DMD Fast Forward. Published on November 8, 2013 as doi:10...

DMD Manuscript #53538

1

PROTEIN RESTORATION IN LOW BIRTH WEIGHT RAT OFFSPRING DERIVED

FROM MATERNAL LOW PROTEIN DIET LEADS TO ELEVATED HEPATIC

CYP3A AND CYP2C11 ACTIVITY IN ADULTHOOD#

Gurjeev Sohi, Eric J. Barry, Thomas J. Velenosi, Bradley L. Urquhart and Daniel B.

Hardy

The Children's Health Research Institute (G.S., D.B.H.) and the Lawson Health Research

Institute (G.S., D.B.H., B.L.U.), Department of Obstetrics & Gynecology (G.S., D.B.H.)

and Physiology & Pharmacology (G.S., E.J.B., T.J.V., B.L.U., D.B.H.), The University

of Western Ontario, London, Ontario, Canada, N6A 5C1

DMD Fast Forward. Published on November 8, 2013 as doi:10.1124/dmd.113.053538

Copyright 2013 by the American Society for Pharmacology and Experimental Therapeutics.

This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on November 8, 2013 as DOI: 10.1124/dmd.113.053538

at ASPE

T Journals on July 3, 2019

dmd.aspetjournals.org

Dow

nloaded from

http://dmd.aspetjournals.org/


2

LOW BIRTH WEIGHT RATS HAVE ELEVATED HEPATIC DRUG METABOLISM

Corresponding Author: Daniel B. Hardy The Department of Physiology & Pharmacology

The University of Western Ontario, London, Ontario, Canada, N6A 5C1 email: [email protected]

telephone: 519-661-2111 ext.84238 fax: 519-661-3827

Number of Text Pages 35 Number of Tables 2 Number of Figures 4 Number of References 59 Number of Words in Abstract 228 Number of Words in Introduction 977 Number of Words in Discussion 1356

Abbreviations: 11�-HSD1, 11�-hydroxysteroid dehydrogenase-1; CAR, constitutive androstane receptor; CVD, cardiovascular disease; cDNA, complementary DNA; P450, cytochrome p450; ER, endoplasmic reticulum; IUGR, intrauterine growth restriction; LP, low protein; MPR, maternal protein restriction; PXR, pregnane x receptor, RT-PCR, reverse transcription-polymerase chain reaction; UPLC-PDA, ultraperformance liquid chromatography with photodiode array detection


at ASPE



Dow

nloaded from



3

Abstract

The World Health Organization has identified hypercholesterolemia to be one of the

major symptoms encompassing the metabolic syndrome. Moreover, epidemiological

evidence indicates that low birth weight offspring are at greater risk of developing the

metabolic syndrome. Previous work in our laboratory demonstrated that maternal protein

restriction (MPR) results in impaired fetal growth and hypercholesterolemia in adulthood.

This was attributed to repression of hepatic CYP7A1, a rate limiting enzyme that

catabolizes cholesterol to bile acids. Another important function of hepatic cytochrome

P450 enzymes is the phase I oxidative metabolism of drugs (i.e. statins for

hypercholesterolemia), which can significantly impact pharmacokinetics. We

hypothesized that MPR offspring may have altered ability to metabolize drugs in

adulthood. To address this hypothesis, Wistar rats were maintained on a 20% protein diet

(Control) or a low 8% protein diet throughout prenatal and postnatal life (LP1) or

exclusively during prenatal life and weaning (LP2). Intriguingly CYP3A and CYP2C11

intrinsic clearance (Vmax/Km) was significantly increased exclusively in LP2 offspring at

postnatal day 130 compared to control or LP1 offspring, as evaluated by testosterone

enzyme kinetics in liver microsomes. The increase in activity was secondary to an

increase in CYP3A23 and CYP2C11 mRNA. Collectively, these findings suggest that a

low birth weight offspring followed by postnatal catch-up growth may have a diminished

response to xenobiotics metabolized by CYP3A and CYP2C11 enzymes.


at ASPE



Dow

nloaded from



4

Introduction

Clinical studies have reported a strong inverse correlation between birth weight and

metabolic risk factors associated with cardiovascular disease (CVD) (Barker, et al., 1989;

Nilsson, et al., 1997; Curhan, et al., 1996; Curhan, et al., 1996; Leon, et al., 1996).

Therefore, the likelihood of prescribing medication for the management of these

metabolic symptoms (i.e. statins for hypercholesterolemia) can be considered to be

greater in these offspring. This may be particularly relevant in cases of low birth weight

offspring, which undergo nutrition-induced accelerated growth in neonatal life and

display an earlier onset of these symptoms (Straka, et al., 1990; Crowther, et al., 1998;

Yajnik, 2000; Eriksson, 2011; Finken, et al., 2006; Martin, et al., 2003). The underlying

reason behind this phenomenon can be explained by the “Predictive Adaptive Response”

hypothesis, which suggests that adverse events during development induce adaptations

suited for survival in a similar predictive environment but can become maladaptive if a

‘mismatch’ to the predictive environment occurs, leading to a “thrifty phenotype” (Hales

and Barker, 2001; Hales and Barker, 1992; Rickard and Lummaa, 2007). Clinically, this

hypothesis has been supported by evidence where accelerated growth due to higher

nutrient exposure in preterm infants results in an increase in markers of the metabolic

syndrome by adolescence (Singhal, et al., 2001; Singhal, et al., 2002; Singhal, et al.,

2003; Singhal and Lucas, 2004; Singhal, et al., 2004). However, very little is known

about drug disposition in adult life of low birth offspring. Moreover, the role of

accelerated growth as result of a nutrition mismatch in postnatal life has not been

examined.


at ASPE



Dow

nloaded from



5

Several clinical and animal studies have observed liver dysfunction in postnatal life of

low birth weight infants (Boehm, et al., 1990; Burns, et al., 1997; Ozanne, et al., 1996;

Rees, et al., 2000; Lillycrop, et al., 2008). Given that approximately 75% of prescribed

drugs are metabolized in the liver (Wienkers and Heath, 2005), liver dysfunction may

alter the pharmacokinetic parameters of several drugs in the postnatal life of these infants.

Amongst the prescribed drugs eliminated by the liver, three fourths of these are

metabolized by the cytochrome P450 (P450) family of enzymes (Wienkers and Heath,

2005). In humans, reduced intravenous midazolam clearance, a measure of hepatic

CYP3A4 activity, has been observed in preterm infants aged between 2 to 15 days (Lee,

et al., 1999; Thummel and Wilkinson, 1998; Thummel, et al., 1996). Interestingly, the

CYP3A4 isozyme is responsible for the metabolism of approximately half of all marketed

drugs (Wrighton, et al., 1996). Yet, it remains to be investigated whether

pharmacokinetics of drugs in the liver are altered in adult life of low birth weight

offspring.

