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Arkansas Children’s Nutrition Center and Department of Pediatrics, University of Arkansas for Medical Sciences, Little Rock, AR 72202
3To whom correspondence should be addressed. E-mail: badgerthomasm{at}uams.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • cytochrome P450 • rats • soy protein • whey protein • casein.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Results of epidemiological studies suggest that consumption of Asian diets that contain high levels of soybean products lowers the incidence of breast, prostate and colon cancers (2
,3
). In addition, experimental studies have reported that the consumption of bovine whey milk proteins may also afford cancer prevention (4
). We have been studying the cancer preventing properties of dietary soy protein isolate (SPI)4
and whey protein hydrolysate (WPH) on 7,12-dimethylbenz-(a)anthracene (DMBA)-induced mammary tumors in Sprague-Dawley rats (5
). While these proteins reduce the incidence of carcinogen-induced mammary gland tumors, the mechanisms behind this protection are not known.
Several of the anticancer properties of soy products have been attributed to phytoestrogens, such as genistein, that are bound to soy proteins. Numerous in vitro studies have revealed that genistein can inhibit the growth of tumor cells and the activities of many enzymes needed for cellular growth and differentiation (6
). We have reported that in soy-fed male rats, dexamethasone-induction of hepatic phase I detoxication enzymes, such as CYP3A, was greater than in casein-fed rats (7
), and 3-methylcholanthrene induction of CYP1A1, a P450 enzyme involved in carcinogen activation, was reduced in rats fed soy (8
). Others have reported that rats fed soy diets had elevated activities of the phase II detoxication enzymes glutathione transferases (GSTs), uridine diphosphate glucuronyl transferase (UDPGT) and NADPH-oxidoreductase (NQO1) (9
,10
). These soy effects on phase I and phase II enzymes would result in lower tissue concentrations of DMBA and may explain the decreased mammary DMBA-DNA adducts measured in soy- or genistein-fed rats (11
).
The anticancer properties of bovine whey protein have been attributed to its ability to elevate cellular glutathione levels, which would also aid in the phase II GST-dependent conjugation of electophiles, such as bioactivated DMBA (4
). Additionally, whey has been reported to enhance the immune system (12
) and its anticancer properties may, therefore, be related to increases in immune surveillance of transformed cells.
DMBA-induced rat mammary carcinoma closely resemble human breast cancer. Similar to those in humans, rat mammary tumors arise from ductal epithelial cells, the occurrence of tumors is preceded by hyperplastic and premalignant lesions, and the histogenesis, morphology and progression of hyperplastic, premalignant and malignant lesions are similar in many respects to those of human breast cancer (13
). Thus, DMBA-induced mammary tumors in rats have proven to be a useful tool for studying the molecular mechanisms involved in initiation, progression, pathogenesis and prevention of human breast cancer. This model is especially well suited for studies of diet and cancer risk.
DMBA is a procarcinogen and requires metabolic conversion to its ultimate carcinogenic metabolite, DMBA-3,4-dihydrodiol-1,2-epoxide, a process that includes two separate oxidations by the microsomal CYP1 enzymes. The first oxidation produces the 3,4-dihydrodiol and is catalyzed by either CYP1A1 or CYP1B1 (14
). The second oxidation produces the highly mutagenic 3,4-dihydrodiol-1,2-epoxide metabolite and is catalyzed by CYP1B1 (15
). CYP1A1 and CYP1B1 are expressed in both the liver and the mammary gland and both enzymes are induced by DMBA (15
). Thus, the extent to which DNA adducts occur after administration of DMBA depends on the level of oxidative metabolism of DMBA, which in turn is determined in part by the activities of CYP1A1 and CYP1B1.
