Abstract
In the present study, we administered two low protein diets (LPDs) to rats during pregnancy and lactation and determined their effect on the ontogeny of select hepatic cytochrome P450 (P450) isoforms in their offspring. The L93 and LM76 LPDs were derived from the American Society of Nutrition recommended AIN93G and a modified version of the AIN76A purified control diets, respectively. The LPDs contained 8% crude protein in the form of casein, whereas the purified control diets contained 19% casein. A regular cereal-based diet (NP) was also included, and, therefore, a total of five groups were tested. Pups in all five groups were weaned onto a regular NP diet on postnatal day 28. Perinatal LPD altered the activities of a number of P450 isoforms in 28-day-old male and female offspring. However, nutritional rehabilitation abolished most of these changes as evidenced by lack of differences between the five groups in the activities of P450 isoforms in either 65- or 150-day-old offspring. Interestingly, 58-day-old female offspring in the LM76 group but not those in the L93 group exhibited shorter hexobarbital sleep time than the purified control group. However, hexobarbital hydroxylase activity and the amount of CYP2C12 protein, an important P450 isoform involved in hexobarbital metabolism in females, were unchanged. This suggests that the decrease in hexobarbital sleep time in this group is not due to an increase in the activity of hexobarbital-metabolizing enzymes. In summary, perinatal LPDs produced transient alterations in activities of select hepatic P450s and resulted in a gender- and diet-dependent long-term alteration in hexobarbital pharmacodynamics.
Cytochrome P450 (P450) is a superfamily of membrane-bound enzymes involved in metabolism of endogenous and exogenous compounds. P450s are present in many organs including the liver, kidney, spleen, adrenal glands, and intestine. The hepatic P450s are known to exhibit zonal expression (Oinonen and Lindros, 1998), with most P450s exhibiting a predominantly perivenous zonation. P450 expression is regulated by hormones such as insulin, thyroid hormone, and growth hormone (Schenkman and Griem, 1993).
Epidemiological studies demonstrate a strong association between low birth weight and high incidence of ischemic heart disease, elevated systolic blood pressure, impaired glucose tolerance, and syndrome X during adulthood (Barker et al., 1993). To study the association in more mechanistic detail, a variety of animal models of intrauterine growth restriction have been developed. A commonly used model involves the administration of low protein diets to rats during the pregnancy and lactation (perinatal) period. There are striking similarities between this model and the epidemiological findings. Maternal low protein diets during pregnancy and lactation decrease birth weight in the pups and result in poor glucose tolerance, hyperlipidemia, and hypertension in the adult offspring.
A careful review of the literature reveals two effects of perinatal low protein diets that are of relevance to hepatic P450 enzymes. First, administration of low protein diets selectively during the perinatal period results in long-term alterations in the morphology of the hepatic periportal and perivenous regions (Burns et al., 1997). In association with these changes, the activity of select cytosolic enzymes located in the periportal and perivenous regions are dramatically altered (Desai et al., 1995). Interestingly, despite the transient nature of the nutritional insult, the alterations in enzyme activity persisted until the offspring were at least 11 months old (when the study was stopped). Because hepatic P450 enzymes are located in the periportal and perivenous zones of the liver, it can be hypothesized that perinatal low protein diets will also permanently alter the activity of these membrane-bound enzymes. Second, administration of maternal low protein diets during pregnancy and lactation produces long-term, possibly irreversible, decreases in the body weight of the offspring (Desai et al., 1997; Cherala et al., 2006). The long-term lowering of body weight could be due to the perinatal low protein diet-mediated alterations in the secretory levels of growth hormone. Additionally, perinatal low protein diets result in long-term alterations in the secretory levels of insulin and thyroid hormones (Desai et al., 1997; Passos et al., 2002). Because this troika of hormones, growth hormone, insulin, and thyroid hormone, are important modulators of hepatic P450 activity (Schenkman and Griem, 1993), it can be hypothesized that perinatal low protein diet-induced alterations in the secretory profile of these hormones will permanently alter the activity of P450 enzymes. However, very few studies have examined this issue comprehensively.
