Invited Review

Toxicologic Pathology, 40: 971-994, 2012

Copyright # 2012 by The Author(s)
ISSN: 0192-6233 print / 1533-1601 online
DOI: 10.1177/0192623312448935

Liver Hypertrophy: A Review of Adaptive (Adverse and

Non-adverse) Changes—Conclusions from the 3rd International
ESTP Expert Workshop
A. P. HALL1, C. R. ELCOMBE2, J. R. FOSTER1, T. HARADA3, W. KAUFMANN4, A. KNIPPEL4, K. KÜTTLER5, D. E. MALARKEY6,
R. R. MARONPOT7, A. NISHIKAWA8, T. NOLTE9, A. SCHULTE10, V. STRAUSS5, AND M. J. YORK11
1
AstraZeneca Pharmaceuticals, Alderley Park, Macclesfield, Cheshire, UK
2
CXR Biosciences, Dundee, UK
3
Institute of Environmental Toxicology, Ibaraki, Japan
4
Merck KGaA, Non-Clinical Development, Toxicology, Darmstadt, Germany
5
BASF SE, Dep. GV/TD, Ludwigshafen, Germany
6
National Toxicology Program Pathology Group, Cellular and Molecular Pathology Branch, National Institute of
Environmental Health Sciences, Research Triangle Park, North Carolina, USA
7
Maronpot Consulting LLC, Raleigh, North Carolina, USA
8
Biological Safety Research Center, National Institute of Health Sciences, Tokyo, Japan
9
Boehringer Ingelheim Pharma GmbH & Co, Biberach/Riss, Germany
10
Bundesinstitut für Risikobewertung, Berlin, Germany
11
GlaxoSmithKline R&D, Safety Assessment, Hertfordshire, UK
ABSTRACT
Preclinical toxicity studies have demonstrated that exposure of laboratory animals to liver enzyme inducers during preclinical safety assessment
results in a signature of toxicological changes characterized by an increase in liver weight, hepatocellular hypertrophy, cell proliferation, and, fre-
quently in long-term (life-time) studies, hepatocarcinogenesis. Recent advances over the last decade have revealed that for many xenobiotics, these
changes may be induced through a common mechanism of action involving activation of the nuclear hormone receptors CAR, PXR, or PPARa. The
generation of genetically engineered mice that express altered versions of these nuclear hormone receptors, together with other avenues of investi-
gation, have now demonstrated that sensitivity to many of these effects is rodent-specific. These data are consistent with the available epidemiolo-
gical and empirical human evidence and lend support to the scientific opinion that these changes have little relevance to man. The ESTP therefore
convened an international panel of experts to debate the evidence in order to more clearly define for toxicologic pathologists what is considered
adverse in the context of hepatocellular hypertrophy. The results of this workshop concluded that hepatomegaly as a consequence of hepatocellular
hypertrophy without histologic or clinical pathology alterations indicative of liver toxicity was considered an adaptive and a non-adverse reaction.
This conclusion should normally be reached by an integrative weight of evidence approach.

Keywords: liver; hypertrophy; adverse; non-adverse; AhR; CAR; PXR; PPARa; weight; fasting; clinical pathology; omics.

The author(s) declared a potential conflict of interest with respect to the research, authorship, and/or publication of this article: Financial support for this
workshop was provided by the ESTP. This article may be the work product of an employee or group of employees of the National Institute of Environmental
Health Sciences (NIEHS), National Institutes of Health (NIH); however, the statements, opinions or conclusions contained therein do not necessarily represent
the statements, opinions or conclusions of NIEHS, NIH, or the United States government.
The author(s) received no financial support for the research, authorship, and/or publication of this article.
Address correspondence to: A. P. Hall, AstraZeneca Pharmaceuticals, Alderley Park, Macclesfield, SK10 4TG, Cheshire, England; e-mail:
peter.a.hall@astrazeneca.com.
Abbreviations: AE, adverse event; AhR, Aryl hydrocarbon receptor; ALP, alkaline phosphatase; ALT, alanine aminotransferase; Arnt, aryl hydrocarbon
receptor nuclear; AST, aspartate aminotransferase; bHLH-PAS, basic helix-loop-helix/Per-Arnt-Sim; CAR, constitutive androstane receptor; CHE, Pseudocholi-
nesterase; CPA, Cyproterone acetate; CYP, Cytochrome P450; DEHP, Bis(2-ethylhexyl)phthalate; ELISA, enzyme linked immunosorbent assay; ESTP, European
Society of Toxicologic Pathology; FXR, Farnesoid X receptor; GD, gestation day; gGT, g-glutamyltranspeptidase; GLDH, glutamate dehydrogenase; GST, glu-
tathione S-transferase; H&E, Haematoxylin and Eosin; IHC, Immunohistochemistry; KO/KI, knock-out/knock-in; LDH, lactate dehydrogenase; LXR, liver X
receptor; MDH, malate dehydrogenase; MGMT, O-6-methylguanine-DNA methyltransferase; mRNA, messenger RNA; MTD, maximum tolerated dose; NOAEL,
no observed adverse effect level; NOEL, no observed effect level; NTP, National Toxicology Program; OATP, organic anion-transporter polyptide; OCT, ornithine
carbomyl transferase; PCA, principal component analysis; PCB, coplanar polychlorinated biphenyls; PCDD, polychlorinated dibenzo(p)dioxins; PCN, pregneno-
lone 16 alpha-carbonitrile; PPARa, peroxisome activated receptor alpha; PPRE, peroxisome proliferator response element; PXR, pregnane X receptor; RT-PCR,
reverse-transcriptase polymerase chain reaction; sER, smooth endoplamic reticulum; STP, Society of Toxicologic Pathology; T3, Tri-iodothyronine; T4, thyroxine;
TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; TSH, thyroid stimulating hormone; UGT, UDP-glucuronosyltransferase; ULN, upper limit of normality.

971
972 HALL ET AL.
INTRODUCTION
Drug and chemically induced liver enlargement in subchro-
nic and chronic toxicology studies in rodents has, for many
years, taxed the toxicology profession in terms of its perceived
relevance to hepatotoxicity, to carcinogenicity in lifetime
bioassays at similar dose levels, and in terms of its relevance
to man (Cohen and Grasso 1981; Elcombe, Rose, and Pratt
1985; Grasso and Hinton 1991). The fact that after more than
50 years of debate (Gilbert and Golberg 1965; Rowe et al.
1959; Weil and McCollister 1963) the issue remains as conten-
tious as ever prompted the European Society of Toxicologic
Pathology (ESTP) to convene an expert opinion group to
discuss the current state of the science. The purpose of the
workshop was to discuss the significance of hepatocellular
hypertrophy in rodents, define more clearly when adaptive
responses become adverse, and understand the long-term
consequences of hepatocellular hypertrophy in order to guide
scientific opinion for risk assessment in man and dose setting
for longer term animal studies.
One critical aspect of hepatic hypertrophy which continues
to pose serious questions for toxicologists is the definition of
what constitutes an adverse hepatic effect, versus a non-
adverse or adaptive effect. This was a major focus of discussion
by the expert opinion group resulting in a consensus opinion
detailed in this article.
HEPATIC HYPERTROPHY—WHAT IS IT?
While to a histopathologist the term hepatic hypertrophy is
well recognized and readily defined histologically, to a toxicol-
ogist, the term can have various connotations including an
increase in the weight of the organ (liver hypertrophy), an
increase in the average size of the hepatocytes (hepatocellular
hypertrophy), and even hepatic enzyme induction (functionally
sometimes referred to as ‘‘work’’ hypertrophy).
In practice, drug- or chemical-induced hepatic hypertrophy
tends to be a combination of all of these parameters in addition
to others including profound changes in the intracellular
enzymes involved not only in phase 1 and 2 drug metabolism
(Crampton et al. 1977b; Lake, Longland, et al. 1976; Maronpot
et al. 2010) but also in other more fundamental cell processes
such as altered oxidative status, fatty acid metabolism, energy
production and utilization, cell turnover and altered hepatocel-
lular cytoplasmic, and nuclear morphology (Cattley and Popp
1989; Crampton et al. 1977b; Grasso et al. 1974; Grasso and
Hinton 1991).
Archetypical changes that often accompany this phenom-
enon are increases in hepatic-derived enzymes (transaminases,
alkaline phosphatase, and g-glutamyltransferase) that may
appear in the plasma following liver enlargement (Ennulat,
Magid-Slav, et al. 2010). Of prime importance for interpreta-
tion of these changes with regard to risk assessment are the sig-
nificant species differences shown in response to chemicals
that induce a classic hypertrophic response in the rodent liver
(Lake 1995; Lake, Brantom, et al. 1976; Rhodes et al. 1986;
Williams and Perrone 1996).
TOXICOLOGIC PATHOLOGY
TABLE 1.—Dose response relationship for liver weight increase for the
PPARa agonist, Fenofibrate (adapted from Price et al. 1986).
Dose (mg/kg/day) Liver weight (% of control)
0 100
13 125
60 157
200 187
Liver Weight Increases
Increases in liver weight in rodents due to exposure to
chemicals can be achieved through a number of mechanisms
and can be accompanied by a range of differing histological
appearances, some of which clearly show cytotoxicity and cell
death, such as carbon tetrachloride and chloroform (Edwards
and Dalton 1942; Eschenbrenner and Miller 1945; Grasso and
Hinton 1991; Reuber and Glover 1968), while other chemicals
such as sodium phenobarbitone, the PPARa (peroxisome acti-
vated receptor alpha) agonists, Nafenopin and Clofibrate, and
trichloroethylene, induce an increase in liver weight without
overt cell necrosis (Lake et al. 1989; Mitchell et al. 1985; Price
et al. 1986). While there is an undoubted relationship between
hepatic necrosis and its consequences on the eventual increased
incidence of neoplasia of the liver (Eschenbrenner and Miller
1945; Grasso and Hinton 1991), this is less clear for chemicals,
or rather for doses of chemicals, that induce liver weight
increases in the absence of overt hepatocellular damage. The
focus of the workshop was on those chemicals that are not tra-
ditionally associated with acute hepatic necrosis but rather
exert their effect by increasing the weight of the liver through
other means.
Organ weight data can provide sensitive indices of toxicolo-
gic change where it can correlate and confirm changes seen
down the microscope. To this end, the STP have recently pub-
lished a position paper (Sellers et al. 2007) that gives careful
guidance to the collection and weighing of organs in routine
toxicology studies. The authors recommend that organ weight
data should be routinely expressed as an organ-to-body weight
ratio to avoid large variations in body weight skewing organ
weight interpretation. In this way, increases in liver organ
weight can be accurately correlated with hepatocellular hyper-
trophy. In rodent studies, where routinely larger numbers of
animals are used, these data can be compared with concurrent
controls and statistically interrogated to derive p values that
may increase confidence through weight of evidence to allow
a judgment of hepatocellular hypertrophy when the histological
changes appear to be relatively small.
This is particularly important since the magnitude of the
increased liver weight can vary considerably between different
chemicals. Liver weight changes may also demonstrate a clear
dose relationship (Table 1; typically between 110% and 150%
of control liver weight). However, it should be noted that
although liver weight increases are correlated with microsomal
enzyme induction, the degree of microsomal enzyme induction
may not be closely correlated with either the magnitude of the
Vol. 40, No. 7, 2012 LIVER HYPERTROPHY—ADVERSE OR NON-ADVERSE? 973