Animal models make it feasible to investigate alterations in pharmacokinetics of a drug to

changes in hepatic P450 enzyme activity. The long-term programming of Cyp3a activity

was recently demonstrated by Tajima et. al. in mice offspring born of mothers receiving a

high fat diet during pregnancy (Tajima, et al., 2012). Specifically, they correlated a

decrease in hepatic Cyp3a activity in six week old offspring with reduced triazolam

substrate clearance (Tajima, et al., 2012). Although an important finding, it remains to be

established whether similar long-term programming of P450 enzyme activities occur

when in utero insults result in low birth weight offspring. In rodent models, low birth


at ASPE



Dow

nloaded from



6

weight offspring undergoing accelerated growth rates due to a postnatal nutrition

mismatch display hypertension (Boubred, et al., 2009), obesity (Desai, et al., 2007),

hypercholesterolemia (Nusken, et al., 2008), insulin resistance (Desai, et al., 2007) and

reduced longevity (Jennings, et al., 1999). Therefore, it would be imperative to also

determine the adverse consequences of altering the postnatal nutrition environment of

low birth weight offspring on long-term programming of hepatic drug metabolizing P450

enzymes.

Maternal protein restriction (MPR) dietary regime is a relevant animal model to study the

developmental origins of adult diseases, since MPR shares features common with

placental insufficiency induced intrauterine growth restriction (PI-IUGR), which occurs

in 8% of pregnancies and produces protein deficiency in the developing fetus (Crosby,

1991; Lamarche, et al., 1998; Ross and Beall, 2008). We and others have previously

demonstrated that MPR (8% protein) during pregnancy and lactation in wistar rats leads

to asymmetric IUGR offspring (Desai and Hales, 1997, Sohi, et al., 2011), which exhibit

impaired liver function in adulthood (Burns, et al., 1997; Ozanne, et al., 1996; Rees, et

al., 2000; Lillycrop, et al., 2005). In addition, we have observed that these offspring

display high circulating and hepatic cholesterol levels exclusively when they are faced

with a nutritional mismatch of a normal (20%) protein diet post-weaning. This was

attributed to the long-term repression of cholesterol 7α-hydroxylase (CYP7A1), which is

rate-limiting for the catabolism of cholesterol to bile acids (Sohi, et al., 2011). It is

noteworthy that decreased CYP7A1 expression is also associated with Endoplasmic

Reticulum (ER) stress in a rat model of hypothyroidism (Zhou, et al., 2009). This is of


at ASPE



Dow

nloaded from



7

great interest considering that we have recently demonstrated that these MPR offspring

exhibit elevated markers of ER stress (Sohi, et al., 2013). Specifically, higher steady-state

levels of phosphorylated eukaryotic initiation factor (eIF)-2 at Serine 51 and glucose

regulated protein Grp78 in the livers were noted at four months of age (Sohi, et al., 2013).

In contrast, MPR derived offspring that were maintained on a low protein diet throughout

life did not display hypercholesterolemia or hepatic ER stress. As P450 enzymes (i.e.

CYP3A23, CYP3A2 and CYP2C11) reside in the ER of the liver and are involved with

Phase 1 of drug metabolism (Avadhani, et al., 2011), we hypothesized that MPR derived

low birth offspring may have an impaired activity of P450 drug-metabolizing enzymes in

adulthood when faced with a nutritional mismatch of a normal (20%) protein diet in

postnatal life. To address this, the effects of a low protein diet throughout life (LP1) or

until lactation (LP2) were compared relative to a normal protein diet throughout life of

the offspring (C).


at ASPE



Dow

nloaded from



8

Materials and Methods

Animals and Dietary Regimes

All procedures were performed in accordance with the guidelines set by the Canadian

Council of Animal Care and upon approval of the Animal Care Committee of the

University of Western Ontario. Female and male Wistar rats at breeding age (250 g) were

purchased from Charles River (La Salle, St.Constant, Quebec, Canada). These rats were

housed in individual cages and maintained at room temperature on a 12-h light, 12-h dark

cycle. For 3 weeks, these rats were left to acclimatize to the animal care facility and their

reproductive cycles were followed. At the onset of proestrus, these rats were mated.

Impregnation was confirmed by the presence of sperm in the vaginal smear the next

morning. Upon confirmation of impregnation (gestation day 1), the rats were fed either a

control diet containing 20% protein or a LP diet containing 8% protein. The LP diet

contained similar fat content and was made isocaloric by a 14% increase in carbohydrates

(Bio-Serv, Frenchtown, NJ, USA). At birth, the litter size was reduced to eight animals

(four females and four males), with weights closest to the litter mean. This ensured a

standard litter size for all mothers. Three different dietary regimes were administered to

these offspring. Offspring derived from a maternal LP diet were either administered the

LP diet throughout postnatal life (LP1) or until the end of weaning (LP2). The LP1 and

LP2 offspring were compared to the Control offspring which received a control diet

throughout prenatal and postnatal life. Food and water was provided ad libitum. The food

intake of these offspring were monitored by measuring their food consumption every

third day, and have been previously published (Sohi, et al., 2011).


at ASPE



Dow

nloaded from



9

At postnatal day 21 (d21), a subset of the offspring were sacrificed and the medial lobe

liver tissue were excised and snap frozen for quantitative RT-PCR. At postnatal day 130

(d130), the pups were also sacrificed and the medial lobe liver tissue was excised and

frozen for quantitative RT-PCR and for testosterone enzyme kinetics in liver microsomes

via ultra performance liquid chromatography. We did not examine the female offspring to

prevent confounding factors related to their estrous cycle and their hormonal profile.

