EXPRESSION PROFILES OF CYTOCHROME P 450 S FOLLOWING SWIMMING EXERCISE IN AGING RATS

Background and Objective Cytochrome P450s (CYPs) are the major drug-metabolizing enzymes responsible for the clearance of approximately 75% of all drugs in clinical use. Drug clearance may be reduced with aging, resulting in increased drug toxicity in the elderly. Here, we evaluated changes in CYP expression following exercise in an aging rat model. Materials and Methods Sixteen male Sprague-Dawley rats were grouped into control (n = 5), short-term exercise (SE) (n = 4), and long-term exercise (LE) (n = 7) groups and changes in CYPs and nuclear receptors (NRs) were measured with aging. Results CYP2C22, CYP3A1, and CYP2C11 mRNAs were up-regulated, whereas CYP26B1 was downregulated in the SE and LE groups compared with the control group. Moreover, mRNA levels of the NRs, constitutive androstane receptor, retinoid X receptor α, peroxisome proliferator-activated receptor, and pregnane X receptor, were significantly increased in the LE group compared with those in the control group. As an indicator of more long-term changes, protein levels of CYP2C11, CYP2B, CYP1A, CYP2C, and CYP2C22 were significantly up-regulated in the LE group compared with the control group. Overall, our data show that CYP and NR expression increased in rats with forced long-term exercise during aging. Conclusion Therefore, we propose that regular swimming exercise may increase CYP levels, resulting in enhanced drug clearance and thereby reducing age-related drug toxicity in elderly individuals.

Cytochrome P450s (CYPs) are found in most living organisms and are mainly localized in the endoplasmic reticulum.However, some CYPs are also found in other subcellular compartments, such as the mitochondria and Golgi body.][3] Approximately two-thirds of human CYPs metabolize endogenous substrates, mediate the biosynthesis of steroids and eicosanoids, and control the degradation of vitamin D. Some CYPs (members of the CYP1-4 families) regulate oxidative reactions of xenobiotics such as drugs and environmental pollutants. 4,5For the metabolism of both endogenous factors and xenobiotics, CYPs require interaction with NADPH-P450 reductase and/or cytochrome b 5 as electron donors. 6YPs can be induced or inhibited by specific dietary components and xenobiotic compounds, including drugs and alcohol; altered CYP expression affects the biotransformation rate of enzymatic substrates. 7or example, grapefruit inhibits the expression of CYP3A4, which is involved in the metabolism of 60-80% of clinically used drugs. 8,9Therefore, drugs metabolized by CYP3A4 may cause adverse effects when taken with grapefruit.Moreover, aging alters the pharmacokinetics of drugs by reducing blood flow to the kidney and liver (20-30%) and through changes in liver mass.Drug metabolism in aging humans is associated with drug toxicity. 10Additionally, aging may alter the expression of many enzymes in the liver as well as that of hepatic NADPH cytochrome P450 reductase and polymeric immunoglobulin receptor.In rats, aging affects CYP2B1 and CYP2B2 levels. 11ging has also been shown to result in decreased antipyrine clearance by increasing the half-life of antipyrine in the plasma. 12YP expression is also affected by endogenous compounds, including hormones, growth factors, and cytokines. 13The levels of many endogenous compounds are altered by aging.Indeed, reduced drug metabolism capacity in elderly people may be a consequence of alterations in lean body mass, body fat, muscle mass, and total body water and reductions in plasma albumin levels and blood flow.5][16] Additionally, aerobic exercise activity increases the hepatic oxidative metabolism of drugs. 17or example, physical exercise enhances the clearance rate of spironolactone and hexobarbital. 18CYP levels were also found to be increased in rats after 14 days of exercise compared with that in sedentary rats. 19,20imilarly, physical activity in elderly individuals shows beneficial effects on oxidative metabolism in the liver, 21 and regular exercise contributes to the health of aged humans.Additionally, aerobic exercise activity increases the hepatic oxidative metabolism of low-clearance drugs. 17Regular physical activity can effectively reduce the risk of age-related disease and mitigate age-related drug toxicity by improving drug clearance. 22herefore, we hypothesized that regular exercise may contribute to proper drug clearance in elderly individuals.In the current study, we examined whether physical activity in elderly rats could improve drug toxicity.