TABLE 2.—Correlation between liver toxicity and carcinogenesis in mouse National Toxicology Program studies (adapted from Allen et al. 2004).

Liver neoplasia liver toxicity Cancerþ Toxþ Cancerþ Tox Cancer Toxþ Cancer Tox Significance value (p)

Hypertrophy 10 17 0 56 <.001
Necrosis 8 19 1 55 <.001
Cytomegaly 4 23 2 54 .084
Increased Liver weight 19 8 17 39 <.001
HYPT þ NEC þ CYM 17 10 2 54 <.001
HYPT þ NEC þ CYM þ LW 25 2 18 38 <.001

Notes: CYM ¼ hepatocellular cytomegaly; NEC ¼ hepatocellular necrosis; HYPT ¼ hepatocellular hypertrophy; DEG ¼ hepatocellular degeneration; LW ¼ increased liver weight.
Significance represents correlation of observation to carcinogenic outcome in a lifetime bioassay.

liver weight increase or the degree of hepatocyte hypertrophy
in rats, dogs, or monkeys (Amacher, Schomaker, and
Burkhardt 1998, 2001; Amacher et al. 2006; Ennulat, Walker,
et al. 2010).
Increases in liver weights have been shown to be associated
with the induction of increased incidences of hepatocellular
neoplasia in 2-year carcinogenicity studies in rodents (Allen
et al. 2004; Carmichael et al. 1997) and while considered to
be of little relevance to man for some chemicals (Butler and
Newberne 1975; Ito and Sugano 1991; McClain et al. 1995;
Stevenson et al. 1990), dose levels of a chemical inducing this
degree of liver weight increase would be considered to carry an
increased risk of inducing hepatic neoplasms in these types of
study. In a survey of 138 chemicals used in the agrochemical
industry, a relative increase in liver weight of 150% of con-
trol values after 1 year of treatment was positively correlated
with the induction of liver tumors in mice (Carmichael et al.
1997). Similarly, in another survey of mouse NTP studies
where correlations between liver weight increases and histolo-
gical parameters and carcinogenesis were assessed, the authors
concluded that ‘‘the best single predictor of liver cancer in mice
was hepatocellular hypertrophy’’ (Allen et al. 2004). This study
demonstrates a highly significant relationship between
increases in liver weight and the future outcome of hepatic neo-
plasia (p < .001; Table 2). In a similar review of the rat, a less
statistically significant relationship (p ¼ .018) between liver
weight and hepatocarcinogenesis was also noted, whereby liver
weight increases alone correctly predicted eight of the eleven
liver carcinogens (but overpredicted twenty-six false positives
and failed to predict three true positives; Allen et al. 2004).
Effect of Fasting on Liver Weights
A potentially important confounder in terms of the evalua-
tion of liver weight changes following chemical treatment is
the practice of fasting animals overnight prior to sacrifice. It
is likely that fasting alters the resulting organ weights in
rodents over those not fasted (Chatamra, Daniel, and Lam
1984; Rothacker et al. 1988). Studies have shown that the
rodent liver weight increases in fed animals are maintained for
up to 8 hr after feeding. Water and glycogen account for the
major portion of this increase with the former comprising over
66% of the increase (Leveille and Chakrabarty 1967). Over-
night fasting will rapidly deplete both components (in
preference to other tissues of the body) and accounts for the
loss of liver weight seen under fasting conditions (Rothacker
et al. 1988). It is currently unclear as to what percentage of
laboratories utilizes fasting, but what was apparent from the
workshop was that there exists considerable inter-laboratory
variation in the practice of overnight fasting of animals prior
to necropsy.
Welfare reasons prohibit the overnight fasting of pregnant ani-
mals but a systematic study looking into the differences between
fasted and non-fasted animals would seem a worthwhile exercise
if the former leads to better discrimination of effects provided ani-
mal welfare guidelines permit this (Animal and Plant Health
Inspection Services [APHIS] 1997; Animal Welfare Information
Center 2005). However, it is clear that overnight fasting does have
some distinct advantages. First, overnight fasting decreases the var-
iation seen in some clinical pathology parameters and therefore
increases the probability of identifying statistically significant
changes between control and treated animals (Matsuzawa and
Sakazume 1994). Second, animals exposed to high levels of a
xenobiotic very frequently experience decreased food intake due
to inanition/toxicity which is often noted in toxicological studies
as a treatment, and dose related, decrease in hepatocellular gly-
cogen storage (Agren, Wilander, and Jorpes 1931; Lockard et al.
1983). It is possible therefore that loss of glycogen in animals
exposed to a xenobiotic might mask statistically significant
increases in liver weight. Since liver weight increases are gener-
ally considered a sensitive measure of hepatocellular hypertro-
phy, small changes in these parameters may not be visible
using H&E stained sections. This is especially so if animals are
not fasted, as a relative loss of glycogen due to toxicity in the
high-dose groups is likely to reduce hepatocellular volume and
obscure the change in dimensions which helps inform the diag-
nosis of hypertrophy when comparing treated with concurrent
control animals (Li et al. 2003). However, there are clearly
well-described changes in gene expression in fasted animals
(Bauer et al. 2004; Lkhagvadorj et al. 2009) and further work
to assess the relative merits of overnight fasting for subsequent
histopathology evaluation would help assess the importance or
otherwise of this effect.
The practice of fasting rodents before necropsy was one that
was debated at the workshop but because of the differing
experiences of the group a recommendation was made for a
comparison dose–response study with a known hepatotrophic
agent, such as sodium phenobarbitone, in the rat where
974 HALL ET AL.
groups would be fasted and the results obtained compared to
non-fasted groups. The NOEL for the histopathology of
hepatocellular hypertrophy would then be compared between
the fasted and non-fasted groups.
CLINICAL PATHOLOGY
The following clinical pathology parameters can help in the
assessment of adverse effects on the liver evolving during
enzyme induction:
Alanine Aminotransferase and Aspartate
Aminotransferase (ALT/AST)
Several studies in rats dosed with liver enzyme–inducing
compounds have been published. In these studies ALT and AST
activity increases were below twofold of the controls: sodium
phenobarbitone (an archetypical mixed CYP 2B/3A inducer)
given to rats at dose levels where centrilobular hypertrophy
was present without accompanying degenerative changes
induced small increases in both hepatic ALT (Boll et al.
1998) and serum ALT and AST (Lake and Evans 1993).
In contrast, administration of the CYP 1A inducer,
3-methylcholanthrene to rats, at dose levels that induced sig-
nificant levels of drug-metabolizing enzymes, failed to show
any increase in serum ALT or AST (Lake and Evans 1993).
Peroxisome proliferators (PPARa agonists and CYP4A indu-
cers), similarly show minimally increased levels of serum
ALT and AST even when given at dose levels that induce
significant increases in liver weight, provided that the only
histological effect seen in the liver is hepatocellular hypertro-
phy (Eacho et al. 1985; Huang and Shaw 2002; Kramer et al.
2003; Peterson et al. 2004).
Increases in serum ALT activity without increases in hepa-
tic ALT activity are thought to be due to damage to, and leak-
age from, hepatocyte cell membranes, resulting in a release of
enzymes from the cytosol into the blood (Amacher 1998).
Alternatively, increased enzyme synthesis, as a consequence
of liver enzyme induction, may also lead to higher ALT levels
in both the liver and the serum. In this scenario, moderately
higher serum ALT levels, especially in mice treated with liver
enzyme inducers, might occur without cell membrane damage
(Strauss, pers. comm. 2011). Boone et al. (1985) concluded that
increases in serum ALT activity in the range of 2 to 4 or
higher in individual or group mean data when compared with
concurrent controls should raise concern as an indicator of
potential hepatic injury unless a clear alternative explanation
is found. Based on the recommendations of regulatory author-
ities, (EMEA 2010; FDA 2009; HED 2002) increases in ALT
activity of two- to threefold should be considered as indicative
of hepatocellular damage. Distinguishing irreversible and
reversible liver injury is pragmatically dealt with by assessing
the magnitude of the transaminase elevation where minimal
and reversible hepatic injury is commonly accompanied by
small increases in transaminase levels of less than twofold
(Kramer et al. 2003; Lassen 2004; Peterson et al. 2004; Satoh
et al. 1982; Solter 2005). However, considerably larger ALT
TOXICOLOGIC PATHOLOGY
increases in humans (30- to 100-fold of the upper limit of nor-
mal) can also be accompanied by full recovery of hepatic func-
tion (Koch et al. 1997).
Alternatively, small decreases in serum ALT might also be
associated with liver enzyme induction. In a rat study, where
sodium phenobarbitone was given at dose levels of 80 mg/kg,
and b-naphthoflavone (a CYP1A inducer) was given at 100