The purpose of this study was to measure the effects of SPI and WPH on the hepatic and mammary expression of CYP1A1 and CYP1B1 enzymes that are important in the bioactivation of carcinogens such as DMBA. In addition, CYP1A2 was also measured because, like CYP1A1 and CYP1B1, its expression is also regulated by the DMBA-activated aryl hydrocarbon receptor (AhR). Thus, important mechanistic information was gained by studying the diet effects on all three AhR-regulated genes.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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All chemicals unless otherwise specified were purchased from Sigma Chemical (St. Louis, MO). Resorufin was purchased from Pierce Chemical (Rockford, IL) and methoxyresorufin was purchased from Molecular Probes (Eugene, OR). Radionuclides were purchased from DuPont NEN (Boston, MA). Rabbit polyclonal antibodies against rat CYP1A1/CYP1A2 and CYP1B1 were purchased from Gentest (Woburn, MA); monoclonal antibodies against CYP1A1 and CYP1A2 were purchased from Amersham Pharmacia Biotech (Arlington Heights, IL); goat polyclonal antibody against AhR was purchased from Novus Biologicals (Littleton, CO); and rabbit polyclonal antibody against ARNT was purchased from Affinity Bioreagents (Golden, CO). Horseradish peroxidase–conjugated anti-rabbit, anti-mouse or anti-goat IgG was purchased from Amersham Pharmacia Biotech or Santa Cruz Biotechnology (Santa Cruz, CA).
Animal treatments.
The experiments received prior approval from our Institutional Animal Care and Use Committee. Animals were purchased from Harlan Industries (Madison, WI). All animals were housed in an AAALAC-approved animal facility with a 12-h light cycle and constant humidity. Animals were killed by decapitation after administration of 90 mg/kg phenobarbital.
Experiments.
Pregnant female Sprague-Dawley rats (gestation day 4) were purchased from Harlan Industries (Indianapolis, IN). They were housed individually in polycarbonate cages and allowed free access to water and pelleted food. They were randomly assigned to three groups and fed one of three semipurified diets made according to the AIN-93G diet formula (16
), except that corn oil replaced soybean oil, and the protein source was casein (New Zealand Milk Products, Santa Rosa, CA), SPI (Protein Technologies International, St. Louis, MO) or WPH (New Zealand Milk Products, Santa Rosa, CA). Diets containing SPI had 430 mg total isoflavones/kg diet, including 276 mg/kg genistein and 132 mg/kg daidzein equivalents. L-Cysteine, L-methionine, L-phenylalanine, L-tryptophan and L-threonine were added to the SPI diet to the levels of the AIN-93G diet, as described previously (17
). For the whey diet, 0.5 g/kg diet L-methionine and 2.1 g/kg diet L-phenylalanine were added to the AIN-93G diet levels. Female offspring were weaned to the same diet as their mothers. Starting at age 32 d, the stages of the estrous cycle were determined by daily vaginal smears. Rats exhibiting two consecutive 4- or 5-d cycles were used in the present study. Starting at age 47 d, females (n = 8–12) in metaestrous were orally gavaged with sesame seed oil or 65 mg/kg body wt DMBA (Sigma) dissolved in sesame oil. Rats were killed 24 h later and livers and the right abdominal no. 4 mammary gland were harvested as described by Russo et al. (13
) for biochemical analysis.
Mono-oxygenase activities.
Ethoxyresorufin-O-deethylase (EROD), a rat CYP1A1 selective activity (18
), and methoxyresorufin-O-demethylase (MROD), an activity suggested to be selective for CYP1A2 (19
), were measured by following the formation of resorufin spectrofluorimetrically at 536 nm (excitation) and 586 nm (emission) according to the method of Burke and Mayer (20
) using an RF-5301PC scanning spectrofluorometer (Shimadzu Scientific Instruments, Columbia, MD) under conditions of linearity for incubation time and protein.
Western immunoblot analysis.