Moreover, the maternal low protein model has been demonstrated to exhibit early life

programming effects in a sexually dimorphic manner, which was not the focus of this

study (Sohi, et al., 2011; Chamson-Reig, et al., 2009; Guan, et al., 2005)

Real Time PCR Analysis

Total RNA from Wistar rat medial lobe liver tissue was extracted at d21 and d130 by the

one-step method of Chomczynski and Sacchi (Chomczynski and Sacchi, 1987) (TRIzol,

Invitrogen, Carlsbad, CA, USA). RNA was treated with deoxyribonuclease to remove

any contaminating DNA. 4 µg of the total RNA was reverse transcribed to cDNA using

random primers and Superscript II RNase H-reverse transcriptase (Invitrogen, Carlsbad,

CA, USA). Primer sets directed against rat CYP2B1, CYP3A23, CYP3A2, CYP2C11,

Car, Pxr, and β-actin were generated via Primer Express software (PE Applied

Biosystems, Boston, MA, USA) based on published sequences (Table 1). The relative

abundance of each transcript was determined by real-time quantitative PCR as previously

published (Hardy, et al., 2006). For the quantitative analysis of mRNA expression, the

Bio-Rad CFX384 Real Time System was employed using the DNA binding dye IQTM

SYBER green supermix (Bio-Rad, Mississauga, Ontario, Canada). The cycling


at ASPE



Dow

nloaded from



10

conditions were 50°C for 2 min, 95°C for 10 min, followed by 45 cycles of 95°C for 15

sec and 60°C for 1 min. The cycle threshold was set at a level where the exponential

increase in PCR amplification was approximately parallel between all samples. All

primer sets produced amplicons of the expected size and sequence. We calculated the

relative fold changes using the comparative cycle times (Ct) method with β-actin as the

reference guide. Over a wide range of known cDNA concentrations, all primer sets were

demonstrated to have good linear correlation (slope=-3.4) and equal priming efficiency

for the different dilutions compared with their Ct values (data not shown). Melt curve

analysis was conducted at the end of the PCR reaction to ensure a single peak for all

primers. Moreover, all primer sets were validated by amplifying cDNA followed by

running the product on an agarose gel to confirm a single band at the expected amplicon

size. Given that all primer sets had equal priming efficiency, the ∆Ct values (primer

internal control) for each primer set were calibrated to the experimental samples with the

lowest transcript abundance (highest Ct value), and the relative abundance of each primer

set compared with calibrator was determined by the formula, 2-∆∆Ct, in which ∆∆Ct is the

calibrated Ct value.

Hepatic Microsome Isolation

Wistar rat liver microsomes were isolated by differential centrifugation using methods

described previously by Velenosi et al. (Velenosi, et al., 2012). Briefly, 0.9% NaCl

solution was used to rinse liver tissue. The rinsed tissue was homogenized in 1.15% KCl

solution containing 1 mM EDTA and was centrifuged at 9000g for 20 min at 4°C. The

subsequent supernatant was centrifuged at 105, 000g for 60 min at 4°C. The microsomal


at ASPE



Dow

nloaded from



11

pellet was resuspended in 100 mM potassium phosphate buffer containing 20% glycerol

at pH 7.4 and protein concentration was determined by colorimetric BCA Protein Assay

(Pierce Corp., Madison, WI, USA). Microsomal protein extract was stored at -80°C for

further analysis.

Hepatic Metabolism of Testosterone by CYP3A and CYP2C11 Enzymes

Metabolic activity of CYP3A and CYP2C11 in hepatic microsomes was determined

using methods previously described by Velenosi et al. (2012) (Velenosi, et al., 2012).

Testosterone was selected as a probe for CYP3A and CYP2C11 enzyme activities based

on previously documented selective metabolism by specific rat P450 isozymes (Velenosi,

et al., 2012; Souidi, et al., 2005). 50 mM potassium phosphate buffer and 2 mM MgCl2

(pH 7.4) with 1 mg/ml hepatic microsomal protein equating to a final volume of 250l

was used for timed enzymatic reactions. Linear rate of production of metabolites was

determined by varying time, protein and relevant substrate concentrations prior to

conducting enzymatic reactions. Formation of testosterone metabolites (6-OH

testosterone and 16-OH testosterone) was determined to be linear at 10 minutes. The

reactions were initiated by addition of 1mM NADPH to microsomal samples containing

varying concentrations of testosterone. The reaction was terminated by addition of 50 l

of ice-cold acetonitrile followed by 15-minute incubation on ice and centrifugation to

pellet precipitated protein (Velenosi, et al., 2012)


at ASPE



Dow

nloaded from



12

Testosterone Metabolite Analysis by Ultra Performance Liquid Chromatography

with Photodiode Array (UPLC-PDA) Detection

Testostereone metabolite analysis was performed by solid-phase extraction followed by

UPLC-PDA using methods previously described (Velenosi, et al., 2012). The solid-phase

extraction cartridge (C18, Strata-X Polymeric Reverse Phase 33 m; Phenomenex,

Torrance, CA) was conditioned according to manufacturer’s specification.

Carbamazepine was used as an internal standard for testosterone quantification. The

analytes and internal standard were passed across the packing of the cartridge by gravity.

The cartridges were then washed with 1 ml of Milli-Q water followed by 1 ml of 50:50

methanol/water. 1 ml of methanol containing 0.1% triethylamine and 0.1% trifluoroacetic

acid was used to elute the analytes into clean glass test tubes. The eluent was dried,

reconstituted in mobile phase and injected on a Phenomenex Kinetex C18 column (1.7

m particle size, 50 x 2.1 mm; Torrence, CA) for testosterone and metabolite separation.

The column was maintained at 40°C in a Waters ACQUITY UPLC H-Class System. The

mobile phase flow and gradient used for each assay was the same as previously described

(Velenosi, et al., 2012). ACQUITY UPLC PDA detector (Waters) was used to detect

testosterone (245 nm) and carbamazepine at (290 nm) for quantification. The interassay

and intraassay coefficients of variation of the analytes was <10%.


at ASPE



Dow

nloaded from



13

Statistics

All results were expressed as the mean of arbitrary values the standard error of the

mean (SEM). The significance of differences (p<0.05) between mean values were

evaluated using the unpaired Student’s t-test for RT-PCR and testosterone enzyme

kinetics in liver microsomes via ultra performance liquid chromatography. One-way

analysis of variance (ANOVA) followed by a Bonferroni’s Multiple Comparison post

hoc test, was used to evaluate significance of differences for results comparing the effect

of all the dietary regimes for Q-RT-PCR analysis.


at ASPE



Dow

nloaded from



14

Results

A switch to a normal protein diet in postnatal life of MPR derived low birth weight

offspring leads to increases in the levels of steady-state hepatic CYP3A23, CYP2C11

and CYP2B1 mRNA at postnatal day 130

In this study, we wanted to investigate the long-term effects of MPR derived low birth

weight offspring on expression and function of major P450 drug metabolizing

cytochrome enzymes in the liver. Q-RT-PCR analysis was conducted to examine the

steady-state mRNA expression of hepatic CYP3A23, CYP3A2, CYP2C11 and CYP2B1

at postnatal day 130. A 1.79, 1.45 and 1.94 fold increase in CYP3A23, CYP2C11,

CYP2B1 respectively was observed in the livers of MPR offspring who were placed on a

normal protein diet post-weaning (LP2) compared to the Control (Figure 1A, C, D).