mg/kg, there were no recorded elevations in serum transami-
nases, but instead (statistically insignificant) decreases in ALT
or AST activities were recorded (Arvela, Reinila, and Pelkonen
1981). In these experiments, the only histopathology recorded
was centrilobular hepatocellular hypertrophy. Small decreases
in ALT/AST activity may usually be regarded as a ‘‘non-
adverse’’ effect because they cannot be correlated with a toxico-
logically relevant finding. Large changes (>50%), however, may
result not only from induction of hepatic drug metabolism but
from deficiencies in pyridoxal 50 -phosphate. This may be con-
firmed or excluded by measuring enzyme activities with and
without pyridoxal-phosphate addition as a cofactor to the reac-
tion mixture (PSD Guidance Document 2007).
Alkaline Phosphatase (ALP)
Increases in ALP activity associated with hepatic microso-
mal enzyme induction, in the absence of accompanying degen-
erative histopathological findings, have been reported in dog
by a number of workers (Conning and Litchfield. 1971; Leeling
et al. 1975; Robertson et al. 1993). These studies correlated an
increase in ALP activity with increased microsomal enzyme
activity and demonstrated that the source of the ALP increase
was of hepatic origin in the absence of histologically detectable
hepatobiliary injury. Increases in total serum ALP activity up to
approximately 2.5-fold (Leeling et al. 1975), 3-fold (Conning
and Litchfield 1971), and 5-fold (Robertson et al. 1993) were
observed during the course of these studies, which were noted
to recover after an 8-day treatment-free period in concordance
with a reduction in hepatic microsomal enzyme activity (Litch-
field and Conning 1972). Additionally, the ALP change reported
by Robertson et al. (1993) was not associated with any other
changes in clinical pathology parameters. Leeling et al.
(1975) concluded that marked changes in ALP levels during
drug treatment should not automatically be assumed to have
toxicological implications.
During the current ESTP-sponsored workshop, case study
material was presented where a dose-related increase in plasma
ALP activity (up to 10-fold) was shown to be of hepatic origin
in the dog and followed for up to 52 weeks. Increases in ALP
activity were evident at 4 weeks (with maximal comparable
levels observed at 13, 26, and 52 weeks). ALP activity corre-
lated with hepatocyte hypertrophy and increases in liver weight
up to 1.5-fold of control values. One individual animal showed
evidence of hepatocellular degeneration and atrophy at week
52, but this change was also associated with moderate increases
in AST (5-fold), ALT (2-fold), and GLDH (glutamate dehydro-
genase; 15-fold). Therefore, it is considered that increases in
circulating ALP activity in the dog, with associated increased
Vol. 40, No. 7, 2012 LIVER HYPERTROPHY—ADVERSE OR NON-ADVERSE?
liver weight and histological hepatocellular hypertrophy but
without hepatocellular degeneration could be interpreted as
an adaptive, rather than an adverse response to chemical
exposure.
g-Glutamyltransferase (gGT)
Induction of hepatocyte gGT has also been reported with
several chemicals, including sodium phenobarbitone which is
known to cause liver weight increases and induce hepatic meta-
bolism as well as various CYPs (Ratanasavanh et al. 1979). In
case studies presented at the workshop, messenger RNA
(mRNA) for hepatic gGT was shown to be induced in a number
of repeat dose (7 days) oral toxicity studies in the rat where
centrilobular hypertrophy, increased liver weights, increased
CYPs (predominantly 2B2 and 3A3), and phase 2 enzymes
activities (predominantly GSTA3 and UGT1A6) were
observed. However, this only translated into increases in circu-
lating gGT activity where marked induction (>50-fold) of
hepatic gGT mRNA was observed. In rats treated with pheno-
barbital for 5 days, liver but not serum gGT activity was
increased, indicating the relative insensitivity of serum-
derived gGT as a marker of hepatobiliary change in laboratory
animals compared to measuring hepatic gGT (Goldberg et al.
1981). In a reported study where kava kava was administered
to rats for 14 weeks, statistically significant increases in serum
gGT were observed in both males and particularly so in
females, which accompanied hepatocellular hypertrophy, liver
weight increases, and significant induction of CYPs (Clayton
et al. 2007). The authors concluded that the changes seen in the
liver, including the increases in serum gGT, were adaptive in
nature.
However, cholestasis due to partial obstruction of intrahepa-
tic bile ducts may also result in similar changes in gGT enzyme
activities. The decision therefore to ascribe increases in gGT to
enzyme induction should be made on a weight-of-evidence
approach with full consideration of increases in liver weight,
the absence of biologically significant increases in transami-
nase activity, as well as the absence of adverse histopathologi-
cal changes.
Bilirubin/Bile Acids
Total bilirubin, as well as bile acid levels, can be reduced as
a consequence of higher conjugation rates and excretion via
bile when hepatic enzyme–inducing chemicals are adminis-
tered (Boiteux-antoine et al. 1989; Echchgadda et al. 2004;
Gógl et al. 1979). An increase in plasma bilirubin levels is gen-
erally not seen following liver enzyme induction, but is usually
indicative of impaired hepatic bile flow, accelerated red blood
cell destruction, or decreased bilirubin metabolism (Boone et
al. 2005; Jonker, Liddleb, and Downes 2011). Additionally,
competitive inhibition of bilirubin conjugation by drugs (such
as Atazanavir that inhibits UDP-glucuronyltransferase isoen-
zyme, 1A1) may lead to a dose-related, asymptomatic, uncon-
jugated hyperbilirubinemia in humans and in rats (Neely et al.
2010; Zucker et al. 2001), which is clearly non-adverse and
975
normally not accompanied by histopathological evidence of
hepatic degeneration (Neely et al. 2010). When accompanied
by increased serum bile acids, increased bilirubin is a reliable
indicator of hepatic toxicity and loss of hepatic function (Levin
and Schwartz 1965).
Other Clinical Pathology Parameters
Global coagulation tests, such as prothrombin time and acti-
vated partial thromboplastin time, can be shortened by drugs/
chemicals that induce hepatic drug metabolism due to
increased synthesis of coagulation factors by liver cells
(Niemegeers et al. 1981; Poulsen, Lerche, and Pedersen
1985). Triglyceride levels can be decreased or increased during
enzyme induction depending on the administered compound
(Amacher, Schomaker, and Burkhardt 1998). In the experience
of the authors, administration of enzyme inducers may also
alter protein levels—the change often being correlated with
changes in lipid levels (Strauss, pers. comm. 2011).
In addition to the parameters discussed above, there are
several other clinical pathology changes that reflect alterations
in the functional/anatomical status of the liver. In addition to
cholesterol, glucose, urea, and ammonia, many enzymes can
be measured that are localized in different intracellular sites
within the hepatocyte. Enzymes such as lactate dehydrogenase
(LDH), sorbitol dehydrogenase (SDH), and a-glutathione
S-transferase (aGST) are located in the cytosol; malate dehy-
drogenase (MDH) is in the cytosol and mitochondria; ornithine
carbamoyl transferase (OCT) and GLDH are in the mitochon-
dria and pseudocholinesterase (CHE) is in vesicles. These
enzymes are not measured routinely, but they may be useful
on occasion to substitute for other more commonly used
enzymes or to provide additional information—for example,
SDH may be used in the guinea pig because of its greater liver
specificity (Clampitt and Hart 1978). Additionally, GLDH and
OCT may be measured to estimate the grade of liver cell
damage, while CHE can be used as an additional marker of the
synthetic capacity of the liver (Giffen et al. 2002; Kutty and
Payne 1994; Litchfield and Gartland 1974; O’Brien et al.
2002).
The measurement of more specific biomarkers of hepatic
damage in liver tissue, serum, or urine may help distinguish
adaptive enzyme induction from adverse liver cell toxicity.
Studies measuring transcriptome profiles in liver tissue
(Ellinger-Ziegelbauer et al. 2011; Kramer et al. 2003; Peterson
et al. 2004) as well as metabolome patterns in plasma (van
Ravenzwaay et al. 2010) and urine (Robertson et al. 2000) are
aimed at fulfilling this aspiration.
If there are no accompanying histopathological changes,
then the default assumption is that any consistent, and signifi-
cant, adverse change in clinical pathology parameters can be
regarded as an adverse effect with relevance to human health.
However, small clinical chemistry changes beyond historical
control ranges can be regarded as adaptive, if sufficient evi-
dence exists that these effects are due to altered metabolism,
if the change by its own is doubtful or of minimal toxicological
976
importance, and the finding alone is not predictive for an
adverse liver effect (Regulation EC No. 1772/2008, Annex 1,
chapter 3.9)—for example—decreases in total bilirubin levels
without any sign of hypoproliferative anemia, accompanied
by decreases in total bile acid concentrations, may be consid-