CYP1, AhR and ARNT were measured in total mammary protein prepared using TRI Reagent (MRC, Cincinnati, OH) according to the manufacturer’s directions and the method of Varela and Ip (21
). Hepatic CYP1 proteins were measured from microsomes. Hepatic AhR and ARNT were measured in total protein prepared by homogenizing 100 mg liver into ice-cold tissue lysis buffer [20 mmol/L Tris, pH 7.4; 0.15 mol/L NaCl; 1 mmol/L EDTA; 1 mmol/L EGDA; 1% Triton X-100; 2.5 mmol/L sodium pyrophosphate; 1 mmol/L ß-glycerolphosphate; 1 mmol/L Na3V04; 1.0 mg/L leupeptin; and 1 mmol/L 4-(2-aminoethyl)benzenesulfonyl fluoride]. Proteins were resolved on 100 g/L polyacrylamide gels and transferred to a Hybond-P membrane (Amersham Pharmacia Biotech). Membranes were blocked 
8 h at room temperature with shaking in Tris-buffered saline plus Tween (TBST) [10 mmol/L Tris-buffered saline, 0.130 mol/L NaCl, 2.7 mmol/L KCl, pH 7.4, plus 100 g/L milk powder containing 50 µL Tween-20]. Membranes were then incubated with primary antibody diluted to 5 mg/L AhR, 1:500 ARNT, 1:4000 CYP1B1, 1:500 polyclonal CYP1A1/CYP1A2 for mammary, and 1:200 monoclonal CYP1A1 or CYP1A2 for liver in TBST plus 50 g/L milk powder over night at 4°C with shaking. After washing three times in TBST, the membranes were incubated for 1 h at room temperature in TBST plus 50 g/L milk powder containing horseradish peroxidase–conjugated secondary IgG a (1:5000–10000). Membranes were washed three times in TBST, and the proteins were visualized using the enhanced chemiluminescence plus system (ECL Plus; Amersham Pharmacia Biotech) and detected by autoradiography. Immunoquantitation was obtained by densitometric scanning of the resulting autoradiographs using a Bio-Rad GS525 molecular imager (Richmond, CA).
Relative reverse transcriptase–polymerase chain reaction of CYP1, AhR and ARNT mRNA levels.
Total RNA was extracted using TRI Reagent (MRC) according to the manufacturer’s directions. First-strand cDNAs were synthesized from total RNA using the Reverse Transcription System (Promega, Madison, WI) according to the manufacturer’s protocol. CYP1A1, CYP1A2, CYP1B1 and ß-actin primer sequences were described previously (22
). The AhR (GenBank accession no. U04860) and ARNT (GenBank accession no. U61184) primer sequences designed using Primer Designer software version 1.01 (Scientific & Educational Software, Durham, NC) were as follows: RAHR-F1, 5'-CAGAAGCA TGCAGAATGAGGAG-3' (AhR forward), and RAHR-R2, 5'-TGACCATGGCTGACTGTGAG TT-3' (AhR reverse); RARNT-F1, 5'-CGTGGCAACTGCAGGATCAGAA-3' (ARNT forward), and RARNT-R1, 5'-CCGGAAGAGACATGGCACTGAA-3' (ARNT reverse). Polymerase chain reaction (PCR) was performed in a total volume of 25 µL of PCR buffer containing 1.1 mmol/L MgCl2, 0.2 µmol/L each primer, 1 unit of RediTAQ DNA polymerase (Sigma) and 2 µL of first-strand cDNA containing deoxynucleoside triphosphates. The amplification cycle number was determined to keep amplification of the desired products in the linear range to avoid the "plateau effect" associated with increased numbers of PCR cycles. The PCR cycle started at 95°C for 5 min followed by a three-step cycling: denaturation at 95°C for 30 s, annealing at 68°C (CYP1A1, CYP1A2, CYP1B1 and ß-actin) or 58°C (AhR and ARNT) for 1 min and extension at 72°C for 1.5 min. Amplification cycle numbers were as follows: liver CYP1A1 and AhR, 25 cycles; liver CYP1A2 and ß-actin, 23 cycles; liver CYP1B1, 27 cycles; liver ARNT, 34 cycles; mammary CYP1A1, CYP1B1, AhR and ß-actin, 25 cycles; mammary CYP1A2, 38 cycles; and mammary ARNT, 37 cycles. This was followed by a final extension step at 72°C for 10 min. The PCR products were fractionated through 20 g/L agarose gel at 120 V and visualized by dual staining with ethidium bromide (Sigma) and cybergreen (Molecular Probes, Eugene, OR). The bands were analyzed by video densitometry; the areas of the peaks were calculated in arbitrary units. The relative value was generated by calculating the ratio of the arbitrary units of the CYP1, AhR or ARNT amplicon to that of ß-actin.
Statistical analysis.
