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Articles

Inducibility of Hepatic CYP1A Enzymes by 3-Methylcholanthrene and Isosafrole Differs in Male Rats Fed Diets Containing Casein, Soy Protein Isolate or Whey from Conception to Adulthood^{1 ,2}

Arkansas Children’s Nutrition Center, Arkansas Children’s Hospital Research Institute and Department of Pediatrics, University of Arkansas for Medical Sciences, Little Rock, AR 72205

³To whom correspondence should be addressed. E-mail: RonisMartinJ{at}UAMS.edu.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Hepatic cytochrome P₄₅₀ (CYP)1A1 and 1A2 enzymes were studied in male Sprague-Dawley rats derived from 5–7 litters fed diets in which the protein source was casein, soy protein isolate or whey. At age 65 d, rats were gavaged with corn oil (vehicle), 40 mg/kg 3-methylcholanthrene (3-MC) or 75 mg/kg isosafrole (ISO). Hepatic expression of CYP1A1 and CYP1A2 mRNA, apoprotein and associated monooxygenase activities were measured 17 h later. No significant dietary effects were observed on basal expression of either enzyme. However, interactions between diet and the two inducers (3-MC and ISO) were observed in soy-fed rats for ethoxy- and methoxyresorufin O-dealkylase activity, CYP1A1 and CYP1A2 apoprotein and mRNA (P < 0.05). The level of induction of CYP1A1 mRNA and apoprotein was lower in rats fed soy diets than in rats fed casein diets (P < 0.05), and the level of induced CYP1A2 mRNA was lower in rats fed soy or whey (P < 0.05) after treatment with the aryl hydrocarbon (Ah) receptor–dependent inducer 3-MC. This was accompanied by a 50% reduction in constitutive levels of the Ah receptor in liver cytosol of soy-fed, relative to casein-fed rats, and a slightly smaller reduction in whey-fed rats. Expression of the Ah receptor correlated with 3-MC–inducibility of CYP1A1 mRNA in rats fed the three diets. In contrast, in rats induced with ISO, which does not bind to the Ah receptor and induces CYP1As via different mechanisms than 3-MC, ethoxyresorufin O-deethylase activity and levels of CYP1A1 apoprotein and mRNA were elevated to a greater degree in soy-fed than in casein- or whey-fed rats (P < 0.05). Moreover, after ISO treatment, induction of methoxyresorufin O-demethylase activity, CYP1A2 apoprotein and mRNA levels was observed only in rats fed soy (P < 0.05). These data suggest potential effects of dietary protein source on metabolism of a wide variety of CYP1A substrates, including environmental and dietary carcinogens, many of which induce their own metabolism.

KEY WORDS: • soy protein isolate • CYP1A • induction • rats • whey protein

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Diet has long been recognized as an important modulator of cancer risk (1) . Epidemiologic studies have linked consumption of traditional Asian diets with a significantly lower incidence of breast, prostate and colon cancers (2) . Asian diets are high in soybean products such as tofu and miso, and soy-containing diets have been reported to provide significant protection against breast cancer in animal models (3 ,4) . It has been suggested that soy-associated isoflavones, primarily genistein and daidzein, are responsible for the cancer chemoprotective effects, and studies with diets containing purified isoflavones have demonstrated significant protection in rodent breast cancer models (3 ,5) . In addition to dietary phytochemicals such as isoflavones, epidemiologic and experimental studies suggest that consumption of bovine dairy products may also confer cancer protection (4 ,6 ,7) . This effect has been linked to a class of proteins that comprise 20% of the total milk protein, namely, the whey fraction (4 ,6 7 8) .

One potential chemoprotective mechanism is the effect of different diets on procarcinogen bioactivation and carcinogen metabolism to reduce cancer initiation. In general, increased metabolism, coupling phase I oxidation with phase II conjugation, will lead to faster clearance of ingested procarcinogens such as polycyclic aromatic hydrocarbons (PAH)⁴ and result in significantly fewer DNA adducts and mutations for a given dose (9) . This could be the result of increased hepatic metabolism, which would increase systemic clearance and reduce circulating concentrations of the carcinogen; alternatively, enhanced local metabolism within the carcinogen target tissue could be responsible (10) . Such a mechanism could explain the decrease in DNA adducts, which was reported in mammary tissue of rats after treatment with the mammary carcinogen 7,12-dimethylbenz[a]anthracene (DMBA) in rats fed soy protein isolate or genistein compared with those fed casein-based diets (11) .