In the current study, we determined the effects of administration of a low protein diet (LPD) to pregnant and lactating rats on the activities of select hepatic P450 enzymes in the offspring at three different ages: prepuberty, young adult, and mature adult. Based on their established importance in drug metabolism, the activities of the following enzymes were measured: CYP2D1, CYP2E1, ethoxyresorufin O-deal-kylase (EROD), pentoxyresorufin O-dealkylase (PROD), the testosterone hydroxylases, and cytochrome P450 reductase (CPR). Additionally, the hexobarbital sleep time test, a measure of the functional status of P450 superfamily, was performed in young adult offspring. The American Society for Nutrition, formerly known as American Institute of Nutrition, recommends two different purified control diets, the AIN93G and the AIN76A (American Institute of Nutrition, 1980; Reeves et al., 1993). The two LPDs in our study were derived from the AIN93G purified diet and from a modified version of the AIN76A purified diet. Both low protein diets contained 8% crude protein in the form of casein, whereas their corresponding purified control diets contained 19% protein. LPDs were made isocaloric to their respective purified control diets by increasing the content of compensatory carbohydrates. The two LPDs differed in the type and concentration of fats and carbohydrates. Our previous study provides detailed compositions of the two low protein diets and compares their effect on reproductive performance in rats and on growth and development in their offspring (Cherala et al., 2006).
It is known that chronic feeding of adult animals with purified versus cereal-based diets produces differences in the activities of P450 enzymes (Wattenberg, 1975). However, the comparative effects of perinatal administration of these diets on the ontogeny of P450 enzymes have not been systematically investigated. Therefore, we incorporated an additional control group of dams fed a regular cereal-based diet and compared the effects of perinatal administration of purified diets and nonpurified diet on the ontogeny of activity of select hepatic P450 enzymes.
Materials and Methods
Materials. Perchloric acid, acetonitrile, diethyl ether, acetic acid, methylene chloride, potassium dibasic phosphate, and EDTA were obtained from Fisher Scientific Co. (Springfield, NJ). Cortexolone and 17β-N,N-diethylcarbamoyl-4-methyl-4-aza-5α-androstan-3-one were obtained from Steraloids (Wilton, NH). Bufuralol and 1-hydroxybufuralol were gifts from Hoffmann-La Roche (Nutley, NJ). Testosterone metabolites (6β-, 2α-, 2β-, 16α-OH, and androstenedione) were gifts from Dr. Peter Harvison (University of the Sciences in Philadelphia, Philadelphia, PA). Mouse anti-rat CYP2C12 antibody was a gift from Dr. Marika Rönnholm (Huddinge University Hospital, Huddinge, Sweden). Peroxidase-conjugated anti-mouse IgG was purchased from Oxford Biochemical Research (Oxford, MI). All other reagents were obtained from Sigma-Aldrich (St. Louis, MO).
Diets. Regular nonpurified diet (NP), the AIN93G purified diet (C93), a modified version of the AIN76A purified diet (M76), and their corresponding low protein formulations, LPD AIN93G (L93) and LPD AINM76A (LM76), were purchased in pellet form from Purina Test Diets (Richmond, IN). The purified diets contained 19% crude protein in the form of casein, whereas the isocaloric low protein diets contained 8% crude protein in the form of casein. Detailed compositions of all five diets used in the study are available in the literature (Cherala et al., 2006).
Experimental Methods.Experiment with dams. The study was approved by the Institutional Animal Care and Use Committee of the University of the Sciences in Philadelphia. Virgin female Sprague-Dawley rats (Charles River Laboratories, Inc., Wilmington, MA) were mated by housing one male rat with two female rats. Day 1 of pregnancy was assigned upon observation of sperm in the daily morning vaginal smears, at which time rats were randomly assigned to one of the following five diet groups: NP, M76, LM76, C93, or L93. Each group consisted of seven to eight pregnant rats, and these rats received their assigned diet throughout pregnancy and lactation.
