Age‐related differences in susceptibility to toxic effects of valproic acid in rats

628 P. ESPANDIARI ET AL.

Published in 2007 by John Wiley & Sons, Ltd. J. Appl. Toxicol. 2008; 28: 628–637

DOI: 10.1002/jat

JOURNAL OF APPLIED TOXICOLOGYJ. Appl. Toxicol. 2008; 28: 628–637Published online 9 November 2007 in Wiley InterScience(www.interscience.wiley.com) DOI: 10.1002/jat.1314

Age-related differences in susceptibility to toxic effectsof valproic acid in rats†

Parvaneh Espandiari,1,* Jun Zhang,1 Laura K. Schnackenberg,2 Terry J. Miller,1 Alan Knapton,1

Eugene H. Herman,1 Richard D. Beger2 and Joseph P. Hanig1

1 FDA, Center for Drug Evaluation and Research, Silver Spring, MD, USA 209932 FDA, National Center for Toxicological Research, Jefferson, AR 72079, USA

Received 23 April 2007; Revised 12 September 2007; Accepted 13 September 2007

ABSTRACT: A multi-age rat model was evaluated as a means to identify a potential age-related difference in liver injury

following exposure to valproic acid (VPA), a known pediatric hepatotoxic agent. Different age groups of Sprague-Dawley

(SD) rats (10-, 25-, 40-, 80-day-old) were administered VPA at doses of 160, 320, 500 or 650 mg kg−−−−−1 (i.p.) for 4 days.

Animals from all age groups developed toxicity after treatment with VPA; however, the patterns of toxicity were dissimilar

within each age group. The high dose of VPA caused significant lethality in 10- and 25-day-old rats. All doses of VPA

caused decrease in the platelet counts (10-, 25-day-old rats) and the rate of growth (40-day-old rats) and increases in the

urine creatine concentration (high dose, 80-day-old rats). VPA induced hepatic and splenic alterations in all age groups.

The most severe lesions were found mostly in 10- and 80-day-old rats. Significant changes in blood urea nitrogen, alanine

aminotransferase and alkaline phosphatase were observed in 10-day-old pups after treatment with low doses of VPA. The

highest VPA dose caused significant decreases in the levels of serum total protein (40- and 80-day-old rats). Principal

component analysis of spectra derived from terminal urine samples of all age groups showed that each age group clus-

ters separately. In conclusion, this study showed that the vulnerability profile of each age group was different indicating

that a multi-age pediatric animal model is appropriate to assess more completely age-dependent changes in drug toxi-

city. Published in 2007 by John Wiley & Sons, Ltd.

KEY WORDS: valproic acid; age-related toxicity; stages of development; biomarkers

* Correspondence to: Dr Parvaneh Espandiari, FDA/CDER/OPS/DAPR,

10903 New Hampshire Ave, Life Sciences Building 64, Room 2086, Silver

Spring, MD 20993, USA.

E-mail: [email protected]

The contents of this paper do not necessarily reflect any position of the Food

and Drug Administration.

† This article is a U.S. Government work and is in the public domain in the

U.S.A.

Abbreviations: Adverse drug reaction (ADR); absorption, distribution,

metabolism and excretion (ADME); alkaline phosphatase (ALP); alanine

aminotransferase (ALT); blood urea nitrogen (BUN); free induction decay

(FID); deuterium oxide (D2O); gastrointestinal (GI); mg kg−1 day−1 (mkd);

principal component analysis (PCA); Sprague-Dawley (SD); total protein

(TP); trimethylsilyl-2,2,3,3-tetradeuteropropionic acid (TMSP); valproic acid

(VPA); white blood cell (WBC).

could be due to the non-linear maturation of certain

pathways responsible for drug absorption, distribution,

metabolism and excretion (ADME) (Faustman et al.,

2000; Pirmohamed et al., 1998). Biological processes in

which differences have been detected between pediatric

and adults are: levels of drug metabolizing enzymes,

stomach pH, gastrointestinal emptying time, levels of

serum albumin and non-adult body H2O:fat ratio, levels

of both biliary activity and renal excretion (Bates and

Balistreri, 2004; Blumer and Reed, 1992; Chuang and

Haber, 1998; Cresteil et al., 1985; Cresteil, 1998; Heyman,

1998; Weaver et al., 1991; Wershill, 1992). One of the

limiting factors in understanding ADRs in pediatric

populations has been the lack of appropriate animal mod-

els that can be used to predict the possible consequences

of exposure to drugs during the early years of develop-

ment. Drug dosage for children is often based on adult

doses that have been normalized to presumed pediatric

body area or weight. This framework for the determina-

tion of pediatric dosing does not take cognizance of

developmental differences, which could cause significant

vulnerabilities in vital target organs.