Statistical analysis of DMBA effects in the three diet groups was performed by two-way ANOVA followed by the Tukey test using the SigmaStat Software (Jandel Scientific Software, San Rafael, CA) with P < 0.05 considered significant. Where just comparing diet effects (Table 3)
, one-way ANOVA followed by Student-Newman-Keuls multiple comparison was used. Data are presented as mean ± SEM
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Body weight, liver weight and yield of microsomal protein for rats fed the three different diets and treated with sesame oil vehicle or DMBA were measured, and there were no significant differences among groups (data not shown). DMBA induced the metabolism of EROD and MROD by eight- and threefold, respectively, in the CAS-fed rats (Table 1
). The DMBA-induced activities of EROD and MROD were lower (P < 0.05) in the SPI-fed rats compared with the CAS-fed rats.
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The constitutive expression of hepatic CYP1A1 mRNA was lower (P < 0.05) in the WPH-fed rats than in the SPI- or CAS-fed rats (Figs. 1
and 2and Table 2
). However, this diet-dependent mRNA difference did not translate to differences in protein expression (Table 2)
. There was no constitutive CYP1B1 mRNA or protein detected in any diet group. The hepatic DMBA-induced mRNA expression for CYP1A1, CYP1A2 and CYP1B1 was lower (P < 0.05) in SPI- and WPH-fed rats compared with CAS-fed rats. At the protein level, there was a decrease (P < 0.05) in DMBA-induced expression for CYP1A1 in the SPI-fed rats compared with the CAS-fed rats with no differences in CYP1A2.
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Diet-dependent effects on liver and mammary gland AhR and ARNT protein expression and mRNA steady-state levels.
Liver and mammary gland AhR and ARNT mRNA expressions were greater (P < 0.05) in the SPI-fed rats compared with the WPH- or CAS-fed rats (Figs. 3
and 4and Table 3
). Hepatic AhR and ARNT protein expressions were not different among diet groups. In the mammary gland, the SPI-fed rats had lower AhR and ARNT protein levels (P < 0.05) and these corresponded to the lower DMBA-induced CYP1 proteins reported in Table 2
. The mammary gland AhR protein level in the WPH-fed rats was greater (P < 0.05) than in the CAS-fed rats, and this corresponded to the reduced expression of CYP1B1 (P < 0.05) mRNA and protein levels in the WPH-fed rats after DMBA treatment (Table 2)
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) or chemically-induced mammary tumors are lower in rats fed diets made with soy flour or soy protein isolate (24
–28
). Similarly, we have recently reported that female Sprague-Dawley rats fed diets containing SPI had an 
25% lower incidence of DMBA-induced mammary tumors compared with CAS-fed rats (5
). Additionally, in the same study, we demonstrated that a diet made with a common milk protein, WPH, had an 45% reduced incidence of DMBA-induced tumors. The mechanisms by which these dietary proteins reduce mammary gland cancer are not known but could be related to the CYP1A1 and CYP1B1 phase I enzyme–dependent metabolism of DMBA. DMBA is a procarcinogen that is metabolized to its ultimate carcinogenic metabolite by CYP1A1 and CYP1B1 enzymes, and DMBA induces its own metabolism by upregulating expression of CYP1A1 and CYP1B1 enzymes (14
,15
). This potential mechanism is supported by our previously reported study that measured a reduction in polycyclic aromatic hydrocarbon-induced hepatic CYP1A1 expression in male rats fed SPI (8
).
In the current report, we measured the constitutive expression and DMBA-induced expression of CYP1A1, CYP1A2 and CYP1B1, because CYP1A1 and CYP1B1 bioactivate DMBA and all three enzymes share regulation through the AhR, thereby allowing insight into potential diet-dependent mechanisms. In female rats fed SPI or WPH diets, DMBA-induced mammary CYP1A1, CYP1A2 and CYP1B1 expressions were significantly lower than in CAS-fed rats. CYP1A1 possesses both EROD and MROD activities (18
), and the decrease in hepatic DMBA-induced EROD and MROD activities may be the result of the reduced CYP1A1 protein expression alone, since CYP1A2 protein was not affected in the liver. However, in the mammary gland, concomitant decreases in CYP1A2 and CYP1A1 mRNA and protein were observed in DMBA-treated rats, suggesting a possible role for CYP1A2 in the mammary gland as well.