ered adaptive provided that the synthetic pathway for these
molecules remains unaffected (Kuipers et al. 1989) since these
changes may be due to an increase in the conjugation rate and
excretion via bile. Other confounding examples include reduc-
tions in global coagulation tests, which result from increased
coagulation factor synthesis as a consequence of enzyme
induction (Poulsen, Lerche, and Pedersen 1985) but, alterna-
tively, could result from an acute phase reaction or
thrombophilia.
A general rule of thumb is that changes affecting a single
clinical chemistry parameter beyond historical control values
can normally be regarded as ‘‘non-adverse’’ when no histo-
pathological correlate can be found (ECETOC Technical
Report No. 85, 2002).
Pitfalls When Interpreting Clinical Pathology
Parameters
Different results in studies with liver enzyme–inducing
compounds administered to various animal species as well as
literature studies of different facilities have to be cautiously
interpreted.
Although serum alanine aminotransferase (ALT) activity in
the rat, mouse, dog, and humans mainly originates from liver
tissue, species-specific differences exist regarding plasma
half-life of the enzyme. In humans, the half-life of ALT in the
plasma is 47 hr, in dogs it is reported to be 17 hr, while in rats it
is approximately 3 to 4 hr (Boyd 1983). Depending on the
enzyme kinetic ALT activity increases may be detectable in
one species but not in the other in repeated dose studies.
g-glutamyltranspeptidase (gGT) and alkaline phosphatase
(ALP) have different tissue distributions in the rat, mouse, and
dog (Clampitt and Hart 1978; Keller 1981). For example, the
hepatic isoform is the main contribution to total circulating
ALP activity in the dog, but has comparatively lower activity
in the rat and hence has lower diagnostic sensitivity in this
species. Moreover, in young animals that are generally used
in regulatory toxicology studies, serum ALP activity derives
mainly from the bone isoenzyme and not from the liver
(Hoffmann et al. 1994). Therefore, in addition to liver enzyme
induction, changes in other organs may need to be assessed.
Induction of the aforementioned enzymes by chemicals also
appears to be species-specific: in dogs, corticoid-induced ALP
isoenzyme activities are often increased (Gaskill et al. 2005),
whereas in mice, hepatic ALP is increased (Kawasaki, Mataki,
and.Takano 1994). In rats, hepatic gGT is induced in response
to liver enzyme inducers (Gallagher et al. 1998; Satoh et al.
1982). In pregnant rats from gestation day 6 (GD6) until
GD20, ALP, instead of gGT activity is increased when enzyme
inducers are administered (Strauss, pers. comm. 2011). In mice,
in contrast to rats, ALT is more often increased when hepatic
HALL ET AL. TOXICOLOGIC PATHOLOGY
enzyme inducers are administered without any histopathologi-
cal indication of liver cell toxicity (Strauss, pers. comm. 2011).
When comparing the above with older published studies, it
is apparent that different methods can lead to widely different
results. There is general agreement that ALT and AST activities
should be measured according to The International Federation
of Clinical Chemistry and Laboratory Medicine (IFCC) methods
without addition of the cofactor, pyridoxal 50 -phosphate. If
decreased transaminase activities are observed, a repetition with
addition of the cofactor should be performed in order to exclude/
confirm alternative reasons for the decreased activity other than
enzyme induction (Evans and Whitehorn 1995).
In addition, one should be aware that commercial gGT kits
for human medicine have a limit of detection of about 3 U/L (at
37 C, i.e., 50 nkat/L, e.g., Roche instruction manual gGT Szasz
mod., 2010-06, V7). Normal rat serum gGT activities are gen-
erally below this limit of detection, so that moderate gGT
increases cannot be measured with these tests. Also, total
bilirubin kits (e.g., Roche instruction manual total bilirubin
Diazo method 2011-5, V8) for human medicine may also not
be adequate for measuring rodent bilirubin levels, particularly
where decreases in bilirubin occur, unless reagents and proce-
dures are appropriately modified.
Serum Enzyme Activity Elevation in Human Studies
Hepatic enzyme inducers, such as sodium phenobarbitone,
have been reported to increase the serum enzyme levels in
human patient populations, and elevations in ALT, AST, and
gGT were seen in patients following treatment with several
anticonvulsant drugs (Aiges et al. 1980; Wall et al. 1992). In
a study of epileptic patients receiving drug therapy, elevations
in gGT were seen in almost all individuals leading the authors
of the study to conclude that this did not indicate hepatic dam-
age, since none of the patients exhibited clinical symptoms.
Instead, these elevations in enzymes merely confirmed that the
patients were receiving the drugs (Hirayanagi, Fujii, and
Teshirogi 1991). Elevated gGT levels, in the absence of other
markers of hepatic damage, were reported to be commonly ele-
vated in patients taking rifampicin (Davis 1989), carbamaze-
pine, zarontin, phenobarbital, and phenytoin (Knight 2005;
Ohta and Toda 2001; Rosalki, Tarlow, and Rau 1971; Vanden-
berghe 1996; Whitfield et al. 1973). Much of this elevated
enzyme activity was considered due to hepatic induction of the
enzyme, and its subsequent loss into the blood, rather than an
indicator of frank toxicity to the liver (Ohta and Toda 2001).
Finally, anti-epileptic drugs in man have also been reported
to increase serum ALT up to 3-fold the upper limit of normality
(ULN), and AST up to 2-fold the ULN in approximately 25%
of the patient cohort studied (Haidukewych and John 1986).
Once again these were not considered to be indicative of hepa-
totoxicity but to be a consequence of hepatic enzyme induction
and hepatocellular hypertrophy. Indeed, liver biopsies taken
from patients on long-term therapy failed to show any histolo-
gical evidence of hepatotoxicity (Jacobsen et al. 1976). How-
ever despite this, it should be noted that there is a correlation
Vol. 40, No. 7, 2012 LIVER HYPERTROPHY—ADVERSE OR NON-ADVERSE?
FIGURE 1.—Gross photograph of a control mouse (left panel, 1a) and treated (right panel, 1b) liver demonstrating an increase in liver volume char-
acterized by a fine reticular pattern after treatment with the enzyme inducer phenobarbital.
between drug-induced hepatic CYP induction and documented
idiosyncratic hepatotoxicity in man (Li 2002).
HISTOLOGICAL ASSESSMENT
The first indication of hepatomegaly is usually noted at
necropsy by the measurement of an increase in organ weight,
and in extreme cases, by the concomitant visual observation
of an increase in liver size (volume; Figure 1). In small animals
where experiments can be powered to gather statistically sig-
nificant data, the assessment of an increase in liver weight is
relatively straightforward. However, in dogs and other larger
animals, interindividual variation combined with smaller group
sizes can make the assessment of meaningful increases in liver
organ weight more uncertain.
Clinical pathology can provide valuable ancillary data to
indicate alterations in liver function; however, the diagnosis
of morphological or structural changes in the liver is ulti-
mately made by a trained pathologist assessing formalin fixed,
paraffin embedded, haematoxylin and eosin (H&E) stained
sections. To this end, the National Toxicology Program (NTP)
for 2-year rat carcinogenicity studies advocates two liver sam-
ples taken at the widest part of the left lobe and right median
lobes (Maronpot et al. 1989; NTP 2006). The RITA trimming
guide recommends to sample in addition the caudate lobe in
rats and mice (Ruehl-Fehlert et al. 2003).
Treatment-related increases in liver weight may result from
a wide variety of causes such as hyperplasia (of any of the resi-
dent cell types), hypertrophy, inflammation, fibrosis, abnormal
977
storage of metabolism products, particles, or cleavage prod-
ucts, neoplasia, and congestion (Carthew et al. 1996; Greaves
2007). Typically, these changes do not occur in isolation, so
in the absence of overt adverse changes such as inflammation,
necrosis, or degeneration, it is important to recognize that
increases in liver weight may be induced by hypertrophy,
hyperplasia, or very frequently a combination of the two
(Maronpot et al. 2010).
An increase in liver cell size can result from accumula-
tion of glycogen, lipid, and water (hydropic degeneration)
or the proliferation of subcellular organelles (typically
smooth endoplasmic reticulum [sER] and/or peroxisomes).
Experiments in which hepatocellular hypertrophy was
induced in mice with phenobarbital suggest that polyploi-
dization is an additional and inevitable component of liver
cell hypertrophy caused by these types of drugs (possibly
as an adaptive response to increased metabolic demands
—RNA and protein synthesis; Böhm and Noltemeyer
1981; Bursch et al. 2004). Morphologically in both rats
and mice, hepatocellular hypertrophy may present as a dif-
fuse change affecting all zones of the liver lobule, or a
regional change usually affecting the centrilobular region,
but frequently extending to the mid-zonal region (and fur-