Since 1967, valproic acid (VPA) has been used as an

anticonvulsant drug for the management of epilepsy and

other seizure disorders (Keane et al., 1982; Chapman

et al., 1982; Simon and Penry, 1975). VPA is one of

Introduction

Age-related differences in organ maturation can influence

the spectrum and intensity of drug activity (Stephenson,

2005; Makri et al., 2004). As a result, age is a critical

factor in the occurrence of adverse drug reactions (ADRs)

in pediatric patients. There are instances where differen-

tial drug toxicity has been observed only in pediatric

patients. For example, acetaminophen is less hepatotoxic

in children (Insel, 1996), whereas valproic acid, chloram-

phenicol and lamotrigine are more toxic in this patient

population (Dreifuss et al., 1987; Kapusnik-Uner et al.,

1996; Guberman et al., 1999). ADRs in pediatric patients

VALPROIC ACID AND STAGES OF DEVELOPMENT 629


DOI: 10.1002/jat

the most frequently prescribed anticonvulsant drugs in

pediatric and adult patients. However, reversible increases

in liver enzymes have been detected in 15–30% of VPA-

treated patients (Eadie et al., 1988). Rarely, VPA causes

fatal hepatotoxicity in children younger than 2 years

old (1:500) (Serrano et al., 1999; Dreifuss et al., 1987)

during the first 6 months of therapy. The pathogenic

mechanism(s) responsible for liver failure in children

induced by VPA treatment may include: an inborn error

in VPA metabolism (Appleton et al., 1990), induction

of VPA reactive metabolites (Kesterson et al., 1984;

Granneman et al., 1984) and inhibition of the beta-

oxidation pathway (Fromenty and Pessayre, 1995).

The present study was initiated in an attempt to develop

a pediatric animal model(s) that could mimic the differ-

ent stages of human development and to use this model

to evaluate changes in drug toxicity profiles as a function

of age. The ultimate goal was to determine whether

this model could be used to detect potential ADRs in the

pediatric population. VPA was used because this com-

pound causes ADRs that are more frequent in infants and

young children than in adults (Serrano et al., 1999; Cloyd

et al., 1993; Dreifuss et al., 1989).

Materials and Methods

Animals

Sprague-Dawley (SD) rats (Harlan, Indianapolis, IN)

10-, 25-, 40- or 80-day-old-rats were used in the present

study. The acclimation period was different for each

age group; 7 days for 33- and 73-day-old and 2 days for

23-day-old (to allow dosing at the youngest age feasible).

In order to obtain 10-day-old rats, pregnant females

(gestation day 15) were allowed to deliver. After birth,

both female and male pups were housed with their dams

and were treated beginning at 10 days of age. Male rats

were used in all age groups except for the 10-day-old

pups where both female and male pups were included

(n = 11–13). In this age group, data from both genders

were combined when analysis showed no female or male

treatment group differences. The animals were housed

in plastic cages and maintained in a controlled environ-

ment (22 °C with a 12 h light–dark cycle). Rats had

access to Purina rodent laboratory chow (Purina Mills,

St Louis, MO) and water ad libitum.

Chemicals

VPA was purchased from Sigma Chemical Co. (St Louis,

Missouri). The drug was dissolved in normal saline.

NMR solvents trimethylsilyl-2,2,-3,3-tetradeuteropropionic

acid (TMSP) and deuterium oxide (D2O) were obtained

from Cambridge Isotope Laboratories (Andover, MA).

Experimental Protocol

Animals were dosed once daily (early morning; same

time each day) with VPA (four consecutive days at 160,

320, 500 or 650 mg kg−1 day−1 (mkd) or with saline in

dose volumes of 5 ml kg−1 body weight (i.p.). Twenty

four hours after the last (fourth) injection, 25-, 40- and

80-day-old rats were anesthetized with isoflurane, termi-

nal blood samples were collected from the abdominal

vena cava and urine was obtained from the bladder. The

animals were then euthanized by exsanguination. Termi-

nal blood also was obtained at different time intervals

(0, 4, 52 and 76 h) for 10-day-old pups treated with the

320 mg kg−1 VPA (n = 11–13 for each group). In 25-,

40- and 80-day-old rats, with the highest dose of VPA,

blood from tails was obtained at time intervals (0, 4, 52

and 76 h) (n = 5 to 7) that were after 1, 3 and 24 h of

the last treatments. All procedures performed during the

course of the study were approved by the Center for

Drug Evaluation and Research Institutional Animal Care

and Use Committee and were in accordance with the

Guide for Care and Use of Laboratory Animals (Institute

of Laboratory Animal Resources, 1996).