Experiments with offspring. Upon birth, pups were sexed, and litter size was noted. All litters were randomly culled to 12 pups (6 male and 6 female) on the day of birth and further randomly culled to 8 pups (4 males and 4 females) on day 4 after birth. Offspring from all diet groups were weaned on day 28 after birth and were housed in isosexual groups according to perinatal diet treatment. Pups from litters in all five groups were weaned on to a nonpurified diet. It is therefore important to note that different dietary treatments were administered only during gestation and lactation. One male and female offspring from each litter in all five groups were randomly chosen and sacrificed on days 28, 65, and 150 after birth. Livers were collected from all sacrificed animals, weighed, snap-frozen in liquid nitrogen and stored at –80°C for subsequent isolation of microsomes and cytosol.
Hepatic Microsome and Cytosol Isolation. Hepatic microsomes and cytosol were prepared using a differential ultracentrifugation method (Shapiro and Szczotka, 1984). The resulting microsomal pellet was suspended in pH 7.4 buffer (50 mM Tris and 0.25 M sucrose), and stored at –80°C. Microsomal and cytosolic protein content was measured with the Bradford method using bovine serum albumin as the standard (Bradford, 1976).
P450 Isoforms and CPR Activities. The catalytic activities of select hepatic P450 isoforms and CPR were measured using established procedures. Appropriate references for assay conditions, substrates used, and products quantitated for each P450 isoform are listed in Table 1. All reactions were performed in the linear range with respect to microsomal protein concentration and incubation time.
Testosterone Hydroxylases Activities. The stereo- and regio-specific hydroxylation of testosterone was used as a marker of activities of multiple P450 enzymes. The assay was conducted as described in the literature (Sonderfan et al., 1987) with minor modifications. Briefly, 0.2 mg/ml microsomal protein was preincubated at 37°C for 5 min in 100 mM dibasic potassium phosphate buffer (pH 7.4) containing 250 μM testosterone, 1 μM17β-N,N-diethylcarbamoyl-4-methyl-4-aza-5α-androstan-3-one, and 1 mM EDTA. The total volume of the reaction mixture was 1 ml. The reaction was initiated by addition of the NADPH regenerating system and stopped after 60 min by addition of 6 ml of methylene chloride. An internal standard (cortexolone) was added to the mixture, and the metabolites and internal standard were extracted into the organic phase by vigorous shaking for 5 min. The organic phase was evaporated to dryness under nitrogen, the residue was reconstituted in the mobile phase, and 50 μl was injected onto a C18 column (Nova-Pak, 3.9 × 300 mm, 4 μm; Waters, Milford, MA) maintained at 45°C. The mobile phase (50:50 v/v water-methanol) was isocratically pumped at 1.2 ml/min, and the eluent was monitored at 247 nm. The activities of testosterone-2α,-6β, and -7α hydroxylases were computed as a ratio of peak area of the corresponding metabolite to that of internal standard and expressed as peak area ratio per minute per milligram of microsomal protein.
Hexobarbital Hydroxylase Activity. The rate of conversion of hexobarbital to 3-hydroxyhexobarbital is a measure of the activity of hexobarbital hydroxylase (Böcker, 1982). A reaction mixture consisting of 0.4 mg/ml microsomal protein, 5 mM hexobarbital, and 0.1 M dibasic potassium phosphate (pH 7.4) was preincubated for 5 min at 37°C. The reaction was initiated by the addition of a NADPH-regenerating system, and the mixture was incubated for 60 min. The reaction was terminated by the addition of dichloromethane and simultaneous placement of tubes on ice. An internal standard (phenobarbital) was added to the mixture, and the metabolite and internal standard were extracted into the organic phase after vigorous shaking for 10 min. The organic phase was evaporated to dryness under nitrogen, the residue was reconstituted in the mobile phase, and 50 μl was injected onto the C18 column (Nova-Pak, 3.9 × 300 mm, 4 μm; Waters). The mobile phase [67:33 v/v, 3.5 mM sodium monophosphate (pH 2.7)/acetonitrile] was pumped at 1.2 ml/min, and the eluent was monitored at 238 nm. The activity of hexobarbital hydroxylase was computed as a ratio of peak area of metabolite to internal standard and expressed as peak area ratio per minute per milligram of microsomal protein.
Hexobarbital Sleep Time (HST). Hexobarbital solution (prepared freshly on the day of the experiment) was administered as a 100 mg/kg i.p. dose. HST was designated as the time between injection and recovery of righting reflex, which was defined as the ability of the animal when placed on its back on a flat surface to turn over on its paws three times in 15 s.