Interestingly, in MPR offspring subjected to protein restriction throughout pregnancy and

postnatal life (LP1), there was no difference in hepatic CYP3A23, CYP3A2, CYP2C11

and CYP2B1 mRNA expression (Figure 1A, B, C, D). Upon comparing the two MPR

dietary regimes, the LP2 offspring displayed a 1.93, 1.27 and 3.04 fold elevation in

hepatic CYP3A23, CYP2C11 and CYP2B1 mRNA expression, respectively, relative to

LP1 (Figure 1A, C, D). There was also a noticeable trend to an increase in CYP3A2

mRNA expression in LP2 offspring compared to Control and LP1 offspring (P<0.06)

(Figure 1B). The average steady-state mRNA levels for the Control group were 25.03,

24.85, 31.96, 20.81, 20.72 cycles threshold for CYP3A23, CYP3A2, CYP2B1, CYP2C11

and -actin, respectively.

We also evaluated whether the two LP dietary regimes differentially affect the

steady state mRNA levels of the nuclear receptors Car and Pxr, considering that they


at ASPE



Dow

nloaded from



15

serve as master transcription factors regulating the transcription of several xenobiotic

detoxification enzymes (Wang, et al., 2012). As depicted in Figure 2, there was no

difference in Car mRNA levels between the LP1 and LP2 offspring. However, there was

a slight increase in hepatic Car mRNA level in the LP2 offspring when compared to

Control.

Elevated expression of hepatic CYP3A23 and CYP2C11 correlates with increases in

their drug metabolizing activity in MPR offspring.

To evaluate whether postnatal 20% protein dietary restoration in MPR derived low birth

weight offspring also impacted long-term function of hepatic CYP3A and CYP2C11

enzymes, testosterone metabolism assay was performed using rat liver microsomes. This

assay is used to determine CYP3A and CYP2C11 enzyme activities, as testosterone

metabolism to 6-OH testosterone and 16-OH testosterone has been previously

observed to be mediated by CYP3A and CYP2C11 enzymes, respectively. 16-OH

testosterone is also an indicator of CYP2B1 under conditions when CYP2B1 is induced

(Williams and Borghoff, 2000; Chovan, et al., 2007). Therefore, we also measured 2-

OH testosterone, given that CYP2C11 also metabolizes testosterone to this metabolite

while CYP2B1 does not (Chovan, et al., 2007). Full enzyme kinetics of these metabolites

was determined for this study. Vmax/Km, a measure of intrinsic clearance for 6-OH

testosterone, 16-OH testosterone and 2-OH testosterone, was significantly elevated by

1.9. 3.25 and 2.14 fold respectively in LP2 offspring compared to the Control offspring

and by 1.8, 4.33 and 2.5 fold respectively in LP2 offspring compared to the LP1 offspring

at postnatal day 130 (Figure 3 A, B, C). There was no difference in Vmax/Km for 6-OH


at ASPE



Dow

nloaded from



16

testosterone, 16-OH testosterone and 2-OH testosterone between LP1 and Control

offspring (Figure 3 A, B, C). The increases in intrinsic clearance of testosterone

metabolites corresponded with the increases in CYP2B1, CYP2C11 and CYP3A23

mRNA expression, respectively (Figure 1D, C, A). The Michaelis-Menten kinetic

parameters for 6-OH testosterone, 16-OH testosterone and 2-OH testosterone are

presented in Table 2.

The expression of the major hepatic P450 drug metabolizing enzymes were

unaltered by MPR at postnatal day 21.

Since an increase in CYP3A, CYP2B and CYP2C11 expression and activity was

observed by adulthood in MPR offspring that received a control protein diet post-

weaning (LP2), we further pursued whether this increase was exclusively due to a switch

in diet post-weaning or was persistent before the normal protein was restored at postnatal

day 21. Interestingly, MPR offspring displayed no significant difference in CYP3A23,

CYP3A2, CYP2B1 and CYP2C11 mRNA levels compared to Control at postnatal day 21

(Figure 4). The average steady-state mRNA levels for the Control group were 24.85,

23.68, 26.45, 33.57, 20.89 cycles threshold for CYP3A23, CYP3A2, CYP2B1, CYP2C11

and -actin, respectively.


at ASPE



Dow

nloaded from



17

Discussion

In this study we present the novel finding that MPR derived low birth weight rat

offspring have elevated CYP3A and CYP2C11 activity in adulthood, exclusively when

faced with a nutritional mismatch of a normal (20%) protein diet in postnatal life. This

was found to coincide with increases in their steady-state mRNA levels. Interestingly,

when low birth weight offspring were maintained on a low protein diet post-lactation,

they exhibited no differences in expression of these P450 enzymes. Collectively, this

study suggests that an inappropriate dietary intervention strategy in IUGR offspring may

augment important hepatic drug-metabolizing enzymes in adult life.

Contrary to what we had initially hypothesized, administering a normal protein diet post-

weaning to maternal low protein diet derived low birth weight offspring (LP2) resulted in

elevated expression of drug metabolizing enzymes CYP3A23 and CYP2C11 in

adulthood. The LP2 offspring displayed corresponding increases in intrinsic enzymatic

activity of these P450 enzymes. Given that testosterone is also a substrate for these P450

enzymes, it is noteworthy that circulating testosterone has been previously reported to be

decreased in these LP2 offspring (Chamson-Reig, et al., 2009). Our data provide

evidence for a potential mechanism behind this observation. However, a previous study

has reported a decrease in testis weight and function in offspring derived from a low

protein diet during pregnancy and lactation (Zambrano, et al., 2005). Therefore, the

possibility of decreases in testosterone synthesis may also contribute to a reduced

circulating testosterone level in the LP2 offspring. Conversely, no differences in the

steady-state mRNA expression and activity of these P450 enzymes were observed when


at ASPE



Dow

nloaded from



18

the IUGR offspring were maintained on a low protein diet (LP1). Moreover, other than a

trend to a decrease in CYP2C11, no significant changes in expression of any of these

P450 enzymes were observed at postnatal day 21, which represents the end of lactation, a

time point where normal protein was restored for LP2 offspring. This goes to suggest that

the postnatal nutritional mismatch of a normal (20%) protein diet is likely the more

significant contributing factor to elevated CYP3A23 and CYP2C11 expression in adult

life, as opposed to the direct actions of the low protein diet itself during pregnancy and

lactation. Since, increases in CYP3A and CYP2C11 activity were consistent with

CYP3A23 and CYP2C11 steady-state mRNA expression in LP2 offspring, we postulated

transcriptional mechanisms to underlie the increases in activity. Specifically, we

attempted to investigate the role of xenobiotic sensing nuclear receptors Pxr and Car

which represent major transcription factors involved with the transcriptional induction of