Animal Care and Overview of the Experimental Design
Sixteen male Sprague-Dawley rats (4-weeks old) were purchased from the Korea National Sport University, in Seoul, Republic of Korea and housed two per cage in environmentally controlled conditions (temperature, 22°C; relative humidity, 51%), with a 12-hour light-dark cycle (lights on 7:00 am to 7:00 pm).The rats were randomly assigned to one of three groups: control (no exercise, evaluated at 50 weeks of age, n=5), swim short-term exercise (exercise for 4 weeks beginning at 46 weeks of age, n=4), and swim long-term exercise (exercise for 20 weeks beginning at 30 weeks of age, n=7; see Table 1).All rats were fed a standard irradiated chow diet (Purina Mills Inc., Gray Summit, MO, USA).Before experimentation, the animals were familiarized to the laboratory conditions by swimming for 10 min/day for 5 days.The study protocol was approved by the institutional ethics review board of the Korea National Sport University.

Exercise Training
Two rats at a time were trained to swim in a 55-gallon container filled with water to a depth of 3 feet.
The temperature of the water was maintained at 36°C.On the first day, the swimming time was 10 min/day, and the time was increased by 10 min each day to a maximum of 1 hour.The groups were trained to swim for 1 hour at the same time (9:00-11:00).Short-term swimming exercise was performed for 4 weeks starting at 46 weeks of age, whereas long-term swimming exercise was performed for 20 weeks starting at 30 weeks of age.After exercise, the rats were dried and placed back into their cages.The rats were sacrificed at 24 hours after the final exercise period.

Liver Microsome Preparation
The rats were anesthetized with an intraperitoneal injection of pentobarbital sodium (60 g/kg body weight [BW]).Rat livers were then harvested, weighed, homogenized in homogenizing buffer (0.1 M potassium phosphate buffer, pH 7.4, containing 0.125 M potassium chloride, 1.0 mM ethylenediaminetetraacetic acid [EDTA], and a protease inhibitor cocktail [Sigma, St. Louis, MO, USA]), and centrifuged at 13,000 × g for 20 minutes at 4°C, yielding the supernatant (S9 fraction).To obtain the microsomal fraction, the postmitochondrial fraction (S9 fraction) was centrifuged for 45 min at 250,000 × g.The resulting microsomal pellet was suspended in 10 mM Tris acetate buffer (pH 7.4) containing 0.1 mM EDTA and 23% glycerol and stored at −80°C.Protein concentrations were determined with a bicinchoninic acid protein assay kit (Thermo Scientific, Inc., Rockport, IL, USA) with bovine serum albumin as the protein standard.

RNA Extraction, cDNA Synthesis, and Quantitative Real-Time Polymerase Chain Reaction (qPCR)
Total RNA was extracted with RNA-Bee reagent (Tel-Test, Friendswood, TX, USA) according to the manufacturer's instructions.Total RNA (1 µg) was used for cDNA synthesis with a High Capacity cDNA Archive kit (Applied Biosystems, Foster City, CA, USA), and qPCR was performed with SYBR Green PCR Master Mix (Applied Biosystems) using an Eppendorf Mastercycler ep Realplex PCR instrument with 45 cycles (Eppendorf, NY, USA).GAPDH mRNA was used as a normalization control.The primer sets used in the qPCR are listed in Table 2. qPCR results were analyzed by the ΔΔCt method, as described by Livak and Schmittgen. 24

Statistical Analysis
All of the descriptive data are expressed in terms of the mean ± standard deviations.Kruskal-Wallis tests were used to examine differences in characteristics between groups, and post-hoc tests (Tukey tests using ranks) were used to determine differences related to groups.All analyses were performed using SPSS version 20.0 (SPSS, Chicago, IL, USA).p < 0.05 was considered significant.