Pathology

At necropsy, sections of liver (from each lobe), spleen,

heart, intestine and kidney were removed, weighed and

retained for pathology and other studies. All tissues were

fixed in neutral buffered formalin, embedded in paraffin

(sectioned at 5 μm) and stained with hematoxylin-eosin

for histological examination.

Blood and Sera Analysis

Hematology and clinical chemistry analysis was performed

on the VetScan HMT and the VetScan analyser, respec-

tively (Abaxis, Inc. Union City, CA). For the 10-day-old

group, the blood of 3 or 4 pups was pooled to obtain a

sufficient amount for the different assays.

Metabonomics Analysis

Spectra were acquired on a Bruker Avance spectrometer

operating at 600.133 MHz for proton (1H) and equipped

with a 5 mm triple resonance cryoprobe. Samples were

prepared by adding 60 μl of sodium phosphate buffer

(pH = 7.4) and 20 μl of a mixture of 100 mM imidazole

and 10 mM trimethylsilyl-2,2,3,3-tetradeuteropropionic acid

(TMSP) in D2O to 120 μl of urine. Samples were loaded

into 2.5 mm Micro NMR tubes (New Era, Vineland, NY).

1D 1H NMR spectra were acquired using a presaturation

pulse sequence that irradiates the water peak during the



DOI: 10.1002/jat

delay time, d1 (2.5 s) at a power level of 58.41 dβ. Sixty

four scans were collected into 32 768 data points with a

proton pulse width of 8.10 μs at 2.6 dβ. A spectral width of

7309.94 Hz was utilized with an acquisition time of 2.24 s.

Metabonomics Processing

Each free induction decay (FID) was processed using a

macro in ACD/Labs 1D NMR Manager (Toronto,

Canada). The FIDs were zero filled to 128K, weighted by

0.3 Hz line broadening, Fourier transformed, and phased

using the simple method. The resulting spectra were

baseline corrected using the ‘SpAveraging’ method

with a box half width of 61 points and a noise factor of

3. The spectra were auto referenced by setting the TMSP

peak to 0.00 ppm. After the spectra were processed and

overlaid in the processing window, the group mode was

selected. Regions to be excluded (regions containing

solvent peaks and urea) were defined prior to integration.

Within the integration mode, the bucket width was set

and the intelligent bucketing option was selected. A

width of 0.04 ppm with a looseness of 50% was chosen.

The whole spectrum was used as the reference. Once

the appropriate parameters were defined, the group of

spectra was integrated, resulting in a table of integrals

that was exported as a text file for statistical analysis.

Additionally, each spectrum was also exported as a jcamp

file for quantitative analysis of individual metabolites.

Statistical Analysis

The number of saline treated rats in each age group was

10–12 (pools from different experiments of the same age)

and for treated VPA groups the number was 5–7. Data

from female and male 10-day-old rats were combined

for cases in which there were no differences between

genders. All data from the whole blood, sera and body,

organ weights were expressed as mean ± SEM and ana-

lysed using ANOVA with post-hoc (Bonferroni Multiple

Comparisons test). For % mortality data, a Fisher Exact

Test for 2-by-2 tables (StatXact 3. software) was used.

P < 0.05 was taken as the level of significance. All sta-

tistical analyses for metabonomics data were performed

using Statistica version 6.0 software (Statsoft, Tulsa, OK).

The text files of integral intensities were imported into

Statistica. Principal component analysis (PCA) based

on covariances was applied to the intelligently bucketed

NMR intensities and separately to the metabolites

identified by Chenomx Eclipse software (Chenomx,

Edmonton, Canada).

Results

Several different ages of SD rats were treated with VPA

at different doses to investigate young vs mature suscep-

tibility to the toxic side effects of VPA.

Mortality

The lethal dose in 40- and 80-day-old rats was different

from that in 25- and 10-day-old rats. Most of the 10-

day-old pups treated with 500 mkd VPA died soon after

the first or second treatment (12/12 male and 11/12

female pups). Likewise, a majority of the 25-day-old

animals treated with 650 mkd died (4/7) after the third

treatment and only one 80-day-old rat died after the third

treatment with 650 mkd. In contrast, no deaths occurred

in the 40-day-old rats at any VPA dose (Fig. 1).