ther) with increasing exposure (Figure 2). Occasionally,
periportal hypertrophy without evidence of centrilobular
hypertrophy is also seen—for example, both fenofibrate
and ciprofibrate induced periportal hypertrophy in the
cynomologus monkey as a result of both peroxisomal and
mitochondrial proliferation (Hoivik et al. 2004).
978 HALL ET AL.
FIGURE 2.—(a) H&E stained low power photomicrograph of a male
B6C3F1 mouse liver treated with 100 mg/kg/day of pentabromodiphe-
nyl oxide for 90 days showing centrilobular hepatocyte hypertrophy
with characteristic enlargement of hepatocytes noted by the decreased
number of nuclei per unit area and pale staining centrilobular eosino-
philic cytoplasm. (b and c). H&E stained low power photomicrograph
of a control male C57Bl mouse liver (left panel, 2b) and a treated
TOXICOLOGIC PATHOLOGY
At the ESTP liver hypertrophy expert group, it was
generally agreed that an increase in liver weight of at least
20% was required to histologically detect a change in hepato-
cyte cell size (corresponding with other publications; Ennulat,
Walker, et al. 2010). However, whereas the assessment of zonal
changes in liver cell size is relatively straightforward, diffuse
changes are considerably more difficult to detect by examina-
tion of standard H&E stained sections without reliance upon
morphometry (Greaves 2007).
In H&E stained sections at low power, hepatocellular hyper-
trophy is usually recognized by a zonal increase in liver cell
size and eosinophilia with a reciprocal decrease in nuclear
density (i.e., a decrease in the number of hepatocyte nuclei per
unit area of tissue; Figure 2).
The staining characteristics of the hepatocyte cytoplasm
vary depending on the mechanism of cellular hypertrophy and
consequently the organelle responsible for the increase in
cytoplasmic volume.
Phenobarbitone and other microsomal drug-metabolizing
enzyme inducers induce hepatocyte hypertrophy through sER
proliferation. This results in the characteristic eosinophilic
‘‘ground glass’’ appearance of hepatocyte cytoplasm (Figure 2).
Chronic administration of certain enzyme inducers, such as
chlordane, can result in extreme hypertrophy characterized
by bizarrely enlarged hepatocytes with significant increases
in hepatocyte ploidy (Figure 3). If electron microscopy is per-
formed, induction by typical microsomal enzyme inducers such
as phenobarbital reveals characteristic stacks of smooth ER that
crowd out other organelles (Figure 4).
Other classes of xenobiotics such as Clofibrate (and similar
hypolipidaemic fibrates) as well as ether/phenoxy herbicides
induce hepatocyte hypertrophy through a different mechanism.
These chemicals activate the PPARa (peroxisome proliferator-
activated receptor alpha) resulting in peroxisome proliferation
(together with sER proliferation). Histologically, these samples
stain with an intensely eosinophilic granular cytoplasm in H&E
sections (Figure 5). Electron microscopy has demonstrated that
the granules are in fact single catalase positive electron dense
vesicles bounded by a single plasma membrane (Figure 6).
MOLECULAR METHODS TO ASSESS LIVER ENZYME INDUCTION
A variety of methods are available to assay drug-induced
hepatocyte enzyme induction (reviewed in Fretland and Mon-
shouwer 2010; Figure 7). Many of these methods such as in
silico modeling, receptor binding assays, and immortalized cell
lines have proven valuable in identifying potential induction
liabilities. They have also proven pivotal in increasing our
C57Bl mouse liver (right panel, 2c) on a 4-week feeding study show-
ing diffuse macrovesicular fatty vacuolation (control) and diffuse
hepatocyte hypertrophy (treated) characterized by a diffuse enlarge-
ment of hepatocytes. Note that diffuse changes tend to be more subtle
in appearance compared to regional changes and require careful com-
parison to concurrent control samples and correlation with liver organ
weight data to accurately identify.
Vol. 40, No. 7, 2012 LIVER HYPERTROPHY—ADVERSE OR NON-ADVERSE?
FIGURE 3.—H&E stained high-power photomicrograph of a male
B6C3F1 mouse liver chronically (2-year carcinogenicity study) treated
with chlordane showing the cellular features of extreme hypertrophy
characterized by a notable increase in cellular size accompanied by
binucleate hepatocytes with enlarged hepatocyte nuclei (polyploidy).
knowledge of ligand–receptor binding interactions. For exam-
ple, in silico modeling of PXR–ligand crystal structure com-
plexes have elucidated the molecular basis for PXR receptor
promiscuity allowing it to interact with such diverse ligand
binding partners as phenobarbital and rifampicin (Watkins et
al. 2001). However, these methods, although still widely used
and of general applicability, possess significant limitations—
for example—immortalized cell lines tend to express the major
CYPs at lower levels than fresh hepatocyte preparations
(Fretland and Monshouwer 2010).
The use of newer techniques and, in particular, the use of
cryopreserved human hepatocytes can now provide more
reliable data to build confidence for human risk assessment and
to predict the effects of novel chemicals on drug-metabolizing
enzyme induction in man (Fretland and Monshouwer 2010).
This type of information can also be supplemented by experi-
ments generated in laboratory species including nonhuman pri-
mates that show considerable sequence homology in their
nuclear hormone receptors—for example, the ligand binding
domain of PXR in the rhesus monkey possesses 96% sequence
identity with the human receptor at the amino acid level
(Moore et al. 2002). More recently, with the advent of huma-
nized mice which express the human form of one or more of
the nuclear receptors PXR (Gonzalez 2007), CAR (Ross
et al. 2010), or PPARa receptor (Morimura et al. 2006), inves-
tigators can compare the role of the human receptor with that of
the rodent receptor to determine the species-specific responses
to prototypic and novel xenobiotics.
Enzyme Induction in Safety Assessment
The determination of enzyme induction is now carried out rou-
tinely during the safety assessment of new chemical entities in the
pharmaceutical sector. In one study where rats were dosed with
979
RO9210, a non-nucleoside reverse transcriptase inhibitor for the
treatment of HIV infection, Zabka et al. (2011) demonstrated by
routine examination of H&E stained slides, centrilobular
hypertrophy with secondary thyroid follicular hypertrophy
and pituitary gland pars distalis hypertrophy. IHC analysis
of formalin fixed paraffin embedded sections of the pitui-
tary and thyroid glands demonstrated thyrotroph hypertro-
phy with granule depletion and thyroid follicular epithelial
cell hyperplasia. Image analysis was able to confirm and
quantify the extent of the centrilobular hypertrophy identi-
fied by histological examination of the H&E stained slides.
IHC analysis of the liver demonstrated increased CYP 2B1/
2 and CYP 3A2 staining intensity with extension of staining
from the centrilobular to the periportal region. Frozen rat liver
samples as well as cultured rat hepatocytes were used to quan-
tify the extent of mRNA induction of drug-metabolizing
enzymes (UGT1A1, UGT 1A2, CYP1A1, CYP 1A2, CYP2B1
3A1, and CYP2B1 3A2) by Taqman quantitative RT-PCR.
These data illustrate the utility of routine histological assess-
ment, IHC and qRT-PCR to assess the nature and extent of
drug-metabolizing enzyme induction to help demonstrate the
adaptive nature of the toxicologic changes induced by
RO2910. Induction of hepatocyte drug-metabolizing enzymes
in this case resulted in increased turnover of T4 (thyroxine) and
secondary thyroid hypertrophy/hyperplasia due to stimulation
of the pituitary–thyroid–endocrine axis. An ELISA method for
total thyroxine (T4), total tri-iodothryonine (T3), and thyroid
stimulating hormone (TSH) confirmed the increase in TSH and
decrease in T4 (with no change in T3).
MECHANISMS OF HYPERTROPHY
Nuclear Hormone Receptors
The nuclear hormone receptors constitute a superfamily of
xenosensing receptors that specifically interact with endogenous
and exogenous chemicals. Receptor–ligand binding results in the
activation of a battery of genes mediating oxidative drug metabo-
lism, conjugation, and transport, which serves to eliminate the
xenobiotic from the organism to maintain homeostasis. The
majority of these nuclear receptors are expressed on the liver
(Bookout et al. 2006) and can be functionally divided into those
that are generally associated with drug metabolism—the preg-
nane X receptor (PXR) and the constitutive androstane receptor
(CAR)—and those that are involved in the metabolic regulation
of endogenous compounds—principally glucocorticoids (gluco-
corticoid receptor), lipid oxyseroids (liver X receptor (LXR)), bile
acids (farnesoid X receptor (FXR)), and lipid metabolism (peroxi-
some proliferator-activated receptor (PPARa)) (reviewed in Plant
and Aouabdi 2009; Table 3).
Peroxisome Proliferator-activated Receptor Alpha
Currently, there are three peroxisome proliferators activated
receptors, termed peroxisome proliferator-activated receptor
alpha (PPARa), PPARg and PPARb/d which heterodimerize
with RXR to transactivate distinct but overlapping gene targets
980 HALL ET AL. TOXICOLOGIC PATHOLOGY