P450 drug-metabolizing enzymes in humans (Willson and Kliewer, 2002). In this study

we observed a slight increase in Car mRNA levels in LP2 offspring when compared to

the Control group. However, there was no difference in Car levels between LP1 and LP2

offspring. We also observed a noticeable increase in the well characterized increases in

expression of the Car target gene CYP2B1 in the LP2 offspring (Yoshinari, et al., 2001;

Honkakoski, et al., 1998). However, it is unlikely that the modest increase in CAR

expression is completely responsible for the observed changes in CYP3A23 and

CYP2C11 expression. Moreover, the role of endocrine factors, which are key regulators

of these P450 enzymes, cannot be ruled out and deserve further consideration. For

instance, corticosteroids have been previously demonstrated to upregulate CYP3A23 and

CYP2C11 (Daskalopoulos, et al., 2012; Huss, et al., 1996). Interestingly, our laboratory


at ASPE



Dow

nloaded from



19

has recently demonstrated that LP2 dietary regime leads to an upregulation of hepatic

11�-hydroxysteroid dehydrogenase-1 (11�-HSD1) (Vo, et al., 2013) . An increase in 11�-

HSD1 would be indicative of an increased conversion of inactive corticosteroids to their

active form. Therefore, it is possible that a potential increase in active corticosteroids may

underlie the upregulation of these P450 enzymes in the livers of LP2 offspring in adulthood.

In support of the main tenet of the “predictive adaptive response” hypothesis, MPR

derived low birth weight rat offspring when faced with a nutritional mismatch in

postnatal life, have been previously reported to display hypercholesterolemia (Nusken, et

al., 2008), visceral obesity (Desai, et al., 2007), hypertension (Boubred, et al., 2009), type

2 diabetes (Desai, et al., 2007) and reduced longevity (Jennings, et al., 1999). Conversely,

in the absence of protein restoration, rat IUGR offspring have been observed to live

longer (Sasaki, et al., 1982) . Given that the liver plays a key role in metabolism, any

alterations in its function can lead to the development of the metabolic syndrome and

reduced lifespan. We recently observed indices of hepatic ER stress exclusively in MPR

derived IUGR offspring, which received restored maternal protein in postnatal life (Sohi,

et al., 2013). Interestingly, an elegant study by Pascual et. al. in 2008 demonstrated that

tunicamycin-induced ER stress in HepG2 cells induced the expression of CYP2B6, the

human ortholog of CYP2B1 (Pascual, et al., 2008). The molecular mechanism behind this

induction was attributed to ER stress activation of liver enriched activating transcription

factor 5 (ATF5), which shares close sequence homology to the more ubiquitously

expressed ATF4. ATF4 activation is known to occur via its selective translation during a

period of global protein translation attenuation due to elevated phosphorylation of

eukaryotic initiation factor 2. Therefore, it is possible that activation of ATF5 due to the


at ASPE



Dow

nloaded from



20

previously observed hepatic increases in phosphorylation of eukaryotic initiation factor

2 (Pascual, et al., 2008), may also cause the induction in CYP2B1 expression observed

in the study. Interestingly, ATF5 has been observed to interact with C/EBP family of

transcription factors, as well as synergistically potentiate the actions of nuclear receptor

Car, which are both established regulators of the CYP2B6 gene (Neuvonen, et al., 2006).

With the use of Alggen PROMO software, we have identified multiple putative

C/EBP binding sites at the promoter of all the P450 enzymes examined in this study.

Therefore, it is conceivable that under conditions of stress (i.e. ER stress), the liver of

LP2 low birth weight offspring responds by elevating hepatic xenobiotic metabolizing

enzymes in an attempt to increase their detoxification capacity.

To date there have been no clinical studies conducted to evaluate the effects of low birth

weight on the pharmacokinetics of drugs in adulthood. This is particularly relevant to

drugs that would likely be used to manage the symptoms of the metabolic syndrome that

are observed in these offspring. For instance, despite strong clinical and animal evidence

linking IUGR to hepatic dysfunction and elevated cholesterol levels in adult life, there is

little known about whether statin pharmacokinetic or pharmacodynamic parameters are

altered. Out of all the statins that target the liver, simvastatin, lovastatin and atorvastatin

are metabolized primarily through CYP3A4, and fluvastatin is metabolized through

CYP2C9 (Neuvonen, et al., 2006). Since, the rat ortholog of CYP2C9 (i.e. CYP2C11)

activities was elevated in adulthood, it is likely that low birth weight rat offspring would

metabolize fluvastatin in the liver at a faster rate. Our data suggest that low birth weight

rat offspring may require larger doses of fluvastatin to maintain efficacy towards


at ASPE



Dow

nloaded from



21

reducing circulating cholesterol levels, a hypothesis which still remains to be tested.

Moreover, in order to completely understand the impact of low birth weight on alterations

in pharmacokinetics of drugs, several additional factors need to be evaluated, mainly

changes in plasma binding protein levels and drug transporter function. It is also

important to determine whether different insults, including infection, stress, placental

dysfunction and inflammation, leading to low birth weight would similarly impact drug

pharmacokinetic measures in adult life.

In summary, this study highlights that low birth weight offspring faced with a nutritional

mismatch of a normal (20%) protein diet in postnatal life have elevated activity of hepatic

phase I drug metabolizing enzymes CYP3A and CYP2C11 in adulthood. Moreover,

maintaining these low birth weight offspring on a low protein diet prevented the increases

in expression of these enzymes. It is plausible that these offspring sense the nutritional

mismatch as an unanticipated insult and consequently respond by increasing their

detoxification capacity. In light of this study, future studies examining a need for

optimizing drug dosing to ameliorate symptoms of metabolic syndrome in IUGR

offspring that display nutritional mismatch induced accelerated growth would be useful.


at ASPE



Dow

nloaded from



22

Acknowledgements

We thank Dr. Lin Zhao (Department of Obstetrics and Gynecology, Western University)

for technical assistance with molecular analysis.


at ASPE



Dow

nloaded from



23

Authors Contributions

Participated in Research Design Study: Sohi, Urquhart, Hardy

Conducted Experiments: Sohi

Contributed new reagents or analytic tools: Urquhart, Hardy

Performed data analysis: Sohi, Barry, Velenosi

Wrote or contributed to the writing of the manuscript: Sohi, Barry, Velenosi, Urquhart,

