Induction of Hepatic Xenobiotic Metabolizing Enzymes in Female ...

50, 10 –19 (1999) Copyright © 1999 by the Society of Toxicology

TOXICOLOGICAL SCIENCES

Induction of Hepatic Xenobiotic Metabolizing Enzymes in Female Fischer-344 Rats following Repeated Inhalation Exposure to Decamethylcyclopentasiloxane (D 5) James M. McKim, Jr.,* ,1 Supratim Choudhuri,* Paul C. Wilga,* Ajay Madan,† Leigh Ann Burns-Naas,* Robert H. Gallavan,* Richard W. Mast,‡ Dennis J. Naas,§ Andrew Parkinson,† and Robert G. Meeks* *Dow Corning Corporation, Health and Environmental Sciences, 2200 W Salzburg Rd., Midland, Michigan 48686; †XenoTech LLC, 3800 Cambridge, Kansas City, Kansas 66103; ‡Everest Consulting Associates, 15 N. Main Street, Cranbury, New Jersey 08512; and §AccuTox Consulting Services, Ltd., Midland, Michigan 48642 Received August 31, 1998; accepted February 28, 1999

Decamethylcyclopentasiloxane (D 5) is a clear, odorless, synthetically-derived silicone fluid that represents one member of a class of compounds that has been in commercial use for about 50 years. The primary use of D 5 has been in personal care products such as shampoos and antiperspirants. D 5 consists of alternating silicon-oxygen bonds connected in a ring (cyclic) arrangement with two methyl groups covalently bonded to each silicon atom (MW, 370 g/mol). The structure of D 5 is shown in Figure 1. Although the physical-chemical properties of D 5 have been well described (Lane and Burns, 1996; Noll, 1968), less is known about its biological effects. Because of the wide range of commercial applications and potential for human exposure, detailed biochemical studies have been initiated to understand the fate and effect of D 5 in animals. In a previous study, oral treatment of female Sprague Dawley rats for 12 days with D 5 (2000 mg/kg/day in corn oil) increased the liver-to-body weight ratio by 64% compared to controls, decreased the amount of total hepatic cytochrome P450, and increased aminopyrine N-demethylase activity (a cytochrome P450 activity associated with phenobarbital inducible enzymes) (Siddiqui 1989). When D 5 was administered orally at 1500 mg/kg/day in a distilled water suspension for 28 days, the liver-to-body weight ratio increased 19% in male and 23% in female rats relative to controls (Stark et al., 1990). In a more recent study, (Burns-Naas et al., 1998), female Fischer344 rats were exposed to 10, 25, 75, and 160 ppm D 5 vapors for 1 month and evaluated for various immunological end points as well as changes in several organ weights. There were no statistically significant changes in organ weights, with the exception of liver, which was increased only at the highest exposure concentration. No overt signs of toxicity were noted in any of these studies. These reports suggest that D 5, has the ability to increase liver size and alter the activity and tissue levels of hepatic cytochrome P450 enzymes. However, the mechanisms underlying these observations, the effects of low doses, alternate routes of exposure, and the individual P450 subfamilies affected by D 5 treatment have not been investi-

Decamethylcyclopentasiloxane (D 5) is a cyclic siloxane with a wide range of commercial applications. The present study was designed to investigate the effects of D 5 on the expression and activity of selected rat hepatic phase I and phase II metabolizing enzymes. Female Fischer-344 rats were exposed to 160 ppm D 5 vapors (6 h/day, 7 days/week, for 28 days) by whole-body inhalation. Changes in the activity and relative abundance of hepatic microsomal cytochromes P450 (CYP1A, CYP2B, CYP3A, and CYP4A), epoxide hydrolase, and UDP-glucuronosyltransferase (UDPGT) were measured. Repeated inhalation exposure of rats to D 5 increased liver size by 16% relative to controls by day 28. During a 14-day post-exposure period, liver size in D 5-exposed animals showed significant recovery. Exposure to D 5 did not change total hepatic P450, but increased the activity of hepatic NADPH-cytochrome c reductase by 1.4-fold. An evaluation of cytochrome P450 (CYP) enzymes in hepatic microsomes prepared from D 5-exposed rats revealed a slight (1.8-fold) increase in 7-ethoxyresorufin O-deethylase (EROD) activity, but no change in immunoreactive CYP1A1/2 protein. A moderate increase (4.2fold) in both 7-pentoxyresorufin O-depentylase (PROD) activity and immunoreactive CYP2B1/2 protein (3.3-fold) was observed. Testosterone 6b-hydroxylase activity was also increased (2.4-fold) as was CYP3A1/2 immunoreactive protein. Although a small increase in 11- and 12-hydroxylation of lauric acid was detected, no change in immunoreactive CYP4A levels was measured. Liver microsomal epoxide hydrolase activity and immunoreactive protein increased 1.7- and 1.4-fold, respectively, in the D 5-exposed group. UDPGT activity toward chloramphenicol was induced 1.8-fold, while no change in UDPGT activity toward 4-nitrophenol was seen. These results suggest that the profile for enzyme induction following inhalation exposure of female Fischer-344 rats to D 5 vapors is similar to that reported for phenobarbital, and therefore D 5 may be described as a weak “phenobarbital-like” inducer. Key Words: decamethylcyclopentasiloxane (D 5); hepatic enzymes; cytochrome P450 inhalation exposure; rat.