A number of in vitro and in vivo rodent studies have suggested that treatment with purified isoflavones or consumption of soy-based diets induces phase II enzymes such as glutathione S-transferases (GST), quinone reductase and UDP-glucuronyl-transferases (UDPGT) in the liver, small intestine and colon (12 ,13) . In addition, our laboratory reported significant increases in expression of hepatic GST activities in liver microsomes from female rats fed diets made with whey protein compared with rats fed casein diets after exposure to DMBA (14) .

In contrast, less information exists on the effects of soy protein diets, whey protein diets or soy-associated isoflavones on the expression and inducibility of phase I cytochrome P₄₅₀ (CYP) enzymes. We previously demonstrated increased hepatic expression and dexamethasone inducibility of CYP3A2 and associated monooxygenase activities in male rats fed diets containing soy protein isolate compared with rats fed casein (15) . However, the effects of soy- and whey-based diets on the expression and inducibility of CYP1 family members, which are important in metabolism of PAH such as DMBA, are not known. A number of in vitro studies on CYP1 expression have been performed with purified isoflavones. However, the results were variable, depending on the cell type examined and the inducer used. In rat hepatoma H4IIE cells, Backlund et al. (16) reported inhibition of omeprazole-mediated induction of CYP1A1 by genistein and daidzein, but no effect on CYP1A1 induction by the aryl hydrocarbon (Ah) receptor ligands 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and benzo(a)pyrene. In contrast, using human keratinocytes, Gradin et al. (17) reported inhibition of TCDD-mediated CYP1A1 induction by genistein. Similar results with genistein were reported by Hukkanen et al. (18) using a human alveolar type II cell–derived A549 adenocarcinoma cell line, but TCDD-mediated CYP1B1-induction was unaffected. It is unclear whether these in vitro data can be extrapolated to predict effects of soy diets in vivo because the concentrations of isoflavones utilized were much higher than those attained in the body after soy consumption, and there are many other phytochemical components in soy in addition to the isoflavones including saponins, phytosterols, phenolic acids and coumarins (3) .

In this study, we examined the effects of a diet made with casein, soy protein isolate high in isoflavones or whey protein on activity, expression and inducibility of hepatic CYP1 family members. We focused on induction of CYP1 family members by the PAH 3-methylcholanthrene (3-MC) and the methylenedioxyphenyl compound isosafrole (ISO). The former represents a prototypical environmental procarcinogen, which induces its own metabolism by CYP1A enzymes primarily via binding to the Ah receptor (19) . The latter is a phytochemical widely found in the human diet in foodstuffs, essential oils and flavors. It has been extracted from carrots, parsnips, parsley, pepper, sesame seeds/oil, nutmeg and sassafras (20) . Isosafrole does not bind the Ah receptor and although it requires the Ah receptor to be present for CYP1A1 induction, it induces CYP1A2 via an Ah receptor–independent mechanism (21 22 23) .

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Chemicals and reagents.

3-Methylcholanthrene, ethoxyresorufin and NADPH were obtained from Sigma Chemical (St. Louis, MO). Isosafrole (97% mixture of cis and trans isomers) was purchased from Aldrich Chemical (Milwaukee, WI). Resorufin was purchased from Pierce Chemical (Rockford, IL) and methoxyresorufin was purchased from Molecular Probes (Eugene, OR). Enhanced chemiluminescence (ECL) kits containing monospecific antibodies toward rat liver CYP1A1 and 1A2, "ECL plus" kits and Hydrobond P blotting membranes were obtained from Amersham Pharmacia Biotech (Piscataway, NJ). Goat polyclonal antibodies directed against N-terminal amino acids 3–22 of the mouse Ah receptor linked to keyhole limpet hemocyanin and which cross-react with the rat Ah receptor (24) were purchased from Novus Biologicals (Littleton, CO). Donkey anti-goat immunoglobulin (Ig)G antibodies linked to horseradish peroxidase and nitrocellulose blotting membranes were obtained from BioRad Laboratories (Hercules, CA).

cDNA probes.

cDNAs against mouse CYP1A1 and CYP1A2 were obtained from the American Type Culture Collection (Manassas, VA).

The experiment received prior approval from the Institutional Animal Care and Use Committee at UAMS. All animals were housed in an AAALAC-approved animal facility at ACHRI, and all animal housing and husbandry conformed to USDA guidelines.

Animals and diets.