Effects of Exercise on CYP mRNA Expression in Rat Livers
First, we evaluated changes in the hepatic expression of CYP mRNAs in rats with or without short-or long-term swimming exercise to determine whether regular exercise contributed to enhanced drug clearance in aged rats.Our qPCR data (Figure 1) showed that both short-and long-term exercise significantly up-regulated CYP2C22, CYP3A1, and CYP2C11 mRNAs and significantly decreased CYP26B1 mRNA compared with the levels in the control.CYP1A2, CYP4F4, CYP2B1, and CYP26A1 mRNA expression was slightly increased compared with that in controls; however, these differences were not significant.Additionally, the expression levels of CYP2B2, CYP26B1, and CYP3A2 mRNAs in livers from rats subjected to swimming exercise were similar to or lower than those of the control.Notably, the expression levels of CYP2B1, CYP3A1, CYP4F4, and CYP2C11 mRNAs were higher in rats after long-term exercise than in rats subjected to short-term exercise.In general, most CYPs examined in this study showed increased expression in both exercise groups.

Effects of Exercise on the mRNA Expression of Nuclear Receptors (NRs) in Rat Livers
NRs are important in the regulation of many CYPs and other phase I and phase II drug-metabolizing enzymes (DMEs).Therefore, we next measured the mRNA levels of NRs.The expression of constitutive androstane receptor (CAR), retinoid X receptor α (RXRMediuα), peroxisome proliferator-activated receptor (PPAR), and pregnane X receptor (PXR) was significantly higher in rats subjected to long-term exercise than in rats in the control group (Figure 2).In addition, CAR, RXRα, and PPARα mRNAs were also significantly up-regulated in rats subjected to short-term exercise compared to the control group.The mRNA levels of aryl hydrocarbon receptor (AhR) and RXR were not altered in either of the exercise groups compared to the control group.Notably, there were no significant differences in NR expression between short-and long-term exercise groups.

Changes in CYP Expression in the Aging Rat Group
Changes in hepatic CYP protein levels in the three groups are shown in Figure 3A and 3B.CYP2C11, CYP2B, CYP1A, CYP2C, and CYP2C22 protein FIG. 1 mRNA expression of CYPs in the exercise groups.Sprague-Dawley rats were grouped and subjected to short-or long-term exercise (or no exercise in the control).Hepatic mRNA levels of CYPs were measured by qPCR.The error bars represent the standard errors.Statistical comparisons among the three different groups were made using one-way ANOVA followed by Kruskal-Wallis tests.*p < 0.05 compared with control rats.FIG. 2 mRNA levels of nuclear receptors in rats.Hepatic mRNA levels of NRs were measured by qPCR.The error bars represent standard errors.Statistical comparisons among the groups were made using one-way ANOVA followed by Kruskal-Wallis tests.*p < 0.05 compared with control rats.levels were significantly increased in the longterm exercise group compared to the control group (p < 0.05).CYP4A, CYP3A, and CYP2E1 protein levels were slightly increased in the long-term exercise group compared to the control group; however, the differences were not statistically significant.Interestingly, CYP2D protein levels were slightly reduced in the long-term exercise group compared with the control group.CYP2C11, CYP2C, and CYP2C22 protein levels were significantly increased in the shortterm exercise group compared with the control group (p < 0.05).In contrast, the protein levels of CYP4A and CYP2E1 were not changed, and that of CYP2B was significantly reduced in the short-term exercise group compared with the long-term exercise group.CYP1A, CYP3A, and CYP2D were slightly up-regulated in the SE group compared with the control group; however, these differences were not statistically significant.In general, CYPs were expressed at higher levels in the long-term exercise group than in the control and short-term exercise groups.