1 To whom all correspondence should be sent at present address: Pharmacia & Upjohn, Inc., Building 300 – 423, 333 Portage Rd., Kalamazoo, MI 49007– 4940. Fax: (616) 833-7722. E-mail: james.m.mckim@am.pnu.com.

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EFFECTS OF D 5 ON RAT METABOLIZING ENZYMES

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light/dark cycle at a temperature of 20 –24°C and a humidity of 30 –70%. The air-exchange rate of the room was 12–15 changes per h. During the course of this study, animals had free access to food (Purinat Certified Rodent Chow #5002) and reverse osmosis-treated water, except during the 6-h exposure period. Body weights and clinical observations were taken daily as an indication of general health. Liver samples were collected from control and D 5 -exposed rats, as well as from day 14-post-exposure animals. Liver samples were frozen in liquid nitrogen and stored at – 80°C until needed. Female rats were chosen for this study because preliminary data suggested that this gender was the most sensitive with respect to liver enlargement.

FIG. 1. Structure of decamethylcyclopentasiloxane (D 5).

gated. Because liver weight was increased only at the 160-ppm exposure concentration in the Burns-Naas study, and this concentration is the highest that can be achieved without generation of aerosols, the present work was undertaken to determine what effect exposure of rats to 160 ppm D 5 vapors might have on the levels of hepatic microsomal cytochromes P450, epoxide hydrolase, and UDP-glucuronosyltransferase enzymes. Once the hepatic enzyme-induction profile for D 5 is known, it may then be possible to place D 5 into a group of agents that produce similar biochemical changes. This basic mechanistic information, when combined with pharmacokinetic, dose-response, and human exposure assessment data, should allow more accurate science-based risk characterization to be elucidated. MATERIALS AND METHODS Chemicals. 7-Ethoxyresorufin, 7-pentoxyresorufin, resorufin, cytochrome c (horse heart), phenylmethylsulfonylfluoride (PMSF), bicinchoninic acid (BCA), sodium cholate, Triton N-101, phenobarbital, 3-methylcholanthrene (3-MC), bovine serum albumin (BSA), and Tween-20 were purchased from Sigma Chemical Co. (St. Louis, MO). Trizma base, sodium dodecyl sulfate (SDS), potassium phosphate (mono- and dibasic), formaldehyde, and methanol were purchased from Fisher Scientific (Pittsburgh, PA). Goat serum was from GIBCO-BRL (Gaithersburg, MD). Goat anti-rabbit secondary antibody (alkaline phosphate conjugate) was from Biosource (Camarillo, CA). Donkey anti-sheep secondary antibody (alkaline phosphate conjugate) and donkey serum were obtained from Jackson Immunochemical (West Grove, PA). BCIP/ NBT substrate was purchased from Kirkegaard Perry Laboratory (Gaithersburg, MD). Reinforced nitrocellulose membranes were from Schleicher and Schuell (Keene, NH). All other chemicals were of highest quality and were obtained either from Sigma Chemical Co. or from Fisher Scientific. Decamethylcyclopentasiloxane (D 5) was obtained from Dow Corning Corporation and was determined to be .99% pure, based on gas chromatography combined with mass spectral analysis. Animals. Female Fischer-344 rats (128 –138 g) from Charles River Breeding Laboratories, Inc., Kingston, New York were maintained on a 12-h

Inhalation conditions. Whole-body inhalation exposures were carried out at WIL Research Laboratories, Ohio. On each day of exposure control animals (0 ppm) were placed into 2.0 m 3 Hazelton 1000 whole-body inhalation chambers while the D 5 (160 ppm) group was placed into 1.0 m 3 Hazelton 1000 whole-body inhalation chambers. The chambers were operated at 12–15 changes per h. A HEPA filter and activated charcoal bed were used to pre-treat room air before it entered the chambers. Test atmospheres were generated with a stainless steel “J-tube” filled with 3-mm glass beads and wrapped with electric heating tape (Miller et al., 1980). The temperature of the J-tube was maintained at 60°C with a Model CN 370 digital temperature controller from Omega Engineering (StanFord, CT). Mass airflow, temperature, relative humidity, chamber negative pressure, and oxygen content were monitored continually and recorded every 30 min. Chamber D 5 concentrations were measured with a Hewlett Packard 5890 Series II gas chromatograph (GC) equipped with a flame inoization detector (FID) and a model 3396A integrator. The GC column was a 30-m 3 0.320-mm DB-5 with 0.25 mm film thickness. D 5 standards were prepared by injecting a known volume of liquid D 5 into a 40 liter Tedlart gas sampling bag. Control animals received filtered room air whereas treated animals were exposed to D 5 vapors at 160 ppm 6 h/day, 7 days/week, for 28 days. Selection of test system and exposure concentration. Previous studies from our laboratory have indicated that female rats were more sensitive to hepatomegaly than males; therefore, female rats were chosen for this study. Due to the physical properties of D 5, the maximum exposure concentration that would reproducibly provide D 5 vapors, and not aerosols, was determined to be 160 ppm. Intra-assay positive control animals. Female Fischer-344 rats were obtained from Charles River Breeding Laboratories Inc., Raleigh, NC. The animals were maintained under conditions similar to those described above. The animals were administered phenobarbital (80 mg/kg/day in 0.9% saline, ip) or 3MC (30 mg/kg/day in corn oil, ip) once daily for 3 consecutive days. Livers were removed on the morning of the fourth day, frozen in liquid nitrogen, and stored at – 80°C until needed. Preparation of hepatic microsomes. Microsomes were prepared from frozen liver by centrifugation, essentially as described by Lu and Levin (1972) and as previously reported by McKim et al., (1998). Total microsomal protein. Microsomal protein concentration was determined by the bicinchoninic acid (BCA) method as described by Smith et al (1985). Samples were analyzed with a Perkin Elmer Lambda II dual beam spectrophotometer at 562 nm. Total P450. Total hepatic P450 content was determined by the reduced carbon-monoxide versus reduced sodium dithionite difference spectra originally described by Omura and Sato (1964), with modifications described by Guengerich (1994). In order to minimize sample turbidity and the resulting interference, microsomal samples were diluted 1:10 with solubilization buffer (0.1 M potassium phosphate, pH 7.7; 20% glycerol; 1 mM EDTA; 0.5% sodium cholate; 0.4% Triton N-101). Samples were analyzed with a dual beam spectrophotometer by scanning wavelengths between 400 and 500 nm. NADPH-cytochrome c reductase activity. NADPH-Cytochrome c reductase activity was measured essentially as described by Phillips and Langdon (1964) and Guengerich (1994). Reduction of cytochrome c was measured by determining ‚Abs 550/min with a Beckman DU-640I spectrophotometer. En-