Body, Liver, Spleen Weight

The rate of growth as indicated by body weight meas-

urements (final body weight to the initial body weight

Figure 1. Percentage survival after 160, 320, 500 and 650 mg kg−1 day−1 (mkd) valproic acid (VPA) treatment of10-, 25-, 40- and 80-day-old Sprague-Dawley rats. * P < 0.05 for tests of treatment means against control



DOI: 10.1002/jat

Figure 2. Effects of treatment with 160, 320, 500 and 650 mg kg−1 day−1 (mkd) valproic acid (VPA) on growth of10-, 25-, 40- and 80-day-old Sprague-Dawley rats. * P < 0.05 for tests of treatment means against control

Figure 3. Effects of treatment with 160, 320, 500 and 650 mg kg−1 day−1 (mkd) valproic acid (VPA) on liver weightof 10-, 25-, 40- and 80-day-old Sprague-Dawley rats. * P < 0.05 for tests of treatment means against control

expressed as percent of control) significantly decreased

with the highest tolerated doses of VPA in all age groups

(Fig. 2). Treatment with the lowest dose of VPA

also caused a significant loss of body weight in the 40-

day-old rats. To evaluate the potential toxic effects of

VPA on liver and spleen, the ratio of these organ weights

to the final body weights of the animals was calculated

and expressed as the percent of control. The liver weight

ratios in 40- and 80-day-old rats were significantly

decreased after treatment with 500 mkd VPA but not at

650 mkd VPA (Fig. 3). Aside from the effect of the

500 mkd VPA in 40- and 80-day-old rats, the other

doses of VPA did not cause biologically significant

changes in the liver weight ratios of any age group

(Fig. 3). In contrast, the relative spleen weights decreased

significantly following treatment with high doses of VPA

(data for 650 mkd not shown) in all age groups. In

addition, significant decreases in spleen weights were

also observed after treatment with 320 mkd VPA in

80-day-old and with 160 mkd VPA in 10-day-old rats

(Fig. 4).

Clinical Chemistry

Liver clinical chemistry values are summarized in Tables 1

and 2. In 10-day-old rats (both genders), blood urea

nitrogen (BUN) was significantly increased (320 mkd

VPA) while alkaline phosphatase (ALP) (160 and 320 mkd

VPA) and alanine aminotransferase (ALT) (160 mkd VPA)

were significantly decreased. Serum total protein (TP)

significantly decreased in 40- and 80-day-old rats given

the highest dose of VPA. The highest doses of VPA

given to 80-day-old rats also caused significant decreases

in the serum level of ALP and ALT. In contrast, these

clinical chemistry parameters were unchanged in VPA-

treated 25-day-old rats (Table 1). The effect of the high-

est dose of VPA (320 mg kg−1 for 10-day-old and

650 mg kg−1 for 25-, 40- and 80-day-old) on ALT was

evaluated at different intervals after treatments (0, 4, 52

and 76 h) (Table 2). The serum level of ALT increased

significantly at 4 h after the first treatment in 10-, 25- and

80-day-old rats; however, there was no elevation of this

enzyme at other time points in any age groups.



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Figure 4. Effects of treatment with 160, 320 and 500 mg kg−1 day−1 (mkd) valproic acid (VPA) on spleen weight of10-, 25-, 40- and 80-day-old Sprague-Dawley rats. * P < 0.05 for tests of treatment means against control

Table 1. Effects of different doses of valproic acid on liver parameters

Age (days) Treatment BUN (mg dl−1) ALT (U l−1) ALP (U l−1) TP (g dl−1)

10 Saline (5 ml kg−1) 24 ± 5.8 53 ± 16 270 ± 17 3.4 ± 0.026160 mkd VPA 24 ± 4.7 20 ± 5.4a 201 ± 21a 5.1 ± 0.16

320 mkd VPA 38 ± 1.4a 50 ± 41 238 ± 115a 3.6 ± 0.16

500 mkd VPA All pups died after 1st or 2nd injections25 Saline (5 ml kg−1) 17 ± 1.6 52 ± 9.3 330 ± 40 4.4 ± 0.14

160 mkd VPA 15 ± 1.5 54 ± 6.6 401 ± 25 4.5 ± 0.15

320 mkd VPA 14 ± 0.57 55 ± 11 343 ± 45 4.3 ± 0.15500 mkd VPA 16 ± 2.7 60 ± 5.3 380 ± 40 4.5 ± 0.06

650 mkd VPA 20 ± 5.0 58 ± 9.6 339 ± 122 4.4 ± 0.28

40 Saline (5 ml kg−1) 14 ± 0.57 47 ± 1.9 371 ± 71 5.0 ± 0.12160 mkd VPA 13 ± 0.50 46 ± 3.0 325 ± 42 5.07 ± 0.10

320 mkd VPA 15 ± 2.2 50 ± 7.9 366 ± 44 5.1 ± 0.20

500 mkd VPA 14 ± 1.4 48 ± 7.0 380 ± 44 5.1 ± 0.05650 mkd VPA 18 ± 2.2 54 ± 11 313 ± 60 4.7 ± 0.4a