Hardy


at ASPE



Dow

nloaded from



24

References

Avadhani NG, Sangar MC, Bansal S and Bajpai P (2011) Bimodal targeting of cytochrome P450s to endoplasmic reticulum and mitochondria: the concept of chimeric signals. FEBS J 278:4218-4229. Barker DJ, Winter PD, Osmond C, Margetts B and Simmonds SJ (1989) Weight in infancy and death from ischaemic heart disease. Lancet 2:577-580. Boehm G, Muller DM, Teichmann B and Krumbiegel P (1990) Influence of intrauterine growth retardation on parameters of liver function in low birth weight infants. Eur J Pediatr 149:396-398. Boubred F, Daniel L, Buffat C, Feuerstein JM, Tsimaratos M, Oliver C, Dignat-George F, Lelievre-Pegorier M and Simeoni U (2009) Early postnatal overfeeding induces early chronic renal dysfunction in adult male rats. Am J Physiol Renal Physiol 297:F943-51. Burns SP, Desai M, Cohen RD, Hales CN, Iles RA, Germain JP, Going TC and Bailey RA (1997) Gluconeogenesis, glucose handling, and structural changes in livers of the adult offspring of rats partially deprived of protein during pregnancy and lactation. J Clin Invest 100:1768-1774. Chamson-Reig A, Thyssen SM, Hill DJ and Arany E (2009) Exposure of the pregnant rat to low protein diet causes impaired glucose homeostasis in the young adult offspring by different mechanisms in males and females. Exp Biol Med (Maywood) 234:1425-1436. Chomczynski P and Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156-159. Chovan JP, Ring SC, Yu E and Baldino JP (2007) Cytochrome P450 probe substrate metabolism kinetics in Sprague Dawley rats. Xenobiotica 37:459-473. Crosby WM (1991) Studies in fetal malnutrition. Am J Dis Child 145:871-876. Crowther NJ, Cameron N, Trusler J and Gray IP (1998) Association between poor glucose tolerance and rapid post natal weight gain in seven-year-old children. Diabetologia 41:1163-1167. Curhan GC, Chertow GM, Willett WC, Spiegelman D, Colditz GA, Manson JE, Speizer FE and Stampfer MJ (1996) Birth weight and adult hypertension and obesity in women. Circulation 94:1310-1315. Curhan GC, Willett WC, Rimm EB, Spiegelman D, Ascherio AL and Stampfer MJ (1996) Birth weight and adult hypertension, diabetes mellitus, and obesity in US men. Circulation 94:3246-3250.


at ASPE



Dow

nloaded from



25

Daskalopoulos EP, Malliou F, Rentesi G, Marselos M, Lang MA and Konstandi M (2012) Stress is a critical player in CYP3A, CYP2C, and CYP2D regulation: role of adrenergic receptor signaling pathways. Am J Physiol Endocrinol Metab 303:E40-54. Desai M, Babu J and Ross MG (2007) Programmed metabolic syndrome: prenatal undernutrition and postweaning overnutrition. Am J Physiol Regul Integr Comp Physiol 293:R2306-14. Desai M and Hales CN (1997) Role of fetal and infant growth in programming metabolism in later life. Biol Rev Camb Philos Soc 72:329-348. Eriksson JG (2011) Early growth and coronary heart disease and type 2 diabetes: findings from the Helsinki Birth Cohort Study (HBCS). Am J Clin Nutr 94:1799S-1802S. Finken MJ, Inderson A, Van Montfoort N, Keijzer-Veen MG, van Weert AW, Carfil N, Frolich M, Hille ET, Romijn JA, Dekker FW, Wit JM and Dutch POPS-19 Collaborative Study Group (2006) Lipid profile and carotid intima-media thickness in a prospective cohort of very preterm subjects at age 19 years: effects of early growth and current body composition. Pediatr Res 59:604-609. Guan H, Arany E, van Beek JP, Chamson-Reig A, Thyssen S, Hill DJ and Yang K (2005) Adipose tissue gene expression profiling reveals distinct molecular pathways that define visceral adiposity in offspring of maternal protein-restricted rats. Am J Physiol Endocrinol Metab 288:E663-73. Hales CN and Barker DJ (2001) The thrifty phenotype hypothesis. Br Med Bull 60:5-20. Hales CN and Barker DJ (1992) Type 2 (non-insulin-dependent) diabetes mellitus: the thrifty phenotype hypothesis. Diabetologia 35:595-601. Hardy DB, Janowski BA, Corey DR and Mendelson CR (2006) Progesterone receptor plays a major antiinflammatory role in human myometrial cells by antagonism of nuclear factor-kappaB activation of cyclooxygenase 2 expression. Mol Endocrinol 20:2724-2733. Honkakoski P, Zelko I, Sueyoshi T and Negishi M (1998) The nuclear orphan receptor CAR-retinoid X receptor heterodimer activates the phenobarbital-responsive enhancer module of the CYP2B gene. Mol Cell Biol 18:5652-5658. Huss JM, Wang SI, Astrom A, McQuiddy P and Kasper CB (1996) Dexamethasone responsiveness of a major glucocorticoid-inducible CYP3A gene is mediated by elements unrelated to a glucocorticoid receptor binding motif. Proc Natl Acad Sci U S A 93:4666-4670. Jennings BJ, Ozanne SE, Dorling MW and Hales CN (1999) Early growth determines longevity in male rats and may be related to telomere shortening in the kidney. FEBS Lett 448:4-8. Lamarche B, Lemieux S, Dagenais GR and Despres JP (1998) Visceral obesity and the


at ASPE



Dow

nloaded from



26

risk of ischaemic heart disease: insights from the Quebec Cardiovascular Study. Growth Horm IGF Res 8 Suppl B:1-8. Lee TC, Charles BG, Harte GJ, Gray PH, Steer PA and Flenady VJ (1999) Population pharmacokinetic modeling in very premature infants receiving midazolam during mechanical ventilation: midazolam neonatal pharmacokinetics. Anesthesiology 90:451-457. Leon DA, Koupilova I, Lithell HO, Berglund L, Mohsen R, Vagero D, Lithell UB and McKeigue PM (1996) Failure to realise growth potential in utero and adult obesity in relation to blood pressure in 50 year old Swedish men. BMJ 312:401-406. Lillycrop KA, Phillips ES, Jackson AA, Hanson MA and Burdge GC (2005) Dietary protein restriction of pregnant rats induces and folic acid supplementation prevents epigenetic modification of hepatic gene expression in the offspring. J Nutr 135:1382-1386. Lillycrop KA, Phillips ES, Torrens C, Hanson MA, Jackson AA and Burdge GC (2008) Feeding pregnant rats a protein-restricted diet persistently alters the methylation of specific cytosines in the hepatic PPAR alpha promoter of the offspring. Br J Nutr 100:278-282. Martin RM, McCarthy A, Smith GD, Davies DP and Ben-Shlomo Y (2003) Infant nutrition and blood pressure in early adulthood: the Barry Caerphilly Growth study. Am J Clin Nutr 77:1489-1497. Neuvonen PJ, Niemi M and Backman JT (2006) Drug interactions with lipid-lowering drugs: mechanisms and clinical relevance. Clin Pharmacol Ther 80:565-581. Nilsson PM, Ostergren PO, Nyberg P, Soderstrom M and Allebeck P (1997) Low birth weight is associated with elevated systolic blood pressure in adolescence: a prospective study of a birth cohort of 149378 Swedish boys. J Hypertens 15:1627-1631. Nusken KD, Dotsch J, Rauh M, Rascher W and Schneider H (2008) Uteroplacental insufficiency after bilateral uterine artery ligation in the rat: impact on postnatal glucose and lipid metabolism and evidence for metabolic programming of the offspring by sham operation. Endocrinology 149:1056-1063. Ozanne SE, Smith GD, Tikerpae J and Hales CN (1996) Altered regulation of hepatic glucose output in the male offspring of protein-malnourished rat dams. Am J Physiol 270:E559-64. Pascual M, Gomez-Lechon MJ, Castell JV and Jover R (2008) ATF5 is a highly abundant liver-enriched transcription factor that cooperates with constitutive androstane receptor in the transactivation of CYP2B6: implications in hepatic stress responses. Drug Metab Dispos 36:1063-1072.