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zyme activity (nmol cytochrome c reduced/min/mg protein) was determined with an extinction coefficient of 0.0295 mM –1cm –1. 7-Ethoxyresorufin O-deethylase (EROD) and 7-pentoxyresorufin O-depentylase (PROD) activity assays. The EROD assay, which is relatively specific for CYP1A1/2, and the PROD assay, which is relatively specific for CYP2B1/2, were performed essentially as described by Prough et al., (1978) and Burke et al., (1985) with minor modifications described by McKim et al., (1998). Testosterone 6b-hydroxylase activity. Hydroxylation of testosterone in the 6b position can be used as an indicator of CYP3A1/2 activity. The products of testosterone hydroxylation catalyzed by liver microsomal enzymes were determined by HPLC, as described by Pearce et al. (1996). Liver microsomes were incubated 8 –10 min at 37°C in 1.0 ml incubation mixtures containing 50 mM potassium phosphate, pH 7.4; 3 mM MgCl 2, 1 mM EDTA, 1 mM NADP, 5 mM glucose-6-phosphate, 1 U/ml glucose-6-phosphate dehydrogenase, 1 mM of the steroid 5a-reductase inhibitor 17b-N,N-diethylcarbamoyl-4-methyl-4-aza-5a-androstan-3-one and 250 mM testosterone. Hydroxylation of lauric acid. Hydroxylation of lauric acid can be used as an indicator of changes in CYP4A activity. This assay was done by combining methods of radiometric partitioning (Giera and Van Lier, 1991) and radiometric HPLC analysis (Romano et al., 1988). This assay measures the rate of conversion of [ 14C]-lauric acid to the water-soluble, hydroxylated metabolites, and was recently reported by Pearce et al. (1996). Briefly, the reaction mixture contained the following in a final volume of 1.0 ml: 400 mg microsomal protein; 1 mM NADP; 5 mM glucose-6-phosphate; 1 U/ml glucose-6-phosphate dehydrogenase; 50 mM potassium phosphate, pH 7.4; 3 mM MgCl 2; 1 mM EDTA; and 4 mM (20 mCi/ml) 14C-lauric acid dissolved in 5 mM sodium carbonate. Following incubation at 37°C, the reactions were terminated by the addition of 1% sulfuric acid, and the 11- and 12-hydroxylated metabolites were extracted in the aqueous phase and analyzed by HPLC. Epoxide hydrolase activity. Epoxide hydrolase activity was determined with cis-stilbene oxide as substrate, essentially as described by Hammock et al. (1985) and Gill et al. (1983). Reaction mixtures (final volume 100 ml) containing 10 mg of liver microsomal protein, 100 mM Tris–HCl buffer (pH 9.0 at 37°C) and 50 mM [ 3H]-cis-stilbene oxide were incubated at 37°C for 5 min. Reactions were stopped with the addition of 200 ml dodecane (McKim et al., 1998). Detection of immunoreactive CYP proteins by Western blot analysis. Microsomal proteins were separated by SDS–polyacrylamide gel electrophoresis (SDS–PAGE), essentially as described by Laemmli (1970), with minor modifications as described by Choudhuri et al. (1996). Western blot analysis was performed according to Choudhuri et al. (1996). Denatured microsomal proteins (0.6 – 6 mg) were electrophoretically resolved in a 10% separation gel and electrotransferred on a nylon membrane overnight. Membranes were blocked at 37°C for 2 h in 2% BSA or 10% donkey serum (for CYP 4A). Incubations in primary antibody were for 4 h at 37°C. For CYP4A immunoblot, the primary antibody (raised in sheep), provided in the Amersham CYP4A Western blotting kit, was used. For all others, about 4 –5 mg/ml of respective primary antibodies (raised in rabbit) was used for each membrane. Incubations in secondary antibody was for 2 h (1:4000 –1:5000 dilution) at 37°C. The secondary antibody used for CYP4A was donkey anti-sheep F(ab9) 2 fragments of alkaline phosphatase conjugate, while that for all others were goat anti-rabbit F(ab9) 2 fragments of alkaline phosphatase conjugate. Blots were developed with the alkaline phosphatase substrate BCIP/NBT. Detection of immunoreactive proteins was accomplished with polyclonal antibodies raised against rat liver microsomal CYP1A1/2, CYP2B1/2, CYP3A1/2, and epoxide hydrolase as described by Parkinson and Gemzik (1991). Analysis of CYP4A immunoreactive proteins was accomplished with primary anti-body provided by Amersham Inc. (Arlington Heights, IL) and described by McKim et al. (1998). UDP-glucuronosyltransferase activity toward 4-nitrophenol and chloramphenicol. UDPGT activity toward 4-nitrophenol, was done essentially according to Bock et al. (1983) and Guengerich (1994). The substrate (4-