In addition to the CYP1A1 effects in the liver, DMBA induced CYP1A1, CYP1A2 and CYP1B1 protein expression in the mammary gland and this induction was lower in SPI- and WPH-fed rats. Mammary gland CYP1 activities were not measured because of the limited tissue available; however, decreased activities for these enzymes would also be expected based on reduced protein expression. CYP1B1 catalyzes the formation of the highly mutagenic DMBA 3,4-dihydrodiol-1,2-epoxide metabolite thought to be responsible for mammary cancer initiation (15
). Such a reduction in relative expression of these phase I enzymes would be expected to decrease the target tissue concentration of the carcinogen and the resultant DNA adducts that lead to mutations and mammary gland cancer. These new observations suggest that the previously reported reduction of mammary tumor incidence in SPI- and WPH-fed rats (5
) could be due in part to a lower concentration of DMBA 3,4-dihydrodiol proximate carcinogen metabolite in the target tissue combined with abridged metabolism of DMBA to its ultimate carcinogenic mutagenic epoxide and fewer DMBA-DNA adducts. This hypothesis is supported by studies conducted in another laboratory that reported a 12% decrease in mammary DMBA-DNA adducts in rats fed an SPI-containing diet (11
).
Previous studies have established that constitutive expression of CYP1A1 in the liver and the mammary gland is very low, whereas CYP1A2 is constitutively expressed in the liver, but not in extrahepatic tissues. CYP1B1 is expressed only in very low levels in the liver and is constitutively expressed in the mammary gland (29
–33
). To determine the mechanisms by which SPI or WPH could affect the expression of CYP1 enzymes in the current study, we measured their steady-state mRNA levels in both liver and mammary gland. We found excellent agreement in the diet effects on the protein and the steady-state mRNA levels for three CYP1 enzymes in DMBA-treated rats. That is, there were corresponding decreases in mRNA levels to match the reduced protein levels in SPI- and WPH-fed rats compared with CAS-fed rats. This suggests that the effects of SPI and WPH were at least in part due to regulation of the gene transcription or mRNA stabilization. Further studies are necessary to determine exactly how these effects occur.
It should be noted, however, that the constitutive expression of the CYP1 mRNAs did not always correlate with observed protein levels for the same CYP1. For example, the reduction in hepatic CYP1A1 and CYP1A2 mRNA in WPH-fed rats did not translate into parallel changes in CYP1A1 or CYP1A2 protein expression. The mechanisms underlying the discrepancies between constitutive mRNA and protein levels are unclear.
The induction of CYP1 expression by PAH such as DMBA has been extensively investigated (31
,33
–36
), and it has been established that the nuclear AhR/ARNT heterodimeric complex acts as a ligand-activated transcription factor (37
) that binds to xenobiotic response elements (XREs) in the regulatory region of CYP1 genes. To further investigate the mechanisms underlying the dietary effects on CYP1 expression, we studied the nuclear AhR/ARNT heterodimeric complex at the time of DMBA treatment. When compared with CAS- or WPH-fed rats, the SPI-fed rats had greater AhR and ARNT mRNA levels but lower AhR and ARNT protein levels just before DMBA treatment in the mammary gland. Thus, SPI may be acting to inhibit the expression of AhR and ARNT proteins, which results in reduced CYP1 expression. It should also be noted that a similar trend occurred with hepatic AhR and ARNT expression in SPI-fed rats. However, WPH-fed rats had essentially the same ARNT protein levels but elevated AhR protein at the time of DMBA treatment, yet had significantly lower levels of CYP1 protein expressed in the mammary gland. Thus, if these AhR and ARNT effects are important in CYP1 expression in the mammary gland, the mechanisms by which SPI and WPH act to reduce CYP1 expression differ substantially, whereby SPI works by inhibiting AhR and ARNT expression and WPH does not.