Virus-free adult breeder female and male Sprague-Dawley rats were purchased from Harlan Industries (Indianapolis, IN). They were housed individually in polycarbonate cages and allowed free access to water and pelleted food. Rats were randomly assigned to three groups and fed one of three semipurifed diets made according to the AIN-93G diet formula (25) , except that corn oil replaced soybean oil and the protein source was casein (CAS; New Zealand Milk Products, Santa Rosa, CA), whey protein (WHEY; New Zealand Milk Products), or soy protein isolate (SPI; Protein Technologies International, St. Louis, MO). Diets containing SPI had 430 mg total isoflavones/kg diet, including 276 mg/kg genistein and 132 mg/kg daidzein. Average daily consumption of isoflavones was 19.3 mg/(kg · d) genistein and 9.2 mg/(kg · d) daidzein, resulting in total isoflavone concentrations of 40 µmol/L in 24-h urine pools (26) . Essential amino acids were added to each diet as listed in Table 1 . After several weeks of consuming the semipurified diets, male and female rats were mated, and pregnant dams were fed their respective diets during gestation. At birth, pups were culled to five males and five females per litter; lactating dams continued to consume their respective diets until the pups were weaned. At weaning, pups had continued free access to their respective diets. Female pups were utilized in a DMBA-induced mammary tumor experiment reported previously (4) and male pups were fed their respective diets until the beginning of the current induction experiment at age 65 d.

At 65 d of age, one male pup from each of 4–6 litters fed CAS, WHEY or SPI diets was gavaged orally with 2 mL of corn oil or 40 mg/kg 3-methylcholanthrene (3-MC) or 75 mg/kg isosafrole (ISO) in 2 mL corn oil at 1600 h. At 0900 h the next morning (17 h later), the rats were killed by decapitation, livers removed and microsomes and cytosol prepared using the differential ultracentrifugation method of Chipman and Walker (27) .

Monooxygenase activities.

Ethoxyresorufin O-deethylase (EROD) a rat CYP1A1 selective activity (28) and methoxyresorufin O-demethylase (MROD) an activity suggested to be selective for CYP1A2 (29) were measured by following the formation of resorufin spectrofluorimetrically at 536 nm (excitation) and 586 nm (emission) (28) using an RF-5301PC scanning spectrofluorometer (Shimadzu Scientific Instruments, Columbia, MD) under conditions of linearity for incubation time and protein.

Western immunoblot analysis.

Western blotting was conducted on pools of liver microsomes derived from 5–7 rats at a concentration of 10 µg protein/well with monospecific primary antibodies directed against CYP1A1 or CYP1A2 (Amersham Pharmacia Biotech) to illustrate differences in mean expression of these apoproteins in vehicle-, 3-MC– and ISO-treated rats fed the different diets. In addition, Western blots were performed on hepatic microsomes from individual rats and blotted onto nitrocellulose membranes for the purpose of immunoquantitation of CYP1A1 and 1A2 apoproteins in rats fed different diets and inducers. Western blot signals were detected using a horseradish peroxidase–linked goat anti-rabbit IgG and ECL according to manufacturers instructions (Amersham Pharmacia Biotech). Additional Western immunoblot analysis was conducted on liver cytosol obtained from individual control rats fed all three diets and treated with the corn oil vehicle to quantitate diet effects on constitutive expression of the Ah receptor. Cytosolic protein (100 µg/well) was loaded and separated by SDS-PAGE on 10% polyacrylamide gels. The separated proteins were blotted onto Hydrobond P membranes (Amersham Pharmacia Biotech) and probed with a goat polyclonal antibody from Novus Biologicals, which was raised against the N-terminal amino acid sequence of the mouse Ah receptor and which also specifically cross-reacts with the rat Ah receptor (24) , at a concentration of 2 mg/L. Donkey anti-goat IgG linked to horseradish peroxidase was used as the secondary antibody at a dilution of 1:10,000. Blots were developed with "ECL plus" (Amersham Pharmacia Biotech). Immunoquantitation was determined by densitometric scanning of the autoradiographs using a GS525 molecular imager (BioRad Laboratories).

Northern analysis.