nitrophenol, 0.5 mM final concentration) was preincubated for 3 min at 37°C in the presence of 0.05% Triton X-100, 5 mM MgCl 2, 0.1M Tris–HCl buffer, and 0.8 mg/ml microsomal protein. The reaction was started with the addition of UDP-glucuronic acid to a final concentration of 3 mM. The mixture was incubated at 37°C for 10 min. Total reaction volume was 0.5 ml. Reactions were terminated by placing 100 ml aliquots of the reaction mixture into 1 ml of trichloroacetic acid, and centrifuging the mixture at 1500 rpm for about 4 min. The amount of glucuronidation was indirectly determined by measuring the amount of unconjugated 4-nitrophenol remaining by removing a 1 ml aliquot and mixing it with 250 ml of 2N NaOH. Samples were quantified by determining their absorbance at 405 nm with a Beckman DU-640I spectrophotometer. UDPGT activity toward chloramphenicol, was performed as described by Young and Lietman (1978) with modifications reported by Arlotto et al. (1986) and Dutton et al. (1981). Briefly, in a final incubation volume of 0.5 ml, radiolabeled [ 14C]-chloramphenicol (2 mM, 0.4 mCi) was incubated with solubilized microsomal protein (0.5 mg/ml) in a reaction mix containing Tris–HCl buffer (200 mM, pH 8.0 at 37°C), MgCl 2 (10 mM), EDTA (1 mM), D-saccharic acid-1,4-lactone (100 mM), UDP-glucuronic acid (4 mM), and CHAPS (0.5 mM). Reactions were started by adding UDP-glucuronic acid and incubated for the required time. Reactions were stopped by adding 6 ml of isoamyl acetate and 1.1 ml water per tube. The 14C-labeled glucuronidated product was extracted in aqueous phase and quantitated by scintillation spectrometry. Enzyme activity (nmol/mg protein/min) was determined based on the fraction of chloramphenicol metabolized (radioactivity recovered in the aqueous phase) and normalizing the value by the amount of protein used and time. Statistical analyses. Statistical analyses were performed with SASt V.6.12. Treatment effects were evaluated by one-way analysis of variance or Students t-test and were considered significant at p , 0.05 level of significance.

RESULTS

In the present study, female Fischer-344 rats were exposed to 160 ppm D 5 vapors. Female rats were chosen because this gender is most sensitive with respect to liver enlargement. A single exposure concentration was tested for two reasons: (1) 160 ppm represents the highest concentration that could be reproducibly generated without contamination from aerosols, and (2) in a previous dose-response experiment (Burns-Naas et al., 1998) only the highest (160 ppm) exposure group showed a statistically significant increase in liver weight. Over the course of the study, the mean daily chamber mass air flow was 413 6 4.5 and 217 6 13 standard liters per minute for the 2.0 m 3 (control) and 1.0 m 3 (160 ppm) chambers respectively. Chamber temperature was 22 6 0.4 and 23 6 0.5°C, relative humidity was 49 6 2% and 51 6 4%, and oxygen content was 21 6 0.1% in the 0 ppm and 160 ppm chambers, respectively. On each day of exposure, the chamber concentration of D 5 was determined between 12 and 15 times. The mean chamber concentration of D 5 over the 28-day period was 160 6 4 ppm. D 5 was not detected on any day in the 0 ppm chamber air. Animals exposed to D 5 vapors (160 ppm) for 28 days showed an average increase in liver size of about 16% relative to controls. During the 2-week post-exposure period, liver size decreased, but remained statistically different from controls (Fig. 2). After 28 days of exposure, the total amount of P450 in hepatic microsomes from D 5-exposed animals was unchanged