FIGURE 4.—(a and b) Transmission electron microscope photograph of control (left panel, 4a) and treated (right panel, 4b) rat hepatocytes showing
hypertrophy after treatment with a COX inhibitor. Treated hepatocytes are characterized by increased amount of sER (sER proliferation) that
crowds out and peripherally compresses other organelles in the cell. Inset shows a higher magnification of the cytosol immediately adjacent
to the nucleus. (Note: Figures 2a, 3, and 4 were previously published in Toxicologic Pathology, 38: 776–95, 2010, and used by kind permission
of Sage Publishing and courtesy of NTP. Figure 4 courtesy of Dr Katsuhiko Yoshizawa [with permission].)

FIGURE 5.— (a and b) H&E stained low-power photomicrograph of Wistar rat liver showing control (left panel 5a) and treated (right panel 5b)
livers from animals treated with a peroxisome proliferating agent (phenoxy herbicide) characterized by centrilobular hepatocyte hypertrophy and
intensely eosinophilic cytoplasm. Inset: oversharpened area of image to demonstrate fine cytoplasmic granularity.
Vol. 40, No. 7, 2012 LIVER HYPERTROPHY—ADVERSE OR NON-ADVERSE? 981

FIGURE 6.—(a and b) Transmission electron microscope photograph of B6C3F1 mouse liver showing control (left panel, 6a) and treated (right
panel, 6b) hepatocytes characterized by centrilobular hypertrophy and electron dense membrane bound granules in hepatocytes after treatment
with a peroxisome proliferator agent (di-isononyl phthalate, DINP).

FIGURE 7.—(a) Immunolocalization of CYP 1A1 in untreated CD1 mouse liver demonstrating centrilobular expression of the enzyme using immu-
nocytochemistry. (b) Immunolocalization of CYP 3A2 in untreated CD1 mouse liver showing a predominantly centrilobular localization of the
enzyme but with random cells throughout the lobule showing expression of the enzyme.