Steady-state mRNA levels of rat CYP1A1 and CYP1A2 were measured using cDNAs for mouse CYP1A1 and CYP1A2. Total RNA was isolated by the TRI Reagent according to the manufacturer’s protocol (Molecular Research Center, Cincinnati, OH). For analysis, total RNA (20 µg) from each sample was separated on a 1.2% agarose, 1 mol/L formaldehyde gel and stained with ethidium bromide to confirm its integrity. The RNA was transferred onto nylon membrane (Hybond-N, Amersham Pharmacia Biotech) by capillary action and bound to the membrane by heat (80°C for 2 h). The cDNAs were labeled with [-³²P]dCTP using the Random Primed DNA Labeling Kit (Boehringer Mannheim, Indianapolis, IN) according to the manufacturer’s instructions and was added at 16.6 MBq/L to hybridization buffer (Rapid-hyb, Amersham Pharmacia Biotech). Hybridizations were performed in roller bottles for 24 h at 42°C. Excess probe was removed by two 15-min washes at room temperature in 150 mmol/L NaCl, 10 mmol/L NaH₂PO₄ and 1 mmol/L EDTA, pH 7.4 (1X SSPE) plus 1% SDS followed by two 30-min washes at 50°C in 0.1X SSPE, 1% SDS. Bands were quantitated by densitometry of the autoradiograph, ethidium bromide–stained gel image (7 S rRNA) or phosphoimager (Bio-Rad) and the ratio of CYP1A1 or 1A2 message/7S RNA was determined and expressed as a percentage of that for the control (30) .

Statistical analysis.

Data are means ± SEM. All data were analyzed using the SigmaStat for Windows program (Jandal Scientific Software, San Rafael, CA). Most data were analyzed by two-way ANOVA followed by the Student-Newman-Keuls test for all pairwise means comparisons to detect differences among groups. An -level of 0.05 was set to determine significance. When variance was heterogeneous, nonparametric analysis by two-way ANOVA of ranks was utilized. Correlation analysis was conducted to compare constitutive Ah receptor expression with induction of CYP1A1 mRNA by 3-MC.

	RESULTS

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General.

General variables, including body weight, liver weight and yield of microsomal protein for rats fed the three different diets and treated with corn oil vehicle, 3-MC or ISO, are presented in Table 2 . Two-way ANOVA of the body weight data from rats fed the three different diets throughout life revealed significant differences (P < 0.001); SPI- and WHEY-fed rats were slightly smaller than CAS-fed rats even though, using the crude method of measuring the food and water remaining in the hoppers or bottles each day as a measure of the previous days intake, no significant differences in food intake were noted among the groups. During the week before the induction experiment, food intakes were as follows: CAS, 63 ± 1 g/(kg · d); SPI, 61 ± 1 g/(kg · d); and WHEY, 62 ± 0.2 g/(kg · d). In addition, there were diet and inducer effects on liver weight and the liver/body weight ratio (P < 0.001). Relative livers weights from WHEY-fed rats were greater than those from CAS- or SPI-fed rats (P < 0.05). However, the yield of liver microsomes/g liver was reduced in the WHEY-fed group of rats compared with those fed CAS or SPI, regardless of treatment (P < 0.05). Therefore, even though WHEY consumption increased relative liver weight and volume, liver membrane protein content was unaffected. In addition, although treatment with 3-MC increased liver weight in rats fed all three diets (P < 0.05), ISO treatment reduced liver weight (P < 0.05).

Data demonstrating the effects of diet and treatment with 3-MC or ISO on hepatic monooxygenase activities dependent on CYP1A enzymes are presented in Table 3 . The data for EROD and MROD are presented as specific activity per milligram microsomal protein and also as activity per 100 g body weight to take into account diet and inducer differences in body weight, liver weight and microsomal yield. Two-way ANOVA of ranks demonstrated diet effects and inducer-associated increases in EROD activity and a diet x inducer interaction with ISO treatment (P < 0.05). Both 3-MC and ISO induced EROD activity relative to that of corn oil–treated controls (P < 0.05). However, ISO induced greater EROD activity in SPI-fed compared with CAS-fed rats (P < 0.05). Two-way ANOVA of ranks demonstrated different effects of 3-MC and ISO treatment on MROD activity and a diet x inducer interaction (P < 0.05). 3-MC treatment induced MROD activity 13- to 17-fold in rats fed all three diets (P < 0.05). However, ISO induced MROD activity (sevenfold) only in SPI-fed rats (P < 0.05).