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FIG. 2. Effects of decamethylcyclopentasiloxane (D 5) on liver weight. Effects observed in female Fischer-344 rats following 28 consecutive days of inhalation exposure to 160 ppm D 5 vapors and after a 14-day post-exposure period. Values represent the mean 6 SEM liver weight relative to controls (N, 10). Values were determined by subtracting the mean control liver-to-body weight ratio from each treated liver-to-body weight ratio, then dividing by the mean control liver-to-body weight ratio, and multiplying by 100. Asterisks indicate values are statistically different from controls (p , 0.05).

compared with controls (Table 1). Contamination with ferrous hemoglobin and conversion of cytochrome P450 to P420 was minimal. Repeated exposure of rats to D 5 produced a slight (1.4-fold), but statistically significant increase in hepatic NADPH cytochrome c reductase activity in D 5-exposed animals (Table 1). Dealkylation of ethoxyresorufin (EROD) was used as an indicator of CYP1A/2 activity. EROD in hepatic microsomes prepared from D 5-exposed rats was increased 1.8-fold relative

to controls. However, when the amount of CYP1A1/2 enzymes were evaluated by Western blot analysis, no increase in CYP1A1 protein was detected (Fig. 3 and Fig. 8). Although not statistically significant, CYP1A2 levels were slightly suppressed. These data suggest that the small increase in EROD activity was due to recognition of ethoxyresorufin as substrate by other CYP enzymes (e.g., CYP2B1 or 2C6). By day 14 post-exposure, EROD activity had returned to control values (Fig. 3). EROD activity in the intra-assay positive control animals treated with 3MC (30 mg/kg) was 12,013 6 1200 pmol/min/mg protein compared to 484 6 13 pmol/min/mg protein in control animals. Changes in the activity and relative abundance of CYP2B1/2 enzymes was measured by dealkylation of pentoxyresorufin (PROD) and Western blot analysis (Fig. 4 and Fig. 8). Exposure to D 5 (160 ppm) produced a 4.2-fold increase in PROD activity and a corresponding increase (3.3-fold) in CYP2B1/2 immunoreactive protein relative to controls. Because CYP2B1 is at or below detectable levels in control microsomes, the fold induction of this specific enzyme could not be accurately determined. Therefore, the Western blot data were combined and presented as an overall change in immunoreactive CYP2B enzymes. On day 14 of the post-exposure period, PROD activity had returned to control levels (Fig. 4). PROD activity in the intra-assay positive control animals treated with PB (80 mg/kg) was 2875 6 78.3 pmol/min/mg protein compared to 28.2 6 1.0 pmol/min/mg protein in control animals. Hydroxylation of testosterone in the 6b position can be used as an indicator of CYP3A1/2 activity. In the present study, testosterone 6b-hydroxylase activity was measured in liver microsomal samples from animals exposed to D 5 for 28 days. Exposure to D 5 resulted in a 2.4-fold increase in hepatic microsomal CYP3A1/2 activity and a corresponding increase

TABLE 1 Summary of Enzyme Activities on Day 28 in Liver Microsomes from Female Fischer-344 Rats Exposed to 0 and 160 ppm Decamethylcyclopentasiloxane (D5) for 4 Weeks Decamethylcyclopentasiloxane (ppm) Enzyme

Assay/substrate

0

160

Total P450 NADPH-cytochrome c reductase CYP1A CYP2B CYP3A CYP4A Epoxide Hydrolase UDPGT UDPGT

Difference spectra Cytochrome c reduction EROD PROD Testosterone 6b-OH Lauric acid 12-OH cis Stilbene oxide Chloramphenicol 4-Nitrophenol

0.6 6 0.04 173 6 5 0.484 6 0.013 0.028 6 0.001 0.288 6 0.016 0.67 6 0.02 9.1 6 0.3 1.24 6 0.14 13.1 6 0.7

0.6 6 0.04 235 6 5* 0.857 6 0.026* 0.117 6 0.013* 0.680 6 0.040* 0.75 6 0.02* 15.0 6 0.4* 2.29 6 0.13* 11.8 6 0.6

Note. Values represent the mean 6 SEM of 8 –10 animals. Activity units are in nmol/min/mg protein except for total P450 which is nmol/mg protein. EROD, 7-ethoxyresorufin O-deethylation; PROD, 7-pentoxyresorufin O-depentylation; UDPGT, UDP-glucuronosyltransferase. * Statistically different from control at p , 0.05.

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detectable increase in immunoreactive CYP4A protein (Table 1). Epoxide hydrolase (EH) activity toward cis-stilbene oxide as substrate, was induced 1.7-fold in microsomes from D 5 exposed animals relative to controls. A corresponding increase (1.4-fold) in EH immunoreactive protein was determined by Western blot analysis (Fig 6 and Fig. 8). When 4-nitrophenol was used as a substrate, no increase in hepatic UDPGT activity was detected in D 5-exposed animals (Table 1). However, when chloramphenicol was used as a substrate UDPGT activity was increased 1.8-fold (Fig. 7).

FIG. 3. Effects of decamethylcyclopentasiloxane (D 5) on hepatic microsomal EROD activity and CYP1A1/2 immunoreactive protein levels. Effects observed in female Fischer-344 rats following 28 consecutive days of inhalation exposure to 160 ppm D 5 vapors and after a 14-day post-exposure period. Activity values represent the mean 6 SEM (N, 10) (top panel). Because CYP1A1 protein levels are below detectable levels, Western-blot data represent only CYP1A2. Immunoreactive protein values (bottom panel) represent the mean 6 SEM (N, 10) of the fold change relative to controls. Asterisks indicate values are statistically different from controls (p , 0.05).