CYP2C12 Western Blot Analysis. Ten micrograms of microsomal protein was electrophoresed on 7.5% SDS-polyacrylamide gels, transferred onto nitrocellulose membranes, and probed with monoclonal mouse anti-rat CYP2C12 antibody (Dhir and Shapiro, 2003). A peroxidase-conjugated anti-mouse IgG was used as a secondary antibody. The signal was visualized using an enhanced chemiluminescence kit (SuperSignal West Dura Extended Duration substrate; Pierce Chemical, Rockford, IL). The optical densities were quantitated using the UVP Bioimaging system and Labworks image acquisition and analysis software (Upland, CA). The densities of samples on a blot were normalized to the density of a control sample that was run on all blots.
Data Analyses. All data are expressed as means ± S.D. In HST experiments, whenever two or more pups from a litter were used, the values of sleep times obtained from the pups were averaged and reported as a single point. As a result, the sample size in each of the groups never exceeded the number of treated dams in each group. Such a conservative method of data reporting and analyses is recommended for analyses of offspring data in multiparous species. It minimizes inflation of the 0.05 level of α and the spurious statistical significance that results as a consequence (Holson and Pearce, 1992; Zorrilla, 1997).
Data were analyzed using Sigma Stat (version 3.1) software. One-way or two-way analysis of variance tests were used on relevant datasets. Wherever appropriate, multiple comparisons were conducted using the Student Newman-Keuls post hoc test. All statistical tests were conducted at a 0.05 level of significance.
Results
The microsomal protein yields in 28-day-old male offspring of both the LPD groups were smaller than those of their respective purified control groups (Table 2). These differences disappeared upon post-weaning nutritional rehabilitation as evidenced by similar microsomal protein yields in 65- and 150-day-old male offspring from the LPD and control groups. There were no differences in the microsomal protein yield between male offspring of purified control groups and the lab chow group at any age. The microsomal protein yield in female offspring showed a pattern similar to that observed in male offspring. Sporadic gender differences were observed in microsomal yields. On day 65, male offspring of both C93 and L93 groups showed a higher yield than the corresponding female offspring. However, 150-day-old male offspring of the LM76, C93, and NP groups showed a lower yield than their corresponding female offspring. Cytosolic protein yields in 28-day-old male and female offspring of both LPD groups were lower than those in the corresponding purified control groups. Similar to microsomal yields, the differences in cytosolic yields in both the LPD groups disappeared by day 65. Gender differences were not observed in cytosolic protein yields (except for the day 28 LM76 group).
As shown in Table.3, 28-day-old male offspring of both LPD groups had lower EROD activities than those of their respective control groups. Also, 28-day-old male offspring of both the purified control groups exhibited lower EROD activities than that of the NP group. However, these differences disappeared by day 65, and the lack of differences persisted on day 150 (supplemental Table 1). EROD activity in the five diet groups in 28-day-old female offspring exhibited a similar pattern. In 28-day-old offspring, EROD activity in males of the LM76 group was markedly greater than that in females. Gender differences were not observed in 28-day-old offspring in the other groups or between any of the groups in 65-day-old offspring. On day 150, EROD activity was significantly higher in males in all five groups (supplemental Table 1).
As shown in Table 3, PROD activities in male and female offspring of both the LPD groups were similar to those of their respective purified control groups at all ages (also see supplemental Table 1). Twenty-eight-day-old male offspring of both the purified control groups showed lower PROD activities than the NP group. PROD activity in 65-day-old male offspring in the M76 group but not in the C93 group was lower than that in the NP group. However, these differences did not persist on day 150 (supplemental Table 1). Similar to their male counterparts, 28-day-old female offspring of both the purified control groups showed lower PROD activities than the NP group. However, these differences disappeared by day 65, and the lack of differences persisted on day 150 (supplemental Table 1). At all ages, the PROD activities of males in all five groups were greater than those in the corresponding females (supplemental Table 1 for day 150 data) with the exception of the NP group on day 28 and the M76 group on day 65.
