeding system. Alternatively, because rats chew the rubber stoppers, it is possible that a CYP1A1/2 inducer is produced in the stomach when ethanol
is present; or the combination of ethanol, a component in the liquid diet, and rubber stopper/stainless steel tubing may generate a CYP1A1/2-inducing compound. The stoppers in Feeding System 1 were composed of : poly 2-chlorobutadiene 1, 3 (Neoprene WRT) (62.50%); N-phenyl-a-naphthylamine (Neozone A) (1.25%); distilled stearic acid (Wecoline 300) (0.31%); magnesium oxide (Maglite Y) (1.25%); 2mercaptomidazoline (NA-22) (0.31%); zinc oxide surface treated with propionic acid (Protox-166) (3.13%); and Intermediate superabrasion oil furnace black (ISAF or Carbon Black) (31.25%). The Carbon Black contains adsorbed polyaromatic hydrocarbons (,0.1% of the carbon black), potent inducers of CYP1A1/2. However, if rubber stoppers are the source of a CYP1A1/2-inducing compound, apparently ethanol is required to render the compound “biologically
230
SMITH ET AL.
used to “reseal” opened alcoholic beverages (wine, beer, liquor) contain rubber components; and some ethanol-containing pharmaceuticals are placed in vials containing rubber stoppers or are delivered through syringes which use black rubber plungers. Data in this study also demonstrate that purified ethanol administered by an all-glass feeding system (Feeding System 2) did not significantly induce hepatic EROD activity (Tables 2 and 3). This contradicts two reports indicating that ethanol administered via a Lieber/DeCarli liquid diet induces CYP1A1 in rats (Roberts et al., 1995) and hamsters (Ueng et al., 1993). Potential explanations for these discrepancies include the facts that rats and hamsters were fed an ethanol diet for 3 weeks (Roberts et al., 1995; Ueng et al., 1993), in contrast to the 2-week period used in this study; highly purified ethanol was not used in the two previous reports (Roberts et al., 1995; Ueng et al., 1993); the feeding system used to administer ethanol in a liquid diet to hamsters was not described (Ueng et al., 1993) and may have contained an ethanol-extractable CYP1A1/2-inducer, like Feeding System 1, and a contaminant may have been responsible for inducing CYP1A1; and studies herein utilized female rats, while the gender of rats in a previous study (Roberts et al., 1995) was not specified, and male hamsters were used (Ueng et al., 1993). Gender differences in ethanol stimulation of
FIG. 4. Means of logarithmically transformed data from Western blot analysis of CYP1A1/2 (A) and CYP2E1 (B) by diet (control or ethanol) and Feeding System 1(m) and Feeding System 2 (v); means 6 standard error.
available"; rats consuming the control diet chew and ingest rubber stoppers but do not exhibit induced CYP1A1/2. In addition, preliminary studies exposed rubber stoppers in Feeding System 1 to water or water containing 6% ethanol; the solutions were then analyzed by GC/MS and by HPLC/ UV. Polyaromatic hydrocarbons were not detected in either 6% ethanol or water solutions. The results of this study are consistent with the conclusion that additional factors may potentiate the myotoxic effects of ethanol (Haller and Drachman, 1980), and additional studies are in progress to identify the mechanism by which ethanol in Feeding System 1 induces CYP1A1/2. In addition, it will be important to determine if ethanolmediated CYP1A1/2 induction, exhibited by Feeding System 1, can occur in humans using ethanol-containing beverages or pharmaceutical drugs. For instance, some devices
TABLE 2 Effect of Feeding Ethanol via System 2 on Hepatic EROD Activity
Sample
EROD (pmol resorufin/min/mg)
C1 C2 C3 C4 Mean
180 138 142 151 153 6 19
E1 E2 E3 E4 ES E6 Mean
105 169 109 138 165 99 118 6 28a
Note. Four rats were fed a liquid diet that did not contain ethanol (C1–4), and six rats were fed an isocaloric liquid diet that contained ethanol (E1–6) for 2 weeks, using Feeding System 2. Liver microsomes were then prepared and assayed for total EROD activity, as described under Methods. Measurements for individual rats are shown above; the mean values 6 standard deviation for the control and ethanol groups are presented. a Not statistically different from control at P , 0.l0.
231
CYP1A1/2 IN RAT SKELETAL MUSCLE TABLE 3 Western Blot Analysis of Hepatic Microsomes from Rats Fed Liquid Diets with Feeding System I or 2 Relative immunoreactivity Feeding System System 2 C1 C2 E1 E2 E3 E4 System 1 C3 E5 E6
CYP1A1/2
CYP2E1
2.6 2.8 2.0 3.2 2.2 2.9
16.1 16.9 31.7 31.4 28.1 36.7
4.2 43.3 32.3
20.0 38.5 38.1
Note. Rats received a control liquid diet (C1–3) or a liquid diet containing ethanol (E1–6) for 2 weeks via Feeding System 1 or 2 (described under Methods). Liver microsomes were then prepared and 100 mg of each sample was subjected to Western blot analysis and probed with primary antibodies specific for CYP1A1/2 or CYP2E1, as described. Relative optical densities of protein bands corresponding to CYP1A1/2 or CYP2E1 were then analyzed by Optimas image analysis, which is depicted above. For CYP1A1/ 2, analysis demonstrated a significant interaction between Feeding System and ethanol containing diet (P , 0.0006) (see Fig. 4). For CYP2E1 there was a small but significant (P 0.331) effect due to Feeding System and a larger (P 0.0002) effect due to ethanol-containing diet, and there was no evidence of an interaction between them (see Fig. 4B).
hepatic benzo[a]pyrene hydroxylase activity have been noted (Lieber et al., 1979); in female rats, feeding ethanol stimulated microsomal benzo[a]pyrene hydroxylase activity, but in male rats feeding ethanol did not stimulate microsomal benzo[a]pyrene hydroxylase. Additional experiments will be required to clarify why an ethanol-containing diet induced hepatic CYP1A1 in several studies, but highly purified ethanol in all-glass feeders did not induce hepatic CYP1A1/2 in this study.
ACKNOWLEDGMENTS
This work was supported in part by a West Virginia University School of Medicine Research Development grant, by NIH/NCI (CA45131-09 to M.R.M.), by ACS (RPG-57-066-01-VM to M.R.M.), and by NIOSH/CDC (R01 OHAR02918 to W.T.S.).
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