(2.9-fold) in CYP3A1/2 immunoreactive protein (Fig. 5 and Fig. 8). Changes in the activity and expression of hepatic CYP4A can be used as a biomarker for peroxisome proliferation. Changes in CYP4A activity can be estimated with the substrate lauric acid. Although CYP4A hydroxylates lauric acid at the 11 and 12 positions, the 12-hydroxylation is the most specific for CYP4A activity. Female Fischer-344 rats exposed to D 5 vapors (160 ppm) showed a small (1.2-fold and 1.1-fold) but statistically significant increase in 11- and 12-hydroxylation of lauric acid respectively. However, Western blot analysis revealed no

FIG. 4. Effects of decamethylcyclopentasiloxane (D 5) on hepatic microsomal CYP2B1/2 activity and immunoreactive protein levels. Effects observed in female Fischer-344 rats following 28 consecutive days of inhalation exposure to 160 ppm D 5 vapors and after a 14-day post-exposure period. Activity values represent the mean 6 SEM (N, 10) (top panel). Immunoreactive protein values (bottom panel) represent the mean 6 SEM (N, 10) of the fold change relative to controls. Asterisks indicate values are statistically different from controls (p , 0.05).


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determined, it may then be possible to predict whether similar effects would occur in humans and whether or not these effects represent adaptive biochemical responses or precursors to chemical toxicity. Repeated inhalation exposure to D 5 at 160 ppm increased liver-to-body weights 16% relative to controls. This change is consistent with that reported in a companion study (BurnsNaas et al., 1998) where D 5 was also administered by wholebody inhalation at 160 ppm. In that study, D 5 increased liver weight by 13% relative to controls. Hepatomegaly observed following inhalation exposure to D 5, and the subsequent recovery following cessation of exposure, is not unique and has been reported for many cytochrome P450-inducing agents. Reversible hepatomegaly in the absence of histopathological

FIG. 5. Effects of decamethylcyclopentasiloxane (D 5) on hepatic microsomal CYP3A1/2 activity and immunoreactive protein levels. Effects observed in female Fischer-344 rats following 28 consecutive days of inhalation exposure to 160 ppm D 5 vapors. Activity values represent the mean 6 SEM (N, 9 to 10) (top panel). Immunoreactive protein values (bottom panel) represent the mean 6 SEM (N, 10) of the fold change relative to controls. Asterisks indicate values are statistically different from controls (p , 0.05).

DISCUSSION

Decamethylcyclopentasiloxane (D 5) represents one member of a family of cyclic siloxanes whose chemical and physical properties make it ideal for many commercial applications. In the present study, the effects of repeated whole-body inhalation exposure of rats to D 5 vapors on the activity and expression of hepatic microsomal phase I and phase II metabolizing enzymes were evaluated. This information should allow D 5 to be placed into a category of chemicals that produce similar biochemical effects and are well-characterized. If the mechanisms by which a chemical produces gross changes in an animal model can be

FIG. 6. Effects of decamethylcyclopentasiloxane (D 5) on hepatic microsomal epoxide hydrolase activity and immunoreactive protein levels. Effects observed in female Fischer-344 rats following 28 consecutive days of inhalation exposure to 160 ppm D 5 vapors. Activity values represent the mean 6 SEM (N, 9 to10) (top panel). Immunoreactive protein values (bottom panel) represent the mean 6 SEM (N, 10) of the fold change relative to controls. Asterisks indicate values are statistically different from controls (p , 0.05).

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FIG. 7. Effects of decamethylcyclopentasiloxane (D 5) on hepatic microsomal UDP-glucuronosyltransferase activity towards chloramphenicol. Effects observed in female Fischer-344 rats following 28 consecutive days of inhalation exposure to 160 ppm D 5 vapors. Activity values represent the mean 6 SEM (N, 9 to 10). Asterisks indicate values are statistically different from controls (p , 0.05).

lesions and hepatic enzyme leakage represents an adaptive rather than adverse response to an exogenous material. In the present study, the magnitude of liver enlargement following exposure to D 5 vapors for 28 days was small (16%) compared to that reported by Siddiqui, (1989). In that study, female Sprague Dawley rats were administered large oral doses of D 5 in corn oil (2000 mg/kg) for up to 12 days. Liver size in those animals increased 64% relative to controls, but returned to control values upon cessation of treatment. In another study, female Sprague Dawley rats treated with D 5 (1500 mg D 5/kg/ day) by oral gavage, as a suspension in distilled water, for 28 days, had an increase in liver size of 23% (Stark et al., 1990). The difference in liver size between these studies was most likely a reflection of the carrier used [corn oil (Siddiqui, 1989) vs. water (Stark et al., 1990)]. The percentage of administered dose absorbed is greatest in the presence of corn oil. In the present study, total hepatic cytochrome P450 in female Fischer-344 rats was not appreciably changed following 28 consecutive days of exposure to D 5 vapors. In contrast, Siddiqui (1989) reported that treatment of rats with large oral doses (2000 mg/kg) of D 5 appeared to decrease the amount of total hepatic P450 in a time-dependent manner. However, the same study also reported a significant increase in aminopyrine N-demethylase activity, which is associated with PB treatment. The apparent differences in total P450 reported here as compared to those reported by Siddiqui (1989) may be a result of the lipophilic nature of D 5, differential rates of metabolism, dose differences, and slow displacement of D 5 from the heme