at ASPE



Dow

nloaded from



27

Rees WD, Hay SM, Brown DS, Antipatis C and Palmer RM (2000) Maternal protein deficiency causes hypermethylation of DNA in the livers of rat fetuses. J Nutr 130:1821-1826. Rickard IJ and Lummaa V (2007) The predictive adaptive response and metabolic syndrome: challenges for the hypothesis. Trends Endocrinol Metab 18:94-99. Ross MG and Beall MH (2008) Adult sequelae of intrauterine growth restriction. Semin Perinatol 32:213-218. Sasaki A, Nakagawa I and Kajimoto M (1982) Effect of protein nutrition throughout gestation and lactation on growth, morbidity and life span of rat progeny. J Nutr Sci Vitaminol (Tokyo) 28:543-555. Singhal A, Cole TJ, Fewtrell M and Lucas A (2004) Breastmilk feeding and lipoprotein profile in adolescents born preterm: follow-up of a prospective randomised study. Lancet 363:1571-1578. Singhal A, Cole TJ and Lucas A (2001) Early nutrition in preterm infants and later blood pressure: two cohorts after randomised trials. Lancet 357:413-419. Singhal A, Farooqi IS, O'Rahilly S, Cole TJ, Fewtrell M and Lucas A (2002) Early nutrition and leptin concentrations in later life. Am J Clin Nutr 75:993-999. Singhal A, Fewtrell M, Cole TJ and Lucas A (2003) Low nutrient intake and early growth for later insulin resistance in adolescents born preterm. Lancet 361:1089-1097. Singhal A and Lucas A (2004) Early origins of cardiovascular disease: is there a unifying hypothesis? Lancet 363:1642-1645. Sohi G, Marchand K, Revesz A, Arany E and Hardy DB (2011) Maternal protein restriction elevates cholesterol in adult rat offspring due to repressive changes in histone modifications at the cholesterol 7alpha-hydroxylase promoter. Mol Endocrinol 25:785-798. Sohi G, Revesz A and Hardy DB (2013) Nutritional mismatch in postnatal life of low birth weight rat offspring leads to increased phosphorylation of hepatic eukaryotic initiation factor 2 alpha in adulthood. Metabolism 62:1367‐1374 Souidi M, Gueguen Y, Linard C, Dudoignon N, Grison S, Baudelin C, Marquette C, Gourmelon P, Aigueperse J and Dublineau I (2005) In vivo effects of chronic contamination with depleted uranium on CYP3A and associated nuclear receptors PXR and CAR in the rat. Toxicology 214:113-122. Straka MS, Junker LH, Zacarro L, Zogg DL, Dueland S, Everson GT and Davis RA


at ASPE



Dow

nloaded from



28

(1990) Substrate stimulation of 7 alpha-hydroxylase, an enzyme located in the cholesterol-poor endoplasmic reticulum. J Biol Chem 265:7145-7149. Tajima M, Ikarashi N, Imahori Y, Okaniwa T, Saruta K, Ishii M, Kusunoki Y, Kon R, Toda T, Ochiai W, Yamada H and Sugiyama K (2012) Consumption of a high-fat diet during pregnancy decreases the activity of cytochrome P450 3a in the livers of offspring. Eur J Pharm Sci 47:108-116. Thummel KE, O'Shea D, Paine MF, Shen DD, Kunze KL, Perkins JD and Wilkinson GR (1996) Oral first-pass elimination of midazolam involves both gastrointestinal and hepatic CYP3A-mediated metabolism. Clin Pharmacol Ther 59:491-502. Thummel KE and Wilkinson GR (1998) In vitro and in vivo drug interactions involving human CYP3A. Annu Rev Pharmacol Toxicol 38:389-430. Velenosi TJ, Fu AY, Luo S, Wang H and Urquhart BL (2012) Down-regulation of hepatic CYP3A and CYP2C mediated metabolism in rats with moderate chronic kidney disease. Drug Metab Dispos 40:1508-1514. Vo T, Revesz A, Ma N and Hardy DB (2013) Maternal protein restriction leads to enhanced hepatic gluconeogenic gene expression in adult male rat offspring due to impaired expression of the liver x receptor. Journal of endocrinology 218:85-97. Wang YM, Ong SS, Chai SC and Chen T (2012) Role of CAR and PXR in xenobiotic sensing and metabolism. Expert Opin Drug Metab Toxicol 8:803-817. Wienkers LC and Heath TG (2005) Predicting in vivo drug interactions from in vitro drug discovery data. Nat Rev Drug Discov 4:825-833. Williams TM and Borghoff SJ (2000) Induction of testosterone biotransformation enzymes following oral administration of methyl tert-butyl ether to male Sprague-Dawley rats. Toxicol Sci 57:147-155. Willson TM and Kliewer SA (2002) PXR, CAR and drug metabolism. Nat Rev Drug Discov 1:259-266. Wrighton SA, VandenBranden M and Ring BJ (1996) The human drug metabolizing cytochromes P450. J Pharmacokinet Biopharm 24:461-473. Yajnik C (2000) Interactions of perturbations in intrauterine growth and growth during childhood on the risk of adult-onset disease. Proc Nutr Soc 59:257-265. Yoshinari K, Sueyoshi T, Moore R and Negishi M (2001) Nuclear receptor CAR as a regulatory factor for the sexually dimorphic induction of CYB2B1 gene by phenobarbital in rat livers. Mol Pharmacol 59:278-284.


at ASPE



Dow

nloaded from



29

Zambrano E, Rodriguez-Gonzalez GL, Guzman C, Garcia-Becerra R, Boeck L, Diaz L, Menjivar M, Larrea F and Nathanielsz PW (2005) A maternal low protein diet during pregnancy and lactation in the rat impairs male reproductive development. J Physiol 563:275-284. Zhou X, Han Y, Liu J, Gao L and Zhao J (2009) Decreased protein and gene expression of hepatic cholesterol 7a-hydroxylase associated with dilated endoplasmic reticulum in chronic hypothyroid rats. Pathol Int 59:729-734.


at ASPE



Dow

nloaded from



30

Footnotes

There is no conflict of interest that could be perceived as prejudicing the impartiality of

the research reported.