(Plant and Aouabdi 2009). PPARa is the major PPAR found in the
liver, but is also distributed and highly expressed in the kidney and
intestine (Bookout et al. 2006) as well as heart and brown adipose
tissue (Xu, Li, and Kong 2005). PPARg is expressed predomi-
nantly in the adipose tissues but also in pancreatic beta-cells,
intestines and spleen whereas PPARb/d is expressed nearly
ubiquitously (Bookout et al. 2006; Greaves 2007).
PPARs regulate lipid and cholesterol metabolism through
induction of (peroxisome proliferator response element
(PPRE)) containing target genes resulting in increased beta-
oxidation of fatty acids (Xu, Li, and Kong 2005). Natural
ligands for PPARa include saturated and unsaturated fatty
acids, eicosinoids, and linoleic acid metabolites. However, a
diverse range of xenobiotics from many classes and structures
are also able to activate PPARa such as the fibrate hypolipidae-
mic agents (clofibrate, fenofibrate, gemfibrozil amongst oth-
ers), methaphenilene, thromboxane synthetase inhibitors,
dehydroepiandosterone, non-steroidal anti-oestrogens, ibupro-
fen, Wy-14,643, diphenyl ether herbicides, and phenoxy herbi-
cides (Greaves 2007).
Peroxisome Proliferator-activated Receptor Alpha
Phenotype
Activation of PPARa by hypolipidaemic agents results in
liver enlargement through peroxisome proliferation (which
982
TABLE 3.—Summary of nuclear receptor actions.
Receptor CYP induction Inducer/Inducers
AhR CYP1A Dioxins, 3-methycholanthrene
CAR CYP2B Barbiturates
PXR CYP3A Rifampicin, dexamethasone,
pregnenolone 16 alpha-carbonitrile
PPARa CYP4A Fibrates
appears morphologically as an increase in peroxisomal volume
density) as well as proliferation of sER and increases in hepatic
cytochrome P450 enzyme activity (Cattley 2003; Walker et al.
1996). In rodents, these changes are highly correlated with
liver tumors which has led to the general view that dose rates
that induce >3 fold increases in peroxisomes and >1.5 fold
increases in liver weight in short term studies are sufficient
to induce hepatocellular neoplasia in long term studies (Cattley
2004; Walker et al. 1996).
Increases in drug-metabolizing enzymes induced by PPARa
activators are typically characterized by increased activity of
CYP4A and acyl CoA oxidase genes (Bell et al. 1991). How-
ever, more recent studies have demonstrated increased activity
of other targets such as human SULT2A1 (Fang et al. 2005)
and human/murine UGT2B4 (Barbier et al. 2003) as well as
decreased bile acid secretion due to decreased activity of cho-
lesterol 7a-hydroxylase (CYP7A1) and sterol 27-hydroxylase
(CYP27 ) (Li and Chiang 2009; Post et al. 2001).
HEPATOCARCINOGENICITY
Humans are regularly exposed to peroxisome proliferating
chemicals either in the form of environmental exposure, or
intentionally through drug treatment. Given the clear associa-
tion between these agents and the induction of hepatocarcino-
genesis in long-term rodent studies, there is a clear concern
over the potential risk to human health.
Hepatocarcinogenesis is thought to primarily result from an
increase in cell division and a reciprocal decrease in apoptosis
induced upon treatment with peroxisomal proliferators result-
ing in hepatocyte hyperplasia and fixation of spontaneous
mutations (Plant et al. 1998). Oxidative damage due to acyl
CoA oxidase induction and escape of H2O2 from peroxisomes
has also been proposed as an indirect mechanism of genotoxi-
city and carcinogencitiy (Conway et al. 1989). However it is
uncertain whether this leads to DNA damage in hepatocytes
and if it is causally related to the development of tumors
(Cattley 2004).
The Role of the PPARa Receptor
The importance of the role of the PPARa receptor in
mediating hepatocarcinogenesis in response to peroxisomal
proliferators was initially raised by the observation that the
relative expression level of the receptor in responder species
HALL ET AL. TOXICOLOGIC PATHOLOGY
Mechanism Histopathology
Arnt heterodimerization and Toxic hepatopathy, hepatocellular hypertrophy,
transcription of target genes multinucleated hepatocytes, fatty change, necrosis
RXR heterodimerization and Ground-glass hepatocyte hypertrophy
transcription of target genes
RXR heterodimerization and Ground-glass hepatocyte hypertrophy
transcription of target genes
RXR heterodimerization and Granular eosinophilic hepatocyte hypertrophy
transcription of target genes
such as the rat and mouse where liver expression is high
compared to non-responsive species such as the guinea pig,
non-human primate, and human where expression (in the
human) is an order of magnitude lower (Choudhury et al.
2000; Palmer et al. 1998).
The relatively recent development of PPARa gene knockout
mice has now confirmed that the PPARa receptor is responsi-
ble for mediating the effects induced by the peroxisome prolif-
erator–inducing agents clofibrate, WY-14,643 and DEHP (i.e.,
hepatomegaly, increases in peroxisomes and enzyme induc-
tion) (Lee et al. 1995; Ward et al. 1998). It is also responsible
for the transient increase in cell proliferation commonly
observed immediately after treatment as well as the more vari-
able low level sustained increases in proliferation occasionally
observed following chronic treatment (Cattley 2004).
Not only do PPARa knockout mice not develop hepatocel-
lular tumors in response to long-term treatment with peroxi-
some proliferators, (Lake 1995; Peters, Cattley, and Gonzalez
1997), but PPARa humanized (PPARa knockout/knockin)
mice also fail to develop tumors in response to these agents
suggesting that the human receptor is structurally different
from the murine receptor (Morimura et al. 2006). Since induc-
tion of cell proliferation (with a concomitant decrease in apop-
tosis) is thought to be the mechanistic basis for the induction of
liver tumors by these non-genotoxic agents, it therefore prob-
able that humans would be resistant to this form of promotion.
Epidemiology and Risk to Man
Studies using human hepatocytes (Bentley et al. 1993; Lake
2009) as well as human biopsy material exposed to gemfibrozil
for up to 27 months (Greaves 2007) have generally concluded
that humans are insensitive and unresponsive to peroxisomal
proliferators at therapeutic dose levels and that humans are not
at an increased risk of developing liver tumors (Cattley et al.
1998; International Agency for Research on Cancer [IARC]
1995). In one study, humans were given oral fibrate therapy for
8 weeks and no differences in peroxisomal acyl CoA oxidase
mRNA transcripts were detected in groups receiving either
fenofibrate, bezafibrate, or gemfibrozil compared to placebo
controls (Roglans et al. 2002).
These epidemiological data, however, have been collected
from relatively small data sets and may therefore not be robust
enough to detect small increases in human liver carcinogenesis.
Vol. 40, No. 7, 2012 LIVER HYPERTROPHY—ADVERSE OR NON-ADVERSE?
Further work over the past decade in nonhuman primates there-
fore has been conducted and confirms that they are relatively
insensitive to the effects of PPARa agonists compared to
rodent species. Exposure to DEHP, diisononyl phthalate, or
clofibrate for 14 days failed to induce increases in peroxisomal
fatty acid beta-oxidation or liver weight in the cynomolgus
monkey (Pugh et al. 2000). In another study conducted in the
nonhuman primate, exposure to K-111, a potent PPARa
agonist elicited only modest increases in lipid beta-oxidation
(up to 3-fold) and peroxisome volume density (1.5- to 2-fold
increase) compared to 50- to 100-fold increase in lipid beta-
oxidation and 10-fold increases in peroxisome volume density
that can occur in rats and mice (Schäfer et al. 2004). Finally,
another recent report demonstrated that exposure to the potent
PPARa/g agonist, ciprofibrate, is capable of eliciting peroxi-
some proliferation (Hoivik et al. 2004) in higher primates but
that this modest induction was associated with transcript pro-
files indicative of an antiproliferative and a pro-apoptotic effect
with no evidence of oxidative stress (Cariello et al. 2005).
Constitutive Androstane Receptor
CAR is an orphan nuclear receptor that forms heterodimers
with RXRa (retinoic X receptor) leading to nuclear transloca-
tion and transactivation of target genes. This can occur even
in the absence of ligand (Honkakoski and Negishi 1998). CAR,
however, is activated by a diverse range of ligands including
1,4-bis(2-(3,5-dichloropyridoxyloxy)) benzene (TCPOBOP)
(murine CAR specific; Wei et al. 2000), CICO (human CAR
specific, Maglich et al. 2003), phenobarbital, and clotrimazole
(the latter two activators of both CAR and PXR, Moore et al.
2000). Activated CAR heterodimers bind to a number of regu-
latory regions of target genes eliciting transactivation of
CYP2B genes.
CAR Phenotype
Activation of CAR by potent drug-metabolizing enzyme
inducers such as Phenobarbitone causes hepatomegaly due to
a profound induction of sER proliferation that occurs in com-
bination with an early and transient hepatocyte hyperplasia/
suppression of apoptosis (Huang et al. 2005) and an enlarge-
ment of the hepatic blood space (Massey and Butler 1979).
Morphological changes are characterized by a centrilobular
hypertrophy that is apparent after 1 week of treatment and can
persist for 80 weeks without overt signs of liver cell damage
(Crampton et al. 1977a).
HEPATOCARCINOGENESIS
Long-term treatment with CAR activators such as TCPO-
BOP and Phenobarbitone results in a strain- and species-
specific induction of liver tumors in rats and mice (Diwan
et al. 1992) that is dependent upon the fixation of somatic
mutations due to the stimulation of S-phase DNA synthesis
leading to hepatocyte proliferation and a decrease in apoptosis
(Hasmall and Roberts 1999). This process is well characterized
983
and appears morphologically as a temporal progression from
centrilobular hepatocyte hypertrophy (with a concomitant
induction of Cyp2b enzymes) to the development of altered
hepatic foci, and ultimately the appearance of adenomas and
carcinomas (Ross et al. 2010). Hand-in-hand with acute
increases in cell proliferation is a CAR-dependent increase in
endoreduplication in which Phenobarbitone and TCPOBOP
stimulate an increase in the ratio of 8N:4N hepatocytes (Huang
et al. 2005), which appears morphologically as an increase in
ploidy/nuclear size. Increases in functional drug-metabolizing
enzyme capacity through cell hypertrophy and hyperplasia act
to promote clearance of xenobiotics and return the organism to
homeostasis. In this respect, increases in hepatocyte ploidy
have been correlated with increased cytochrome P450 activity
(Rajvanshi et al. 1988).
Of note, recovery from Phenobarbitone or TCPOBOP treat-
ment results in complete reversal of changes marked by a return
to normal liver cell size and an associated increase in apoptosis
(Huang et al. 2005). Reversal may even extend to regression of
a proportion (30%) of liver tumors (adenomas and adenocarci-
nomas) observed after the withdrawal of treatment with chlor-
dane in B6C3F1 mice (Malarkey et al. 1995).
The Role of the CAR Receptor
It is now well established that the CAR, in a similar manner
to other nuclear receptors, mediates the acute and chronic
effects induced by Phenobarbitone and TCPOBOP. CAR
knockout mice are resistant to the enlargement of liver cell size
and induction of drug-metabolizing enzymes/transporters as
well as the acute and chronic increases in DNA synthesis (and