80 Saline (5 ml kg−1) 18 ± 0.81 38 ± 5.1 215 ± 28 5.4 ± 0.24

160 mkd VPA 20 ± 4.2 41 ± 12 224 ± 18 5.6 ± 0.37320 mkd VPA 14 ± 2.4 36 ± 2.6 195 ± 57 5.3 ± 0.14

500 mkd VPA 21 ± 0.71 28 ± 2.9a 161 ± 17a 5.1 ± 0.10

650 mkd VPA 22 ± 3.2 34 ± 11a 143 ± 46a 4.5 ± 0.2a

Effects of treatment with valproic acid (VPA) on liver parameters of 10-, 25-, 40- and 80-day-old Sprague-Dawley rats (n = 5–7). For 10-day-old pups, blood

from 3 to 4 pups pooled together. a P < 0.05 for tests of treatment means against control. Results expressed as mean ± SEM. ALP, alkaline phosphatase;

ALT, alanine aminotransferase; BUN, blood urea nitrogen.

Table 2. Effects of treatment with valproic acid on ALT

Group (h after 10 days 25 days 40 days 80 daystreatment) old old old old

Saline 0 53 ± 17 65.5 ± 9.2 57 ± 8.4 54 ± 7.7

VPA 4 93 ± 23a 102 ± 8.2a 74 ± 11 103 ± 20a

VPA 52 86 ± 4.3 59 ± 16 73 ± 18 51 ± 7.01

VPA 76 61 ± 10 58 ± 9.6 54 ± 11 34 ± 11a

Effects of treatment with valproic acid (VPA) (320 mg kg−1 for 10-day-old and 650 mg kg−1 for 25-, 40- and 80-

day-old Sprague-Dawley rats) on alanine aminotransferase (ALT) (n = 5–7). For 10-day-old pups, blood from 3 to 4

pups pooled together. a P < 0.05 for tests of treatment means against control. Results expressed as mean ± SEM.

Hematology

The platelet count decreased after treatment with VPA

in 25-day-old animals (all doses), in male and female

10-day-old pups (160 and 320 mkd) and in 80-day-old

rats (500 and 650 mkd) (Table 3). Decreases in white

blood cell (WBC) count were observed in almost all age

groups given the highest tolerated dose of VPA. A decline

in WBC was also detected in 10-day-old (160 mkd) and

in 80-day-old rats (500 mkd) (Table 3). The red blood

cell count increased after treatment with the highest VPA

dose in 25- and 40-day-old rats (Table 3).



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Table 3. Effects of different doses of valproic acid on blood components

Age Platelets White blood Red blood(days old) Treatment (m mm−3) cells (m mm−3) cells (M mm−3)

10 Saline (5 ml kg−1) 191 ± 33 5.6 ± 1.9 3.74 ± 0.31

160 mkd VPA 105 ± 15a 3.2 ± 0.4a 3.9 ± 0.13

320 mkd VPA 100 ± 16a 3.6 ± 1.5a 3.6 ± 0.33

500 mkd VPA All pups died after 1st or 2nd injection

25 Saline (5 ml kg−1) 985 ± 23 5.7 ± 1.8 3.9 ± 0.17

160 mkd VPA 817 ± 23a 6.8 ± 1.8 3.6 ± 0.98

320 mkd VPA 882 ± 93a 5.8 ± 1.2 3.9 ± 0.35

500 mkd VPA 778 ± 53a 4.8 ± 2.5 3.9 ± 0.25

650 mkd VPA 684 ± 57a 3.3 ± 0.6a 4.2 ± 0.17a

40 Saline (5 ml kg−1) 731 ± 91 11 ± 2.2 4.9 ± 0.11

160 mkd VPA 767 ± 34 11 ± 2.3 4.7 ± 0.05

320 mkd VPA 719 ± 11 9.9 ± 1.5 4.9 ± 0.17

500 mkd VPA 674 ± 94 8.9 ± 3.1 4.7 ± 0.19

650 mkd VPA 920 ± 90 4.8 ± 0.8a 5.2 ± 0.27a

80 Saline (5 ml kg−1) 708 ± 22 8.9 ± 1.9 7.1 ± 0.60

160 mkd VPA 778 ± 26 7.3 ± 0.6 7.1 ± 0.42

320 mkd VPA 707 ± 55 5.7 ± 2.2 7.01 ± 0.06

500 mkd VPA 415 ± 91a 3.1 ± 0.8a 7.38 ± 0.19

650 mkd VPA 401 ± 95a 2.7 ± 0.7a 6.7 ± 0.19

Effects of treatment with valproic acid (VPA) on blood components of 10-, 25-, 40- and 80-day-old Sprague-Dawley rats (n = 5–7). For 10-day-old pups,

blood from 3 to 4 pups pooled together. aSignificantly different from control (P < 0.05). Results expressed as mean ± SEM. aP < 0.05 for tests of treatment

means against control. m mm−3, 103/cubic millimeter; M mm−3, 106/cubic millimeter.