This work was supported by the Canadian Institutes of Health Research [MOP #111001]

to DBH and a Natural Science and Engineering Research Council Discovery Grant to

BLU.


at ASPE



Dow

nloaded from



31

Figure Legends

Figure 1: Quantitative RT-PCR mRNA level analysis of A. CYP3A23, B. CYP3A2, C.

CYP2C11, D. CYP2B1 in the livers of rat offspring derived at postnatal d 130. RNA was

extracted and reverse transcribed for quantitative RT-PCR. mRNA level expression was

assessed via Q-RT-PCR using primers specific for CYP3A23, CYP3A2, CYP2C11,

CYP2B1 and -actin. The relative levels of each mRNA transcript were normalized to

that of the levels of each -actin mRNA transcript. Results were expressed as the mean ±

SEM. * Control vs LP2 Significant difference (P < 0.05), # LP1 vs LP2 Significant

difference (P < 0.05); n = 7–8/group, where each n represents an offspring derived from a

different mother.

Figure 2: Quantitative RT-PCR mRNA level analysis of A. Pxr and B. Car in the livers

of rat offspring derived at postnatal d 130. RNA was extracted and reverse transcribed for

quantitative RT-PCR. mRNA level expression was assessed via Q-RT-PCR using

primers specific for Pxr, Car and -actin. The relative levels of each mRNA transcript

were normalized to that of the levels of each -actin mRNA transcript. Results were

expressed as the mean ± SEM. * Control vs LP2 Significant difference (P < 0.05); n = 7–

8/group, where each n represents an offspring derived from a different mother.

Figure 3: Michaelis-Menten plots of A. 6-OH testosterone, B. 16-OH testosterone and

C. 2-OH testosterone after incubation of rat liver microsomes with 1mM NADPH and

various concentrations of testosterone. Liver microsomes were extracted and timed

enzyme reaction was performed for testostereone metabolite analysis via solid-phase

extraction followed by UPLC-PDA detection. Each data point on the curves were


at ASPE



Dow

nloaded from



32

expressed as the mean ± SEM. n = 5–6/group, where each n represents an offspring

derived from a different mother.

Figure 4: Quantitative RT-PCR mRNA level analysis of A. CYP3A23, B. CYP3A2, C.

CYP2C11, D. CYP2B1 in the livers of rat offspring derived at postnatal d 21. RNA was

extracted and reverse transcribed for quantitative RT-PCR. mRNA level expression was

assessed via Q-RT-PCR using primers specific for CYP3A23, CYP3A2, CYP2C11,

CYP2B1 and -actin. The relative levels of each mRNA transcript were normalized to

that of the levels of each -actin mRNA transcript. Results were expressed as the mean ±

SEM. n = 7–8/group, where each n represents an offspring derived from a different

mother.


at ASPE



Dow

nloaded from



33

Table 1: Real Time PCR Primers

Gene Primer (5’-3’) Reference No.

CYP3A23 FWD ACC AGT GGA AGA CTC AAG GAG ATG T XM 003751127

REV TCA CAG GGA CAG GTT TGC CTT TCT XM 003751127

CYP3A2 FWD GCT CTT GAT GCA TGG TTA AAG ATT TG BC 089765

REV ATC CAC AGA CCT TGC CAA CTC CTT BC 089765

CYP2C11 FWD CCC TGA GGA CTT TTG GGA TGG GC BC 088146

REV GGG GCA CCT TTG CTC TTC CTC BC 088146

CYP2B1 FWD GCT CAA GTA CCC CCA TGT CG XM 003750353

REV ATC AGT GTA TGG CAT TTT ACT GCG G XM 003750353

Car FWD CCT TTT CCG TTC CCT GAC CA AB 204900

REV AGG CAG AAC GTA GTG TTG AGT AB 204900

Pxr FWD TGC ACA CAG GTT CCT GTT CCT GA AF 151377

REV GGG GTG CGT GTC CTG GAT GC AF 151377

β-actin FWD ACG AGG CCC AGA GCA AGA NM 031144

REV TTG GTT ACA ATG CCG TGT TCA NM 031144


at ASPE



Dow

nloaded from



34

Table 2: Michaelis-Menten kinetic values for P450 probe substrates in Control and LP2 offspring rat liver microsomes at Day 130. 1

6β-OH Testosterone 16α-OH Testosterone 2α-OH Testosterone

Km

(µM)

Vmax (pmol/min/mg

protein)

Vmax/Km

(µl/min/mg protein)

Km (µM)

Vmax

(pmol/min/mg protein)

Vmax/Km


Km (µM)

Vmax

(pmol/min/mg protein)

Vmax/Km


Control 172 ± 18 182 ± 27 0.97 ± 0.16 80 ± 12 665 ± 58 8 ± 2 76 ± 17 466 ± 31 7 ± 1

LP1 185 ± 47 155 ± 16 1.03 ± 0.17 181 ± 85 600 ± 45 6 ± 2 66 ± 10 322 ± 25† 6 ± 2

LP2 123 ± 19 214 ± 21 1.85 ± 0.22**# 43 ± 8 873 ± 83# 26 ± 6*## 43 ± 7 569 ± 41*## 15 ± 3*#

Data are presented as means ± SEM. 2 *P < 0.05 LP2 compared with Control. 3 **P < 0.001 LP2 compared with Control. 4 #P < 0.05 LP2 compared with LP1. 5 ##P < 0.001 LP2 compared with LP1. 6 †P < 0.05 LP1 compared with Control.7

This article has not been copyedited and form

atted. The final version m

ay differ from this version.

DM

D Fast Forw

ard. Published on Novem

ber 8, 2013 as DO

I: 10.1124/dmd.113.053538

at ASPET Journals on July 3, 2019 dmd.aspetjournals.org Downloaded from



at ASPE



Dow

nloaded from


DMD Fast Forward. Published on November 8, 2013 as doi:10...

Documents

Transcript of DMD Fast Forward. Published on November 8, 2013 as doi:10...