As shown in Table 4, CPR activity in 28-day-old male offspring in only the LM76 group but not in the L93 group was significantly greater than its purified control. CPR activity in 28-day-old male offspring of the M76 group was lower than that in the NP group. CPR activities in 65- and 150-day-old (supplemental Table 1) male offspring in all five groups were similar. CPR activities in 28-day-old female offspring of the LPD groups were similar to those of their respective purified control groups. However, CPR activities in 28-day-old female offspring in both the purified control groups were lower than that in the NP group. These differences disappeared by day 65, and the lack of differences persisted on day 150 (supplemental Table 1). Gender differences (activity in males greater than that in females) were present in all five groups on day 150 (supplemental Table 1) but not on days 28 and 65 (the 28-day-old NP group being an exception).
CYP2E1 activity in 28-day-old male and female offspring of the LM76 group but not the L93 group was higher than that in its purified control group (Table 4). Also, 28-day-old male and female offspring in the M76 group exhibited lower activity than corresponding animals in the NP group. CYP2E1 activities in male offspring of all five groups and also in female offspring of all five groups were similar on days 65 and 150 (supplemental Table 1). However, CYP2E1 activity exhibited a female predominance in all groups at all ages (the LM76 group on day 28 being an exception) (supplemental Table 1 for day 150 data). There were no differences in the activities of CYP2D1 among the five diet groups of either gender at any age (Table 4). Male predominant expression of CYP2D1 was observed in the NP group only on day 65 and in all groups on day 150 (supplemental Table 1).
The effect of perinatal diets on the regio- and stereo-selective metabolism of testosterone is presented in Table 5. Testosterone 6β-hydroxylase activities in 28-day-old male offspring did not differ between LPD groups and their respective purified control groups. However, the M76 group showed lower activity than the NP group. By day 65, no differences were observed between male offspring in any groups, and the lack of differences persisted on day 150. Twenty-eight- and 150-day-old (supplemental Table 1) female offspring showed a pattern similar to that of males. In 65-day-old female offspring, the activity in the LM76 group was significantly higher than that in the respective purified control group. Testosterone 6β-hydroxylase activity exhibited a male predominance on day 65 (Table 5) and day 150 (supplemental Table 1) but not on day 28.
Twenty-eight-day-old male offspring in both the LPD groups showed lower testosterone 2α-hydroxylase activities compared with their respective purified control groups (Table 5). These differences disappeared upon nutritional rehabilitation as evidenced by similar activities on day 65. There were no differences between male offspring in either purified control group and the NP group at any age. Unlike their male counterparts, 28-day-old female offspring in the LM76 group but not those in the L93 group showed lower activity compared with its purified control group. Testosterone 2α-hydroxylase activity exhibited male predominance both in 65-day-old (Table 5) and 150-day-old (supplemental Table 1) offspring. Interestingly, on day 28 gender differences were only observed in the two LPD groups with males exhibiting lower activity than females.
Testosterone 7α-hydroxylase activity in 28-day-old male offspring in the LM76 group was higher than that in its purified control group (Table 5). In 28-day-old female offspring, there were no differences between the LPD groups and their corresponding purified control groups. At this age, the M76 group showed lower activity than the NP group. There were no differences in activity between any groups in 65-day-old (Table 5) and 150-day-old (supplemental Table 1) male or female offspring. Testosterone 7α-hydroxylase activity was female predominant in the NP group on day 28 and in all groups starting day 65 (supplemental Table 1).
In all five diet groups, hexobarbital sleep time in 58-day-old female rats was longer than that in corresponding male rats (Table 6). There were no differences in HST between any groups in 58-day-old male offspring. On the other hand, 58-day-old female offspring in the LM76 group exhibited a shorter HST than those in the respective purified control group. The microsomal hexobarbital hydroxylase activity in 65-day-old female offspring was similar in all diet groups. Also, the amount of CYP2C12 protein, one of the major P450 enzymes involved in hexobarbital metabolism in female rats, was similar in all diet groups.
The ontogeny of activity of select hepatic P450 isoforms in the NP group specifically is summarized in supplemental Table. 2.
Discussion
In the present article, we examined the effects of perinatally administered LPDs on the ontogeny of activity of select hepatic P450 enzymes in male and female rats. Simultaneously, we compared the long-term effects of perinatal administration of purified diets and a nonpurified diet on the activities of these hepatic P450 enzymes.