group by carbon monoxide (CO) in the assay described by Omura and Sato (1964). For example, it is not uncommon for highly lipophilic compounds to remain with the microsomal fraction during isolation. The microsomes subjected to the carbon monoxide vs. reduced sodium-dithionite difference spectra assay for total P450 may have a considerable amount of D 5 in close proximity to the cytochrome enzymes. This could increase the time required for CO to bind P450, resulting in an apparent decrease in P450 content. In the present study, considerably lower doses were given, and microsomal samples were scanned until a maximum absorbance at 450 nm had been obtained. Moreover, the fact that total hepatic P450 observed in the present study did not change is reflective of the small changes observed in specific P450 enzyme activities. Classical CYP inducers such as 3-methylcholanthrene and b-naphthoflavone increase the expression of CYP1A1/2 enzymes by more than 20-fold in rat liver microsomes (Moorthy et al., 1993; Parkinson 1996; Thorgeirsson et al., 1979). Levels of CYP1A1 are undetectable in control liver microsomes, while CYP1A2 is readily measurable. Female Fischer-344 rats exposed to D 5 vapors by whole-body inhalation in the present study, showed a modest (1.8-fold) increase in hepatic CYP1A1/2 activity, but this small change in activity was not accompanied by an increase in CYP1A1 protein. The increase in EROD activity was most likely due to weak substrate recognition by other CYP enzymes such as CYP2B1 and CYP2C6 (Burk et al., 1994). Although not statistically significant, there was an apparent suppression in CYP1A2 protein levels; this has been reported for other compounds such as phenobarbital (Dragnev et al., 1995). Based on these data, D 5 would not be considered a 3MC-type inducer of cytochrome P450. In contrast to CYP1A1/2 enzymes, exposure to D 5 did increase CYP2B1/2 enzyme activity 4-fold relative to controls. This modest increase in CYP2B1/2 activity was accompanied by a concomitant increase in the relative abundance of CYP2B1/2 proteins. Induction of this group of CYP enzymes is typically the result of exposure to phenobarbital-type compounds. Steroidal agents, such as dexamethasone and pregnenolone 16a-carbonitrile, are the major prototypical inducers of CYP3A enzymes (Schuetz et al., 1984). However, phenobarbital and other CYP2B inducers can produce small to moderate increases in CYP3A1/2 a response consistent with the phenobarbital-induced pleiotropic response (Nims et al., 1993, Parkinson, 1996). Exposure to D 5 produced a small (2.4-fold) increase in CYP3A1/2 activity and immunoreactive protein. As expected, phenobarbital-treated rats produced a moderate (4fold) increase while 3MC-treated rats showed a small (1.5fold) increase. These results for phenobarbital and 3MC are consistent with those reported by others (Dragnev et al., 1995; Lubet et al., 1992), and suggest that the effects of D 5 on CYP3A enzymes are not consistent with potent inducers of


FIG. 8. Representative Western blots of CYP1A, CYP2B, CYP3A, and epoxide hydrolase. The amount of protein loaded onto each gel was as follows: CYP1A1/2, 6 mg of protein from control and D 5 microsomes, and 1 mg of protein from 3MC microsomes. CYP2B1/2, 6 mg of protein from control and D 5 microsomes, and 1 mg of protein from phenobarbital microsomes. CYP3A1/2, 6 mg of protein from control and D 5 microsomes, and 0.6 mg of protein from pregnenolone 16a-carbonitrile (PCN) microsomes. Epoxide hydrolase, 6 mg of protein from control and D 5 microsomes, and 2 mg of protein from from phenobarbital and 3MC microsomes. PCN microsomes were purchased from XenoTech LLC and used as positive controls. Each blot represents hepatic microsomal protein from 5 control and 5 D5-treated animals.

CYP3A enzymes, but are consistent with the effects observed for phenobarbital. Peroxisome-proliferating agents are characterized by their ability to bind to the cytosolic receptor PPAR, and then to DNA to elicit changes in gene expression (Gibson et al., 1993). To date, all agents classified as peroxisome proliferators increase the expression of CYP4A1 in rodent liver (Gibson et al., 1993). Therefore, changes in the activity and abundance of this enzyme can be used as a bio-indicator for peroxisome proliferation. 12-Hydroxylation of lauric acid is catalyzed primarily by CYP4A enzymes. Thus, large changes in lauric acid 12hydroxylase activity can be indicative of changes in CYP4A activity. The small change of 1.2-fold, observed following D 5 exposure in this study, combined with the observation that CYP4A1/2 immunoreactive protein levels did not increase relative to controls, suggests that D 5, under the conditions of this study, is not a peroxisome-proliferating agent. Several different UDP-glucuronosyltransferase (UDPGT) enzymes have been identified, some of which are inducible by various chemicals. For example, UDPGT activity towards 4-ni-