The results of Western immunoblot analysis using monospecific antibodies directed against rat CYP1A1 and 1A2 are shown in Figures 1 , 2 , 3 . The differences in 3-MC and ISO induction of CYP1A1 in CAS- and SPI-fed rats are illustrated in Figure 1 (upper panel) and differences in CYP1A2 are shown in Figure 1 (lower panel). Immunoquantitation of CYP1A1 expression in individual rats is shown in Figure 2 . In agreement with the EROD activity data, two-way ANOVA revealed induction of CYP1A1 apoprotein by both 3-MC and ISO in rats fed all three diets (P < 0.05). However, there were also diet x inducer interactions (P < 0.05). A lower level of 3-MC induction of CYP1A1 was observed in SPI-fed rats compared with those fed CAS or WHEY protein diets (P < 0.05). In contrast, after ISO treatment, CYP1A1 apoprotein induction was greater in SPI-fed rats than that observed in CAS- or WHEY-fed rats (P < 0.05). Thus, the ratio of CYP1A1 apoprotein expression after 3-MC and ISO treatment was shifted from 6:1 in CAS-fed and WHEY-fed rats to 5:7 in SPI-fed rats. Immunoquantitation of CYP1A2 is shown in Figure 3 . In agreement with the MROD activity data, two-way ANOVA revealed diet and inducer differences and diet x inducer interactions on expression of CYP1A2 apoprotein (P < 0.05). Although 3-MC induced CYP1A2 apoprotein to approximately the same degree in rats fed all three diets, treatment with ISO resulted in CYP1A2 apoprotein induction only in SPI-fed rats (P < 0.05).

View larger version (17K): [in this window] [in a new window]   Figure 1. Western immunoblots illustrating the effects of soy protein isolate (SPI) consumption and induction by 3-methylcholanthrene (3-MC, lanes 2 and 5) and isosafrole (ISO, lanes 3 and 6) on expression of cytochrome P450 (CYP)1A1 (upper panel) and 1A2 (lower panel) apoproteins in male rat liver microsomes compared with corn oil (lanes 1 and 4). Samples represent pools containing 10 µg of hepatic microsomal protein derived from casein-fed rats (CAS, n = 6) or SPI-fed rats (n = 5).

View larger version (16K): [in this window] [in a new window]   Figure 2. Immunoquantitation of cytochrome P450 (CYP)1A1 cross-reactive apoprotein expression in hepatic microsomes derived from individual rats fed casein (CAS), soy protein isolate (SPI) or whey and induced with 3-methylcholanthrene(3-MC) or isosafrole (ISO). Data are presented as means ± SEM for rats derived from 4–6 litters/group. The litter is the experimental unit. Bars not sharing a letter differ significantly P < 0.05.

View larger version (16K): [in this window] [in a new window]   Figure 3. Immunoquantitation of cytochrome P450 (CYP)1A2 cross-reactive apoprotein expression in hepatic microsomes derived from individual rats fed casein (CAS), soy protein isolate (SPI) or whey and induced with 3-methylcholanthrene (3-MC) or isosafrole (ISO). Data are presented as means ± SEM for rats derived from 4–6 litters/group. The litter is the experimental unit. Bars not sharing a letter differ significantly P < 0.05.

Northern analyses were conducted using total mRNA isolated from the liver (Figs. 4 , 5 , 6 ). Differences were observed in responses to the two inducers and for each inducer, among rats fed different diets (P < 0.05). 3-MC and ISO induced CYP1A1 mRNA (P < 0.05). However, the level of induction was greater in the liver of rats fed CAS-based diets than in rats fed SPI-based diets (Fig. 5 ; P < 0.05). In contrast, the level of induction of CYP1A1 mRNA by ISO was greater in SPI-fed rats than in rats fed CAS or WHEY (P < 0.05), and the ratio of CYP1A1 mRNA expression after 3-MC and ISO treatment was shifted from 6:1 in CAS-fed rats to 1:1 in SPI-fed rats. CYP1A2 mRNA was highly induced by 3-MC treatment in rats fed all three diets. However, greater CYP1A2 mRNA was expressed in CAS-fed 3-MC–treated rats than in rats fed SPI or WHEY diets (Fig. 6 ; P < 0.05). In contrast, although a small but significant induction of CYP1A2 mRNA was observed after ISO treatment in CAS- and WHEY-fed rats, a greater degree of CYP1A2 mRNA expression was observed in ISO-treated, SPI-fed rats (P < 0.05).

View larger version (40K): [in this window] [in a new window]   Figure 4. Cytochrome P450 (CYP)1A1 and CYP1A2 steady-state mRNA expression in livers from rats fed diets made with casein (CAS), soy protein isolate (SPI) or whey with or without 3-methylcholanthrene (3-MC) or isosafrole (ISO) treatment.

View larger version (16K): [in this window] [in a new window]   Figure 5. Densitometric quantitation of cytochrome P450 (CYP)1A1 mRNA expression in livers from rats fed different diets, casein (CAS); soy protein isolate (SPI) or whey (WHEY), with or without 3-methylcholanthrene (3-MC) or isosafrole (ISO) treatment. Data represent means ± SEM for Northern blots conducted using total hepatic mRNA and corrected for loading by using the ratio of CYP1A1 signal to that of 7S RNA. Data are derived from rats in 4–6 litters/group. The litter is the experimental unit. Bars not sharing a letter differ significantly, P < 0.05.