Table 4. Hepatic alterations in rats treated with different doses of valproic acid

Age (days)

10

10

25, 40, 80

25

40

80

Light microscopic changes detected in liver tissue from 10-, 25-, 40- and 80-day-old Sprague Dawley rats (n = 5 for 25-, 40- and 80-day-old and 11–13 for

10-day-old pups) treated with 320, 500 or 650 mg kg−1 day−1 (mkd) valproic acid (VPA).aNumerator: the number of rats showing the lesion; denominator: the number of rats treated.

Lesion morphologyNon-necrotic lesions

(10/13)a Mild to moderate inflammation

(3/13)a Mild edema, hemorrhage,

cytoplasmic vacuolization

Treated pups died after 1st or 2nd injection

None

(5/5)a Hepatocyte degeneration (vacuolization),

mild hemorrhage, edema

(5/5)a Hepatocyte degeneration (vacuolization),

mild inflammation

(2/5)a Hemorrhage with destruction of

endothelial and sinusoidal cells

Valproic acid(mkd)

320

500

320 and 500

650

650

650

Hepatocyte necrosis

None

None

(1/5)a Small foci of central lobular necrosis

(1/5)a Small foci of central lobular necrosis

(5/5)a Hepatocyte necrosis (more severe

in central lobule than in periportal or

midzonal lobules)

Pathology

The only gross changes observed at necropsy were

found in 25-day-old rats given high doses of VPA. These

rats had a pronounced round distended abdomen. The

stomach and cecum of these rats were swollen and

packed with food. Light microscopic examination in-

dicated that VPA caused morphological alterations

mainly in the liver and to a lesser extent in the spleen.

No significant changes were found in other organs (heart,

lung and kidney). The main features of liver toxicity in-

cluded hepatocyte degeneration (cytoplasmic vacuoles)

and necrosis, inflammation and edema (Table 4). Central

lobular necrosis was noted in the groups of 25–40

day-old rats receiving 650 mkd VPA. In the 80-day-old

animals, central lobular necrosis was usually more severe

than the periportal lobular necrotic lesion. In some in-

stances areas of hemorrhage were noted in association

with the necrosis. The extravasation of red blood cells

at these sites appeared to be due to the destruction of

endothelial and sinusoidal cells. Splenic tissue alterations

induced by the 500 and 650 mkd doses of VPA in the

25-, 40- and 80-day-old-rats were primarily limited to

severe atrophy in the T cell areas. Treatment with

320 mkd VPA (320 mkd) caused moderate splenic atrophy

only in 10- and 25-day-old animals.



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Figure 7. Effects of treatment with 160, 320, 500 and 650 mg kg−1 day−1 (mkd) valproic acid (VPA) on glucose level inurine samples from 25-, 40- and 80-day-old Sprague-Dawley rats. * P < 0.05 for tests of treatment means against control

Figure 6. Effects of treatment with 160, 320, 500 and 650 mg kg−1 day−1 (mkd) valproic acid (VPA) on creatine levelin urine samples from 25-, 40- and 80-day-old Sprague-Dawley rats. * P < 0.05 for tests of treatment means against control

Figure 5. Principal component analysis (PCA) of NMRspectra of control urine samples from 25-, 40- and 80-day-old Sprague-Dawley rats (n = 9–11, control datafrom two different animal studies)

Metabonomic Analysis

PCA analysis of NMR spectra from control rat terminal

urine samples (all age groups except the 10-day-old pups)

(n = 9–11, control data from two different animal stud-

ies) clearly showed that each age group clusters separately

from each other (Fig. 5). Since the PCA indicated metabolic

differences between each age group, further evaluation of

the terminal serum and urine samples from each age group

was performed separately. Altered serum glucose and

lactate levels were observed in 40-day-old rats but those

changes were not reflected in the PCA plots as differen-

tiating between controls and rats dosed with higher

levels of VPA. The PCA plots of control urine showed

that each group was metabolically different and citrate

was higher in the 25-day-old rats. The average urinary

creatine level increased in all three age groups dosed with

650 mkd VPA, but it was significant only in the 80-day-old

rats (Fig. 6). The urinary glucose level was significantly

decreased at the 320 mkd VPA level in 25-day-old rats

(Fig. 7). The serum glucose level was significantly de-

creased in at least one VPA dose level for all three age

groups with 40-day-old showing changes at the lowest

dose levels (160 or 320 mkd) (Fig. 8).