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trophenol is induced following treatment with 3MC, while UDPGT activity towards chloramphenicol is induced following treatment with phenobarbital (Bock et al., 1983). D 5 did not increase UDPGT activity towards 4-nitrophenol, an observation consistent with minimal effects on CYP1A1/2 enzymes, but did produce a small increase in UDPGT activity towards chloramphenicol. The pleiotropic response in gene expression resulting from phenobarbital exposure has been well-described and is characterized by a substantial increase in CYP2B1/2 enzymes with minor increases in CYP3A1/2, epoxide hydrolase, and UDPGT. In the present study, repeated inhalation exposure of female Fischer rats to 160 ppm D 5 vapors for 28 consecutive days resulted in a moderate increase in CYP2B1/2 (4-fold), CYP3A1/2 (2-fold), epoxide hydrolase (2-fold), and UDPglucuronosyltransferase (2-fold). The overall enzyme induction profile for hepatic CYP enzymes, epoxide hydrolase, and UDPGT enzymes reported here is consistent with the classical pleiotropic response described for phenobarbital (Nims et al., 1993). Many drugs and chemicals have been described as “phenobarbital-like” inducers. Some examples include loratadine, doxylamine succinate, and oxazepam (Bookstaff et al., 1996; Griffin et al., 1996; Langer et al., 1991; Parkinson et al., 1992; Skare et al., 1995). Each of these compounds has been shown to increase liver size, induce CYP2B1/2, and produce hepatic tumors in mice or rats in 2-year bioassays. Comprehensive epidemiology studies have reported that humans treated chronically with phenobarbital for epileptic seizures did not have an increase in tumor incidence related to phenobarbital (Clemmesen and Jensen 1978; Olsen et al., 1990, 1989, 1993; Shirts et al. 1986; Whysner et al., 1996). Because of the substantial amount of mechanistic data in the rat regarding the effects of phenobarbital and phenobarbital-like compounds, combined with the epidemiological data obtained in humans, phenobarbital and phenobarbital-like compounds have not been classified as human carcinogens. The changes in rat liver microsomal enzyme expression, measured following exposure to D 5, are nearly identical to those reported in an earlier study in which another cyclic siloxane, octamethylcyclotetrasiloxane (D 4), was tested (McKim et al., 1998). The major difference observed between the two agents was the greater magnitude of CYP2B induction (. 20-fold) reported for D 4 after repeated exposure to 700 ppm during a 4-week study. It is unclear at this time whether the difference in the magnitude of response to D 4 and D 5 can be attributed to tissue distribution, dose, or the physical properties of the compounds themselves. However, it is clear that when rats are exposed to D 4 or D 5, the alterations in hepatic enzyme expression are consistent with those produced by phenobarbital. Thus, under the experimental conditions described in this study, and based on the enzyme induction profile (“finger print“) obtained (Fig. 9), D 5 may be described as a weak phenobarbital-type inducer in the female Fischer-344 rat liver.

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MCKIM ET AL. Choudhuri, S., Zhang, X. J., Waskiewicz, M. J., and Thomas, P. E. (1996). Differential regulation of cytochrome P450 3A1 and P450 3A2 in rat liver, following dexamethasone treatment. J. Biochem. Toxicol. 10, 299 –307. Clemmesen, J., and Hjalgrim-Jensen, S. (1978). Is phenobarbital carciongenic? A follow-up of 8078 epileptics. Ecotoxicol. Environ. Saf. 1, 457– 470. Dragnev, K. H., Nims, R. W., Fox, S. D., Lindahl, R., and Lubet, R. A. (1995). Relative potencies of induction of hepatic drug-metabolizing enzyme genes by individual PCB congeners. Toxicol. Appl. Pharmacol. 132, 334 –342. Dutton, G. J., Leakey, J. E. A., and Pollard, M. R. (1981). Assays for UDP-glucuronosyltransferase activities. Methods Enzymol. 77, 383–391. Gibson, G. G., Chinje, E., Sabzevari, O., Kentish, P., and Lewis, D. F. V. (1993). Peroxisome proliferators as cytochrome P450 inducers. In Peroxisomes: Biology and Importance in Toxicology and Medicine (G. Gibson and B. Lake. Eds.), pp. 119 –133. Taylor & Francis, London. Giera, D. D., and Van Lier, R. B. (1991). A convenient method for the determination of hepatic lauric acid v-oxidation based on solvent partition. Fundam. Appl. Toxicol. 16, 348 –355.

FIG. 9. The hepatic microsomal enzyme induction profile or “finger print” obtained by comparing the relative increase in enzyme activities following treatment with the classical inducer phenobarbital, octamethylcyclotetrasiloxane (D 4), and decamethylcyclopentasiloxane (D 5). Values for D4 were taken from McKim et al., 1998. The induction responses for D 4 and D 5 are qualitatively identical to that obtained for phenobarbital; however, D 5 appears to be less effective than D 4 with regard to enzyme induction.

ACKNOWLEDGMENTS The authors express their sincere appreciation to Mr. Patrick W. Langvardt and Dr. Thomas H. Lane for providing technical advice and to Dr. Glenn Sipes, University of Arizona and Dr. Jay Goodman, Michigan State University for critically reviewing this manuscript.

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