View larger version (17K): [in this window] [in a new window]   Figure 6. Densitometric quantitation of cytochrome P450 (CYP)1A2 mRNA expression in livers from rats fed different diets, casein (CAS), soy protein isolate (SPI) or whey (WHEY), with or without 3-methylcholanthrene (3-MC) or isosafrole (ISO) treatment. Data represent means ± SEM for Northern blots conducted using total hepatic mRNA and corrected for loading by using the ratio of CYP1A2 signal to that of 7S RNA. Data are derived from rats in 4–6 litters/group. The litter is the experimental unit. Bars not sharing a letter differ significantly, P < 0.05.

Western analysis of Ah receptor expression was conducted in liver cytosol from rats fed all three diets and treated with the corn oil vehicle (Figs. 7 and 8) . Specific antisera directed against the N-terminal of the mouse Ah receptor recognized two apoprotein bands at 104 and 106 kDa (Fig. 7 ). This is consistent with the two molecular weight variants of the rat receptor reported by Poland et al. (24) . There was considerable interindividual variability in the level of Ah receptor expression, which is consistent with the variability in constitutive receptor expression in Sprague-Dawley rat liver reported by Pollenz et al. (31) . Mean Ah receptor expression was reduced 56% in rats fed SPI relative to those fed casein and 33% in rats fed WHEY (Fig. 8 ). Due to the small sample size and large interindividual variability, the reduced expression in SPI-fed rats was not significant (P = 0.13). However, an excellent correlation (r² = 0.0995; P = 0.015) was observed between mean constitutive expression of the Ah receptor in hepatic cytosol and 3-MC–mediated induction of hepatic CYP1A1 mRNA in rats fed the three diets (Fig. 9 ).

View larger version (49K): [in this window] [in a new window]   Figure 7. Western immunoblots illustrating interindividual variability and diet effects on aryl hydrocarbon (Ah) receptor expression in cytosol from the livers of individual rats fed casein (CAS), soy protein isolate (SPI) or whey (WPH), 17 h after treatment with corn oil vehicle. Samples were loaded at 100 µg cytosolic protein/well.

View larger version (10K): [in this window] [in a new window]   Figure 8. Immunoquantitation of aryl hydrocarbon (Ah) receptor cross-reactive apoprotein expression (upper 106 kDa band in Fig. 7 ) in hepatic cytosol derived from individual rats fed casein (CAS), soy protein isolate (SPI) or whey (WHEY), 17 h after treatment with corn oil vehicle. Data are presented as means ± SEM for rats from 4–6 litters/group. The litter is the experimental unit. ADU = arbitrary densitometric unit

View larger version (16K): [in this window] [in a new window]   Figure 9. Correlation analysis comparing the mean expression level of the liver cytosolic aryl hydrocarbon (Ah) receptor in rats fed different diets, casein (CAS); soy protein isolate (SPI) or whey (WHEY), after treatment with corn oil vehicle and mean expression of cytochrome P450 (CYP)1A1 mRNA in livers from the same groups 17 h following induction with 3-methylcholanthrene (3-MC).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

One potential protective mechanism underlying dietary effects might be an alteration in procarcinogen activation and detoxification. This might involve inhibition of cytochrome P₄₅₀–catalyzed procarcinogen activation. An example is the inhibition of nitrosamine-induced carcinogenesis by diallyl sulfide and other components of garlic through inhibition of CYP2E1 (35 36 37) . Alternately, a similar effect might be produced by induction of phase II conjugation in the absence of effects on phase I systems, such as that observed after dietary selenium treatment (10) and treatment with many chemical electrophiles (9) . In general, coupled increases in hepatic phase I activation and phase II conjugation would also result in chemoprotection as the result of increased clearance of the carcinogen and reduced concentrations in the target tissues. The end result of any such metabolic changes would be fewer DNA adducts in the carcinogen target tissue. Such a decrease was observed for mammary DNA adducts after DMBA treatment in rats fed soy protein isolate and diets containing genistein (11) . Thus, we are interested in the effects of soy and whey diets on the expression and inducibility of cytochrome P₄₅₀ enzymes known to be important in carcinogen metabolism; in this study, we focused on CYP1A1 and CYP1A2, which are important in the metabolic activation of PAH such as DMBA and aromatic amines produced in cooking such as PhIP and MeIQ (38) .