DISCUSSION
Exercise controls steroid hormone levels.Both acute and regular exercise alters steroid hormone levels.Exercise is important in preventing and treating agerelated diseases and sarcopenia. 25The expression and activity of many enzymes, including DMEs, usually decrease with aging. 26,27Such reduced levels of DMEs can lead to reduced levels of drug clearance, causing enhanced drug toxicity in the elderly. 28,29Aminopyrine N-demethylase activity was previously shown to be increased by 130% with increased age. 30In this paper, we show that the expression levels of some CYPs increased with swimming exercise.Other studies have reported that swimming exercise increases antipyrine clearance by excretion. 31,32It was previously reported that CYP450 27B1, which metabolizes vitamin D, was significantly up-regulated between 1 and 3 hours after the end of exercise.Resistance exercises that improve vitamin D metabolism in skeletal muscle increased the levels of CYP450 27B1. 33CYP450 levels were increased in young and middle-aged rats with 8 weeks of moderate-intensity running exercise.Exercise has been shown to increase deacetylation of the toxic metabolite p-aminophenol in the kidney by 54% in young rats and by 26% in middle-aged rats. 34Exercise has also been shown to improve renal phase I drug metabolism without affecting the phase II processes. 34In addition, regular exercise increases the concentrations and activity of CYPs in the liver, 6,35 and cytochrome P450s, cytochrome b5, and NADPH cytochrome reductase are induced during exercise. 20he half-life of antipyrine in serum is significantly reduced in top athletes compared with that in physical education students. 6Additionally, sprinters and long-distance runners show significantly increased antipyrine clearance than sedentary subjects. 31,36aximal exercise has been shown to alter the activity of GH, IGF-1, and CYP1A2.High-intensity exercise in young ice hockey players increased the GH concentration, with a concomitant increase in CYP1A2 mRNA levels.The expression of CYP1A2 is related to the amount of exercise in athletes. 37These changes may be the result of increased blood flow throughout the body or increased expression of some CYPs in the liver, as observed for CYP2Cs, CYP2Bs, and CYP1A proteins in our study.Moreover, although CYP1A2 protein levels have been shown to decrease with aging, 38 we found that CYP1A protein levels were increased in the swimming exercise group compared with the control group.
A previous analysis of the hepatic expression and activity of CYPs in 3-, 12-, 26-, and 104-week-old rats showed that the levels of total CYPs, microsomal proteins, and cytochrome b5 changed with age in rats. 38For example, CYP1A2, CYP2B1, and CYP2E1 levels were increased in 3-week-old rats compared with those in 12-, 26-, and 104 week-old rats.Moreover, CYP2C11 and CYP3A2 expression has been shown to decrease with age. 39,40Similarly, our study confirmed that long-term regular exercise could delay the age-related decreases in some CYPs and DMEs.
The present results show that CAR, PXR, RXR, and PPAR mRNA levels were increased in the exercise groups compared with the control group.Phase I and phase II DMEs are regulated by CAR and PXR, which have important roles in the metabolism of fatty acids, lipids, and glucose in energy metabolism. 41xercise training increased Cyp7a1 and LXR mRNA expression in hepatic tissue.Regular aerobic exercise increases Cyp7a1 levels, increases the effective control of cholesterol in the liver, and prevents atherosclerosis by increasing HDL levels. 42PXR plays an important role in the regulation of CYP3A4, a major DME in humans. 43In a previous study, swimming exercise for 8 weeks was found to increase mRNA and protein expression of PPAR in elderly mice, and the expression of 3-hydroxyacyl CoA dehydrogenase and carnitine palmitoyl transferase-I, target genes of PPAR, was also increased in the heart. 44Thus, our findings support that exercise may prevent the increased drug toxicity observed in elderly individuals.

FIG. 3
FIG. 3 Expression of CYPs after exercise.A. Immunoblots of CYPs from hepatic micosomes.B. Quantification of immunoblots.Statistical comparisons among the groups were made using one-way ANOVA followed by Kruskal-Wallis tests.*p < 0.05 compared with control rats.

TABLE 1
Schematic Diagram of the Experimental Design

TABLE 2
Primer Sets used for qPCR