In the mammary gland, WPH caused a nearly 100% increase in constitutive AhR protein expression and this may account for the increased expression of CYP1B1 measured in WPH-fed rats. This is supported by in vitro molecular biology studies demonstrating that the AhR regulates both constitutive as well as inducible CYP1B1 expression (38
). The mechanism for the increased AhR expression may result from the action of growth factors and/or growth factor–like peptides in WPH. Numerous studies have reported that whey contains several hormones and growth factors including insulin, bombesin, prolactin, insulin-like growth factor (IGF)-1, IGF-2, platelet-derived growth factor and fibroblast growth factor (39
,40
). Growth factors such as platelet-derived growth factor and basic fibroblast growth factor have been reported to induce AhR expression (41
). Alternatively, these WPH growth factors and/or peptides derived from whey hydrolysis may coregulate the decreased DMBA induction of CYP1 with no direct effect on the AhR or ARNT levels, but with an effect on the expression or activity of transcriptional coactivators or other transcription factors that regulate the CYP1 genes. The WPH used in this study contains peptides that are hydrolysate products from the bovine milk protein and from milk-derived growth factors. In addition, further hydrolysis of protein and peptide products by the gastrointestinal tract will likely yield other peptides. We have hypothesized that some of the proposed health benefits of whey protein are due to the actions of these peptides. For example, in preliminary studies, we have determined that fractionated peptides alter the cell signals that lead to changes in proliferation (42
).
A potential mechanism for these soy effects might be attributed to the soy-associated isoflavones, such as genistein, that have been characterized as estrogens (phytoestrogens) because of their affinity for estrogen receptor (ER)-
and ERß (43
,44
). Soy phytoestrogens may play a role in the CYP1 effects measured here since there is evidence that estrogens can inhibit the induction of CYP1A1 and CYP1B1 (45
–48
). Estrogens may downregulate the protein expression of AhR and/or ARNT, thereby reducing AhR-induced gene transcription. Evidence for this has been reported for ARNT where decreased mRNA expression was lowest at estrous during the cycle in female rats, suggesting that hormones such as estrogen may downregulate ARNT expression (49
). Alternatively, both the ER and AhR interact with the same nuclear coactivators during transcriptional regulation (50
–53
). One possible mechanism for ER-AhR "crosstalk" and antagonism may involve ER binding all of the available nuclear coactivators needed for trancriptional regulation by the AhR. Thus, both in vivo and in vitro studies suggest that estrogen can antagonize DMBA induction of CYP1A1 and CYP1B1, and this may occur by several mechanisms.
In summary, the results reported here demonstrate that DMBA inducibility of CYP1 is dependent on the source of the dietary proteins. Factors found in SPI and WPH possibly interact with the AhR-ARNT pathway leading to decreased induction of the CYP1 protein. Rats fed SPI diets had lower DMBA-induced CYP1A1 hepatic activity compared with those fed WPH or CAS. In addition, there were decreases in all three mammary gland CYP1s in DMBA-induced SPI-fed rats compared with the CAS-fed rats. Because there were larger decreases in DMBA-induced mammary gland tumors in WPH-fed rats compared with SPI- or CAS-fed rats (5
), the decreases in CYP1s in the SPI-fed rats appear to only partly contribute to the reduction in tumors measured. Additional cancer prevention may be derived from diet-induced increases in mammary gland differentiation. We have measured increased mammary gland differentiation in WPH-fed rats compared with both SPI- and CAS-fed rats (54
). Thus, in the WPH-fed rats, the combined decreases in mammary DMBA-induced CYP1 expression, coupled with increased differentiation, may render the mammary gland in these rats more resistant to DMBA-induced mammary tumors, as compared with the SPI- or CAS-fed rats. The decreased mammary tumors in the SPI-fed rats relative to CAS-fed rats may be solely due to SPI-dependent inhibition of the CYP1 genes. The effects of WPH and SPI on mammary gland differentiation and CYP1 expression are currently under investigation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 To whom reprint requests should be addressed. E-mail: jcrowlands{at}salvitas.com.
4 Abbreviations used: AhR, aryl hydrocarbon receptor; ARNT, AhR-nuclear translocator; CAS, casein; DMBA, 7,12-dimethylbenz-(a)anthracene; ER, estrogen receptor; EROD, ethoxyresorufin-O-deethylase; GST, glutathione transferase; IGF, insulin-like growth factor; MROD, methoxyresorufin-O-demethylase; PCR; polymerase chain reaction; SPI, soy protein isolate; TBST, Tris-buffered saline plus Tween; WPH, whey protein hydrolysate.
Manuscript received July 5, 2001. Initial review completed August 1, 2001. Revision accepted September 13, 2001.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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