Discussion

The FDA Guidance for Industry: Non-clinical Safety

Evaluation of Pediatric Drugs (Feb. 2006) has stressed

the importance of utilizing models that include animals of



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Figure 8. Effects of treatment with 160, 320, 500 and 650 mg kg−1 day−1 (mkd) valproic acid (VPA) on glucose levelin serum samples from 25-, 40- and 80-day-old Sprague-Dawley rats. * P < 0.05 for tests of treatment meansagainst control

different ages, for studies that are intended to evaluate

drug safety in the pediatric population. The guidance also

notes that many of the developmental changes found

in animals are similar to those occurring during human

development. Thus, preclinical studies which include

diverse maturation stages would seem to offer a good

opportunity to identify potential age-related differences

in organ-susceptibility to drug toxicity.

In the present study 10-day-old pups experienced the

most toxicity following exposure to VPA. Most of the

pups died following treatment with the two highest VPA

doses. In contrast, no lethality occurred in 40-day-old

rats at any dose of VPA. However, all doses of VPA did

suppress body weight gain at this age. The 10-day-old

pups treated with the lower doses of VPA developed

hepatic lesions in association with changes in the serum

levels of ALP, ALT and BUN. The changes in liver

morphology and serum levels of liver function markers

were more pronounced in the 10-day-old pups than in

any other age group. These alterations occurred in the

absence of any significant change in liver weight. Differ-

ences in the level of liver enzyme activity are known to

exist between adults and infants. In many instances, liver

enzyme systems are not up to full metabolic capacity in

infants compared with adults. Experimentally, the func-

tional level of some hepatic phase 1 enzymes in 0- to 1-

year old infants is reported to be comparable to the level

of these enzymes found in 4- to 17-day-old rats (Leeder

and Kearns, 1997; Waxman et al., 1989; Peng et al.,

1991). In vitro studies have shown that VPA can directly

alter the activity of certain liver enzymes. For example,

VPA inhibits the activities of CYP 2C9 as well as

CYP3A4 enzymes in human liver microsomal prepara-

tions (Wen et al., 2001).

In this study, the serum levels of ALT increased 4 h

after treatment with the high doses of VPA in 10-, 25-

and 80-day-old rats (320 and 650 mg kg−1 respectively).

However, liver morphology was evaluated only at the

end of the experimental period (76 h after initiation of

VPA treatment). At this time, hepatic cellular patho-

logy was detected even though serum ALT levels had

decreased to normal or near normal values. It appears

that increased serum levels of ALT are indicative but not

necessarily sustained throughout the time course of VPA-

induced hepatic injury. In addition, it is possible that for

10-day-old pups, the VPA-induced elevation of ALT is

also a reflection of immature liver function, a key factor

that might be responsible for the increased susceptibility

to hepatotoxicity observed in the present study and clini-

cally, in infants (Anderson, 2002; Serrano et al., 1999;

Dreifuss et al., 1989).

Experimental VPA treatment has been reported to

induce a variety of hepatocyte alterations, including

microvesicular steatosis (Lewis et al., 1982; Kesterson

et al., 1984; Tong et al., 2005). In the present study,

cytoplasmic vacuoles were detected in hepatic cells after

treatment with the highest dose of VPA in all age groups.

However, we were not able to determine whether these

vacuoles contained lipid material. The earlier studies,

which reported steatosis, differed from the present study

in that rats were treated with higher doses of VPA and/

or longer treatment periods. For example, in the study

reported by Lewis et al. (1982) microvesicular steatosis

was observed only in rats 48 h after treatment with a very

high dose of VPA (750 mkd). In another study reported

by Kesterson et al. (1984), fatty liver was detected

in VPA treated rats after 4 or 5 days of treatment with

700 or 600 mkd VPA, respectively. Tong et al. (2005)

reported fatty liver after 4 days of treatment with

500 mkd in only one out of four rats; however, this ratio

was increased (four out of five) by day 10 after the same

dose of VPA treatment.

The 10-day-old pups were also found to be the most

sensitive to splenic alterations elicited by VPA. Even the

lowest VPA dose (160 mkd) caused a significant decrease

in relative spleen weight ratios. A similar decline in

spleen weight ratios was found in 80-day-old rats after

treatment with the 320 and 500 mpk doses of VPA. In



DOI: 10.1002/jat

contrast, the 25- and 40-day-old rats showed significant

changes in the spleen weight ratio only after treatment

with the 500 mkd VPA dose. Splenic histopathology was

also noted, in addition to changes in the spleen weight

ratio. In this instance the 320 mkd dose of VPA caused

moderate splenic atrophy in the 10- and 25-day-old rats.