In this study, we demonstrated for the first time that consumption of soy protein isolate results in significant effects on the inducibility of hepatic CYP1A enzymes in a male rat model compared with rats fed diets containing the milk proteins, casein or whey. Specifically, SPI-fed rats had reduced induction of EROD and MROD activity and a significantly reduced induction of CYP1A1 mRNA, apoprotein and CYP1A2 mRNA by the PAH 3-MC compared with rats fed casein. The reduction of CYP1 enzyme family induction might contribute to the cancer chemoprotection we reported in female rats fed SPI relative to CAS-fed rats after treatment with the PAH procarcinogen DMBA because, similar to other PAH, DMBA induces its own metabolism and CYP1 family enzymes are involved in DMBA activation (14 ,39 40 41) . We are currently verifying the diet effects on DMBA-induced CYP1A genes in female rats.

In contrast, induction of CYP1A1 mRNA, apoprotein and activity by the methylenedioxyphenyl phytochemical isosafrole was significantly enhanced in rats fed SPI compared with those fed CAS. Moreover, although a small increase in CYP1A2 mRNA was observed with ISO in CAS- and WHEY-fed rats, significantly greater induction of CYP1A2 mRNA and significant induction of CYP1A2 apoprotein and activity after ISO treatment was observed only in SPI-fed rats. These data have important implications compared with the patterns of CYP1A1 and CYP1A2 induction by 3-MC and ISO reported for rats fed nonpurified "chow" diets as opposed to semipurified diets such as those used in the current study. The 3-MC induction of CYP1A1 and CYP1A2 observed in CAS- and WHEY-fed rats appears to resemble that reported for "chow"-fed rats (29) , whereas the significantly greater induction of CYP1A2 than CYP1A1 by ISO reported in "chow"-fed rats (29 , 42) was not observed in the current study in rats fed any of the semipurified diets. In fact, little or no induction of CYP1A2 by isosafrole was observed in either the CAS- or WHEY-fed rats. Thus, this may indicate the presence in nonpurified rat diets of dietary factors that enhance the inducibility of CYP1A2 by some prototypical inducers such as ISO compared with purified diets containing milk proteins. Among these dietary factors may be soy-associated phytochemicals such as the isoflavones, genistein and daidzein. Rats fed SPI-containing diets had significantly greater CYP1A2 induction after ISO treatment than rats fed CAS or WHEY, and soy is a common component of many nonpurified rat diets.

The mechanisms underlying the effects of diet on CYP1A enzyme induction remain unclear. The current experiment utilized only one dose of inducer and measured hepatic CYP1A effects at only one time point. Comprehensive dose and time-course experiments will be required to characterize fully the different responses in rats fed the three diets. In addition, although rate-limiting essential amino acids were supplemented, it is possible that differences in intake of essential or nonessential amino acids might alter hepatic metabolism. The effects of SPI diets may result from the soy protein itself or from associated phytochemicals such as isoflavones, saponins, phytosterols or polyphenols. Moreover, it is not clear whether the diet effects represent metabolic imprinting as the result of exposure during early development or are direct effects of diet consumption during induction. We are planning comprehensive feeding studies with phytochemical-stripped SPI and casein supplemented with soy-associated phytochemicals during different developmental periods to address these important issues.

Induction of CYP1A1 by 3-MC is thought to be mediated via binding to the Ah receptor, which then dissociates from a cytosolic complex with HSP90 and other proteins, translocates to the nucleus where it forms a heterodimer with the transcription factor ARNT and the heterodimer, and then activates CYP1A1 gene expression by binding to a response element on the CYP1A1 promoter known as the XRE (19) . Our data demonstrate significant interindividual variability in Ah receptor expression in Sprague-Dawley rat liver. This is consistent with the data of Pollenz et al. (31) and may be the result of genetic variability because Sprague-Dawley is an outbred strain of rats. We found a >50% decrease in mean constitutive Ah receptor expression in liver from SPI-fed rats and a smaller decrease in those fed WHEY. Ah receptor expression was significantly correlated with 3-MC–mediated induction of hepatic CYP1A1 mRNA in rats fed the three diets, suggesting that diet-associated alterations in Ah receptor expression could explain the effects of soy on CYP1A inducibility.

In vitro studies from other laboratories using keratinocyte and alveolar adenocarcinoma cell lines with purified soy-associated isoflavone genistein have reported inhibition of Ah receptor–mediated CYP1A1 induction by TCDD; it was suggested that the effect was the result of tyrosine kinase inhibition by genistein and altered phosphorylation of HSP90 in the cytosolic Ah receptor complex, resulting in impaired translocation of the Ah receptor into the nucleus (17) . A similar study in a liver-derived hepatoma cell line reported no effect of genistein or daidzein on TCDD or benzo(a)pyrene–induced CYP1A1 induction (16) . However, the concentration of genistein utilized in all of these in vitro studies (50–100 µmol/L) was substantially higher than the plasma genistein concentration that can be achieved in these rats after SPI consumption (0.5 µmol/L total genistein including conjugates, of which <20% appears to be present as the aglycone) (34) .