Twenty-eight-day old offspring of both LPD groups exhibited lower microsomal and cytosolic protein yields. Our earlier studies have shown that 28-day-old offspring of low protein diet-fed dams also exhibited decreased levels of serum albumin and total serum protein (Cherala et al., 2006). The dams of these offspring were exposed to LPDs during the first 28 days after parturition and exhibited biochemical characteristics of protein deprivation (Cherala et al., 2006). Therefore, the lower microsomal and cytosolic protein yields in their 28-day-old pups could be a direct consequence of protein-energy malnourishment of the dams. It is known that the type and amount of protein in maternal diets affects microsomal protein yields in the offspring (Ronis et al., 1999).
Although perinatal low protein diets altered hepatic P450 isoform activities in 28-day-old rats, they did not produce a consistent pattern of effects. Perinatal LPDs decreased (EROD, CYP2E1, and testosterone 2α-hydroxylase), increased (CPR and testosterone 7α-hydroxylase), and did not affect (PROD, CYP2D1, and testosterone 6β-hydroxylase) catalytic activities of hepatic CYP enzymes in 28-day-old rats. The literature demonstrates that chronic administration of LPD to adult animals resulted in consistent decreases in the activities of a similar group of hepatic P450 enzymes (Zhang et al., 1999; Cancino-Badias et al., 2003). The differences in the findings could be attributed to the differences in the compositions of the diets used, mode of diet administration (direct feeding in adult animals versus predominantly breast milk-derived nutrition in 28-day-old animals), and age of the animals. Most of the hepatic P450 isoforms examined in the current study exhibit large fluctuations in their activities in rats during the first 28 days (prepubertal period) of life. This fact combined with the multiplicity of acute effects of LPDs on membrane fluidity, cellular protein levels, periportal and perivenous morphology, and secretory profiles of hormones involved in CYP regulation could account for the inconsistency of effects of LPDs in 28-day-old rats.
The primary objective of the study was to determine whether low protein diets during the perinatal period could produce alterations in hepatic P450 enzyme activity that persist long after termination of the nutritional insult. Our day 65 data show that a period of postweaning nutritional rehabilitation abolished most of the changes observed in day 28 P450 enzyme activity. In contrast with our results, other investigators have shown that perinatal LPDs produce long-term, possibly permanent, alterations in the activities of periportally (phosphoenolpyruvate carboxykinase and carbamoylphosphate synthetase) and perivenously located enzymes (glucokinase and glutamine synthetase) (Desai et al., 1995). All of these affected enzymes are located in the cytosol, whereas hepatic P450s are membrane bound. Our results therefore suggest that perinatal LPD cannot produce long-term alterations in the activities of membrane bound enzymes. On the other hand, other transient prenatal and neonatal stimuli such as phenobarbital, monosodium glutamate, and testosterone treatment do produce long-term alterations in the activities of many of the hepatic P450 enzymes examined in this study (Bagley and Hayes, 1983; Pampori and Shapiro, 1994; Agrawal and Shapiro, 2000). The reasons for the inability of perinatal LPDs to produce long-term alterations in CYP enzyme activities are presently unclear.
Interestingly, perinatal administration of the LM76 low protein diet decreased HST in 58-day-old female rats. HST is used as a functional measure of the activity of P450 enzymes and a decrease in sleep time suggested a persistent increase in the activity of isoforms responsible for hexobarbital metabolism. In 65-day-old female offspring of the LM76 group, we observed a significant increase in the activity of testosterone 6β-hydroxylase, which is known to be completely constituted of CYP3A1 in female rats (Schenkman and Griem, 1993). A review of the literature indicated that CYP3A1 plays an insignificant role in hexobarbital metabolism and the two major isoforms responsible for hexobarbital metabolism in adult female rats are CYP2C6 and CYP2C12 (Ryan and Levin, 1990). In uninduced adult rats EROD activity is catalyzed primarily by CYP2C6 (Burke et al., 1994). In our studies EROD activity was unaltered in 65-day-old female rats, indicating that the decrease in HST is not due to an alteration in CYP2C6 activity. In the absence of a CYP2C12 catalytic assay in our laboratory, we estimated its protein expression in 65-day-old female rats. CYP2C12 protein expression did not differ between any groups, suggesting that CYP2C12 status also cannot account for the observed decrease in HST in 65-day-old female rats in the LM76 group. Therefore, in this group of rats, we finally measured hexobarbital hydroxylase activity as a relatively nonspecific marker of hexobarbital metabolism. Surprisingly, there were no alterations in its activity either. Our results suggest that the decrease in HST is not due to changes in hexobarbital metabolism and is possibly due to alterations in other determinants of drug action such as drug distribution or receptor status.