The 500 mkd dose of VPA, which was not tolerated in

10-day-old pups, produced severe splenic damage in the

25-, 40- and 80-day-old rats. Clearly, the spleen is a

major target organ for VPA toxicity and the two young-

est age groups appear to be the most susceptible.

Therapy with VPA has caused bone marrow suppres-

sion and reduced platelet and red blood cell counts (Gidal

et al., 1994; May and Sunder, 1993; Ozkara et al., 1993;

Devilat and Blumel, 1991). VPA induced significant

decreases in white blood cell counts in all age groups.

The white blood cell decreases detected in the 10-day-old

pups were induced by lower VPA doses than those VPA

doses causing this effect in the other age group rats.

Changes in red blood cell counts were not a consistent

finding at any age in the present study. The levels of

platelets decreased in both 10- and 25-day-old rats treated

with the lowest doses of VPA (160 or 320 mkd). The

10- and 25-day-old rats were the most sensitive and the

40-day-old rats the most resistant to changes in platelet

counts after treatment with all doses of VPA (160–

650 mkd). Clinical studies have detected changes in

platelet counts following treatment with VPA (Verrotti

et al., 1999). Prolonged bleeding as a result of the inhi-

bition of platelet aggregation has also been reported with

VPA therapy (Verrotti et al., 1999; Tohen et al., 1995;

Gidal et al., 1994; Delgado et al., 1994). VPA treatment,

in Wistar rats, suppressed the synthesis of platelet

cyclooxygenase and lipoxygenase (Szupera et al., 2000).

The potential for age-related platelet dysfunction could

be an important consideration for pediatric patients that

undergo surgery.

The PCA of spectra derived from terminal urine sam-

ples of all age groups given saline showed that each

age group clusters separately with citrate being higher in

the 25-day-old animals. This is a strong indication that

each age group is metabolically different from the others.

Glucose concentration in the urine was significantly

lower at an intermediate dose in the 25-day-old, but the

significance of this in terminal urine is unclear and does

not appear to represent a major change. What is signifi-

cant is the sensitivity of the 40-day-old rats as evidenced

by significantly lowered levels of serum glucose at all but

an intermediate dose of VPA. This group is more sensi-

tive than the 25- or 80-day-old and may be indicative

of the beginning of glucose regulation problems that are

more pronounced at the two highest dose levels in young

adults. Finally, elevation of creatine levels at the highest

dose in the 80-day-old rats may be a hint that kidney

problems caused by VPA are beginning to emerge. Glu-

cose levels in serum after 4 days of dosing with VPA

were significantly lowered in at least one VPA dosing

level for each age group. In comparison, a separate study

indicated that 600 mg kg−1 VPA administration in adult

mice resulted in altered glucose concentrations in urine

samples at 12 and 24 h and in aqueous liver tissue

extracts at 12 h after VPA administration, which recov-

ered by 24 h (Schnackenberg et al., 2006). Proteomics

analysis in the same study found that two proteins in-

volved in the conversion of glycogen to glucose were up-

regulated following dosing with VPA. These combined

proteomic and metabonomic studies on mice indicated

a perturbation in the glycogenolysis pathway following

administration of valproic acid (Schnackenberg et al.,

2006). While metabonomic analysis in our study was

only done on terminal samples, the results indicate that

there was a perturbation in glucose consistent with the

previously reported results of other investigators.

In conclusion, the present study examined the spectrum

of VPA-induced toxicity in a multi-age rodent model.

Findings indicated that the pattern of toxicity induced by

VPA in the different aged SD rats was quite dissimilar;

each age group was different from the 80-day-old adults

as well as from each other. In this study, the 10-day-old

pups were the most sensitive age group to the toxic

effects of VPA, a finding which seems to correlate

with clinical reports indicating that infants younger than

2 years treated with the drug experience a high incidence

of adverse effects (Serrano et al., 1999; Cloyd et al.,

1993; Dreifuss et al., 1989). The incidence of ADRs in

the pediatric population is a major health issue and can

occur as frequently as in adults (Impicciatore et al., 2001;

Easton et al., 1998). The present study demonstrated that

a multi-age animal model could be useful in identifying

toxicity as it varies across different pediatric stages and

thus possibly serves as the basis for evaluating pediatric

drug safety in conventional non-clinical studies.

Acknowledgement—We wish to thank Drs James L. Weaver andWilliam Rodriguez of the Food and Drug Administration for theirvaluable advice. Additional thanks go to Dr Koorus Mahjoob for histechnical assistance with statistical analysis.

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Age‐related differences in susceptibility to toxic effects of valproic acid in rats

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