Although significant decreases in CYP1A2 mRNA were also observed in SPI- and WHEY-fed rats after 3-MC induction, no effects were observed at the apoprotein level. It is possible that diet effects on post-translational stabilization of CYP1A2 by 3-MC might cancel out any reduction of CYP1A2 mRNA because PAH have been suggested to lengthen CYP1A protein half-life in addition to increasing their synthesis (43) .

ISO does not bind to the Ah receptor (44) . However, induction of CYP1A1 by ISO does appear to require the Ah receptor to be present because ISO has been reported to be incapable of inducing CYP1A1 in DBA mice possessing a low affinity Ah receptor mutation (22) or in Ah receptor knockout mice (23) . Activation of the Ah receptor pathway must be indirect, and it appears that consumption of SPI may stimulate these as yet uncharacterized pathways. A possible role for components of the Ah receptor pathway such as receptor activation, nuclear translocation or heterodimerization with ARNT in soy effects on CYP1A1 induction by isosafrole in vivo remains to be determined.

ISO induction of CYP1A2 expression has been reported to be Ah receptor independent (22 ,23) . Methylenedioxyphenyl compounds such as ISO also form inhibitory complexes with cytochrome P₄₅₀, such as CYP1A2, resulting in post-translational stabilization effects in addition to transcriptional regulation (20 21 22 23) . There was no spectral evidence for residual ISO/P₄₅₀ complexes remaining 17 h post-treatment in liver microsomes from our ISO-treated rats, and CYP1A2 apoprotein and MROD activity were correlated. The effects of SPI-containing diet consumption reported here on ISO induction of CYP1A2 must be the result of interactions with as yet uncharacterized, Ah receptor–independent pathways.

In conclusion, we demonstrated significant effects of soy consumption on the inducibility of CYP1A enzymes by 3-MC and ISO compared with rats fed the milk-derived proteins, casein or whey. These data suggest that there may be a significant interaction among soy, methylenedioxyphenyl phytochemicals, PAH and other dietary components that are known CYP1A inducers and that diet may contribute significantly to interindividual differences in hepatic CYP1A1 and CYP1A2 expression. The CYP1A1, 3-MC induction data are consistent with an impairment in the activation of PAH procarcinogens in rats fed soy because PAH consumption induces its own metabolism. This may contribute to the mechanism underlying cancer prevention after dietary soy consumption. In contrast, the relative lack of effect of whey consumption on CYP1A activities or inducibility suggests that the anticarcinogenic effects of whey are not mediated via dietary-induced changes in this pathway.

	ACKNOWLEDGMENTS

 
The authors wish to acknowledge the excellent technical assistance of Terry Fletcher, David Irby, Cynthia Mercado, Drew Holder, Shanda Sharp, Greg Hird and Ling He in the conduct of these studies. The soy protein isolate utilized in these studies was the gift of Protein Technologies (St. Louis, MO).

	FOOTNOTES

 
¹ Presented in part at Experimental Biology 99, April 1999, Washington, DC [Ronis, M.J.J., Mercado, C., Hird, G., Rowlands, J. C., Hakkak, R. & Badger, T. M. (1999) Effects of dietary consumption of soy protein isolate on expression and inducibility of CYP1A enzymes in rat liver. FASEB J. 13: A583 (abs.)].

² Funded in part by the U.S. Department of Agriculture/Agricultural Research Service under CRLS No. 6251-51000-002-02S.

⁴ Abbreviations used: Ah, aryl hydrocarbon; CAS, casein protein; CYP, cytochrome P₄₅₀; DMBA, 7,12-dimethylbenz[a]anthracene; ECL, enhanced chemiluminescence; EROD, ethoxyresorufin O-deethylase; GST, glutathione S-transferase; Ig, immunoglobulin; ISO, isosafrole; 3-MC, 3-methylcholanthrene; MROD, methoxyresorufin O-demethylase; PAH, polycyclic aromatic hydrocarbon; SPI, soy protein isolate; TCDD, 2,3,7,8 tetrachlorodibenzo-p-dioxin; UDPGT, UDP-glucuronyl-transferase; WHEY, whey protein.

Manuscript received October 2, 2000. Initial review completed November 13, 2000. Revision accepted January 16, 2001.

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