We comprehensively characterized the ontogeny of the P450 isoforms from day 28 to day 150, and to ensure consistency we compared our results from the NP group with those from the literature (see supplemental Table 2). In conformity with the literature, the male offspring in the NP group showed no change in the activities of PROD, CYP2E1, and testosterone 6β-hydroxylase from day 28 to day 150, whereas activities of testosterone 7α-hydroxylase and EROD decreased and activities of testosterone 2α-hydroxylase and CYP2D1 increased over that same time period (Imaoka et al., 1991; Schenkman and Griem, 1993; Chow et al., 1999; Johnson et al., 2002). In female offspring in the NP group, there were decreases in the activities of EROD, CYP2E1, testosterone 6β-hydroxylase, testosterone 2α-hydroxylase, and testosterone 7α-hydroxylase between day 28 and day 65 (Waxman et al., 1989; Imaoka et al., 1991; Schenkman and Griem, 1993). Additionally, because we measured the activities of the P450 isoforms in both male and female offspring, we were also able to determine gender differences in CYP activity in young adult rats (65-day-old) in the NP group (Tables 3, 4, and 5) and compare our results to the literature. In conformity with the literature, we observed male-predominant activity of testosterone 2α-hydroxylase and testosterone 6β-hydroxylase whereas activities of CYP2E1 and testosterone 7α-hydroxylase were female-predominant (Schenkman and Griem, 1993; Mode and Gustafsson, 2006). Therefore, despite the inability to demonstrate effects of a perinatal low protein diet on hepatic P450 enzymes in adult animals, it is apparent that most of our data on ontogeny and gender differences in the activity of these enzymes is in concordance with the literature.
Results from our study indicate that the activities of most of the P450 enzymes in the purified diet groups are lower than those in the nonpurified diet group. These results confirm literature reports and are hypothesized to occur because of components in purified diets that exert an inhibitory effect on hepatic P450 enzymes (Rosenberg, 1991). The purified and nonpurified diet compositions differ in sources of protein, fat, carbohydrate, and micronutrients. It is established in the literature that perinatal administration of different proteins (casein, soy protein isolate, or whey) results in different levels of constitutive and inducible levels of various P450 enzymes (Ronis et al., 1999, 2001). In our study, casein is the source of protein in all purified diet groups, whereas a mixture of nonpurified sources of protein is used in the nonpurified diet. Such differences in protein source could account, in part, for the differences in P450 enzyme activities between the purified and nonpurified diet groups.
In summary, perinatal LPDs alter the activity of select P450 enzymes in prepubertal offspring, but not in adult animals. Perinatal LPD produced long-term alterations in the pharmacodynamics of hexobarbital, and this effect was diet- and gender-dependent.
Acknowledgments
The authors acknowledge the assistance of undergraduate researchers Mahmud Ansari, Josephat Waigwa, and Sheetal Patel in the conduct of the animal studies.
Footnotes
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
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doi:10.1124/dmd.106.013748.
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ABBREVIATIONS: P450, cytochrome P450; EROD, ethoxyresorufin O-dealkylase; PROD, pentoxyresorufin O-dealkylase; CPR, cytochrome P450 reductase; LPD, low protein diet; NP, nonpurified diet; C93, AIN93G purified control diet; L93, low protein diet based on the AIN93G purified diet; LM76, low protein diet based on the modified version of the AIN76A purified diet; M76, Modified version of the AIN76A purified control diet; HST, hexobarbital sleep time
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↵ The online version of this article (available at http://dmd.aspetjournals.org) contains supplemental material.
- Received November 7, 2006.
- Accepted March 27, 2007.
- The American Society for Pharmacology and Experimental Therapeutics