reciprocal p53-mediated decreases in apoptosis) that leads to
hepatocyte proliferation and ultimately hepatocarcinogenesis
(Huang et al. 2005).
Epidemiology and Risk to Man
Liver enlargement after 1 year of treatment with nongeno-
toxic xenobiotics in rodents correlates with an increase in liver
tumors in chronic toxicology studies (Carmichael et al. 1997;
Ross et al. 2010). In a review of 138 pesticide carcinogenicity
studies in mice, induction of liver tumors was very closely cor-
related at 1 year with a mean relative liver weight of 150% and
the presence of significant liver pathology (Carmichael et al.
1997).
In humans, liver enlargement associated with an increase in
cytochrome P-450 levels is seen following exposure to drug-
metabolizing enzyme inducers such as Phenobarbital and other
anti-epileptic drugs (Pirttiaho et al. 1978) as well as alcohol
(Pirttiaho et al. 1982). Therefore, clear concerns are raised for
the safety of human health and the risk of hepatocarcinogenesis
in people exposed to these types of agents.
Whereas treatment of CAR knockout mice clearly demon-
strated the dependence of hepatocarcinogenesis on the pres-
ence of the CAR receptor, it has only recently been
demonstrated that the human CAR receptor is relatively resis-
tant to the mitogenic effects of CAR pathway activation and,
984 HALL ET AL.
therefore, less likely to induce liver tumors through this
mechanism. This observation is based on the resistance of cul-
tured human hepatocytes to induction of replicative DNA
synthesis (Hirose et al. 2009; Lake 2009; Parzefall et al.
1991) as well as similar observations in huPXR/huCAR recep-
tor humanized mice.
In these later studies, treatment of humanized mice (huPXR/
huCAR) with either chlordane or Phenobarbitone resulted in
marked liver weight increases, hepatocellular hypertrophy, and
induction of cytochrome P450 in agreement with clinical data
derived from human liver samples. However, no increases in
replicative DNA synthesis were observed either by direct mea-
surement of BrdU labeling index or by microarray analysis of
cell proliferation associated gene expression changes (Ross
et al. 2010). These experiments also demonstrated species differ-
ences in response to Phenobarbitone and chlordane in that the
human receptor had greater sensitivity for Phenobarbitone-
mediated increases in Cyp2b10 and Cyp3a11 compared to the
mouse receptors, but the converse was true for chlordane.
These data are in agreement with a number of epidemiolo-
gical studies that demonstrate no linkage between hepatocarci-
nogenesis and chronic exposure to Phenobarbitone, phenytoin,
and other anticonvulsants at exposures similar to those that
produce liver tumors in rodents (IARC 2001; Olsen et al.
1989; Whysner, Ross, and Williams 1996).
The mechanistic basis underlying the differences in sensitiv-
ity between the rodent and human receptor to induction of pro-
liferation in response to CAR activation is still unclear.
Microarray analysis of huPXR/huCAR mice has shown that
these mice are resistant to the upregulation of the polo-like
kinase (Plk1)-mediated cell proliferation pathway induced by
Phenobarbitone treatment (Elcombe et al. 2010; Ross et al.
2010). Upregulation of this pathway in wild-type mice implies
that structural differences in the human receptor may account
for critical differences in the types of responder genes transac-
tivated by CAR activation and induction of proliferation.
PREGNANE X RECEPTOR
PXR Promiscuity and Cross-talk
PXR, like CAR, plays a central role in xenobiotic clearance.
CAR and PXR are structurally well conserved with a high
degree of similarity in their DNA-binding domains but signif-
icant divergence in their ligand binding domains (Kliewer et al.
2002). This enables considerable ‘‘cross-talk’’ between PXR
and CAR due to the sharing of ligands, coregulators, or
DNA-binding elements resulting in a battery of genes coordi-
nately regulated by both nuclear receptors. To this end, Ueda
and Maglich identified sixty-nine genes regulated by CAR
(Ueda et al. 2002) and forty genes regulated by PXR (Maglich
et al. 2003) with many of them being coregulated by both PXR
and CAR (Plant and Aouabdi 2009).
Not only can PXR interact with other nuclear hormone
receptors to provide functional redundancy in xenobiotic meta-
bolism, but it is also regulated by a number of other receptors
including PPARa, glucocorticoid receptor, and CAR (reviewed
TOXICOLOGIC PATHOLOGY
in Plant and Aouabdi 2009) to help integrate an organism’s
response to xenobiotic exposure.
This functional cross-talk is also reflected in the wide range
of structurally diverse ligands that PXR can bind due to the
markedly flexible pocket domain (Kliewer, Goodwin, and
Willson 2002). This results in activation by numerous xenobio-
tics from different chemical series such as steroids, antibiotics
(including the prototypic inducer rifampicin), antimycotics,
bile acids, aflatoxins (Ratajewski et al. 2011), and the herbal
antidepressant St. John’s wort (Kliewer, Goodwin, and Willson
2002). As with other nuclear hormone receptors, species differ-
ences exist in sensitivity to different PXR activators. However,
since human and mouse PXR only share 80% amino acid
homology in their ligand-binding domain compared to 96%
amino acid identity in the DNA-binding domain, mouse and
human PXR differ markedly in their responses to PXR ligands
(Lehmann et al. 1998; Ma, Idle, and Gonzalez 2008). For
example, human PXR is not activated by pregnenolone 16
alpha-carbonitrile (PCN, a potent inducer of murine CYP3A
and PXR) whereas rifampicin is a selective activator of human
PXR but not murine PXR (Bertilsson et al. 1998).
PXR Phenotype
PXR is a master regulator of CYP3A transcription (Bertils-
son et al. 1998; Gibson, El-sankary, and Plant 2002) as well as
other genes involved in phase I oxidation, phase II conjugation,
and phase III transport. The growing list of PXR target genes
include, in addition to CYP3A family members, CYP2B6
(Wang et al. 2003), Cyp7a1 (cholesterol 7 a-hydroxylase; Stau-
dinger et al. 2001), UDP-glucuronosyltransferase 1A1 (Hartley
et al. 2004), intestinal P-glycoprotein (Geick, Eichelbaum, and
Burk 2001), OATP2 (Hartley et al. 2004), and MRP2 (Fromm
et al. 2000).
Activation of PXR by PCN and cyproterone acetate (CPA),
a synthetic steroid with anti-androgenic and contraceptive
activity, results in liver hyperplasia and hypertrophy
(Japundzić et al. 1974) in line with the response seen with other
nuclear hormone receptors. This effect is visible within 3 days
of treatment in rats (Schulte-Hermann et al. 1980) and is
accompanied by a decrease in the frequency of binucleated
hepatocytes and a concomitant increase in nuclear ploidy
(Schulte-Hermann, Hoffmann, and Landgraf 1980). As with
other nuclear hormone receptor agonists, long-term administra-
tion of CPA results in hepatocarcinogenesis in the mouse
(Schuppler and Günzel 1979) and rat (Tucker, Kalinowski, and
Orton 1996).
Role of the PXR Receptor
The generation of PXR knockout mice demonstrated that
the PXR receptor is necessary for the induction of cellular
hypertrophy and hyperplasia in response to PCN (as well as
other PXR dependent functions such as OATP2 upregulation
and bile acid excretion; Staudinger et al. 2001). This is consis-
tent with similar observations made in CAR and PPARa
knockout mice and underlines the central coordination of
Vol. 40, No. 7, 2012 LIVER HYPERTROPHY—ADVERSE OR NON-ADVERSE?
hepatocellular proliferation through the nuclear hormone
receptor superfamily in response to a diverse range of
xenobiotics.
Epidemiology and Risk to Man
The risk that PXR activators pose to the development of
liver tumors in man has not been extensively researched. Iso-
lated reports have noted that CPA is hepatotoxic in man (Savi-
dou et al. 2006) but despite its possible mutagenic potential,
long-term follow-up of a group of fifty-three patients revealed
no increase in liver cell carcinomas (Regidor et al. 2000).
Instead, attention has focused on the role that PXR plays in
drug–drug interactions through enhanced PXR-mediated meta-
bolism resulting in either decreased exposure/efficacy or
increased toxicity through metabolic activation (reviewed in
Ma, Idle, and Gonzalez 2008). More recent research has also
highlighted the role of PXR in bile acid homeostasis, PXR-
mediated hepatic steatosis, steroid hormone homeostasis, and
inflammatory bowel disease (Ma, Idle, and Gonzalez 2008).
ARYL HYDROCARBON RECEPTOR
Aryl hydrocarbon receptor (AhR) is a member of the bHLH-
PAS (basic helix-loop-helix/Per-Arnt-Sim) gene family of
transcription factors (reviewed in Schwarz and Appel 2005).
It is activated by polychlorinated dibenzo(p)dioxins (PCDD)
and coplanar polychlorinated biphenyls (PCBs) resulting in
nuclear translocation, heterodimerization with its binding part-
ner Arnt (AhR nuclear translocator), and transcription of target
genes (Landers and Bruce 1991).
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a potent
activator of AhR, and like other AhR receptor agonists, results
in induction of multiple drug-metabolizing enzymes such as
CYP1A1, CYP1A2, CYP2B1, and UGT1A6 (Schwarz and
Appel 2005). In parallel with hepatocyte drug-metabolizing
enzyme induction, there is liver enlargement due to hepatocyte
hypertrophy, multinucleated hepatocytes, fatty change, necro-
sis, and increased cellular replication with suppression of apop-
tosis resulting in the eventual outgrowth of enzyme altered
hepatic foci and hepatocarcinogenesis (Bock and Köhle
2005; Kasai et al. 2009; Maronpot et al. 2010; Yoshizawa
et al. 2007).
Role of AhR
TCDD and other polyhalogenated dibenzodioxins as well as
dibenzofuranes and dioxin-like PCBs are non-genotoxic. Stud-
ies in AhR-receptor-deficient mice have demonstrated that the
acute toxic effects of TCDD are mediated via the AhR receptor
(Fernandez-Salguero et al.1996). Similar work in mice mutated
for the AhR receptor has also demonstrated that promotion of
hepatocarcinogenesis by TCDD is dependent upon sustained
activation of the AhR receptor (Beebe et al. 1995). In line with
this, mice that have constitutively active AhR receptor show
increased promotion and yield of liver tumors following
N-nitrosodiethylamine initiation (Moennikes et al. 2004).
985
Epidemiology and Risk to Man
TCDD was first classified as a human carcinogen in 1997
(IARC 1997) and since then epidemiological studies have
linked dioxin exposure to cancers in man (Ma et al. 2006;
Schwarz and Appel 2005). However, the carcinogenic effect
of TCDD and dioxin-like molecules is weak and there is some
uncertainty as to their true carcinogenic effect (Schwarz and
Appel 2005). This uncertainty is partly due to the binding char-
acteristics of the human AhR receptor which shows approxi-
mately a 10-fold lower affinity to TCDD than laboratory
animal strains (Connor, and Aylward 2006). At this stage,
therefore, it is not possible to definitively define what the
adverse effects or risk is to human health, especially with
regard to hepatocarcinogenesis from exposure to xenobiotics
that induce liver enlargement through this mechanism. This
view is in line with that from a recent AhR Receptor expert
panel workshop that concluded that the mode of action for AhR
mediated carcinogenesis could not be excluded for humans
(Elcombe, pers. comm. 2011).
THE APPLICATION OF NEWER TOXICOGENOMIC TECHNOLOGIES
Advanced technologies such as microarray analysis and
quantitative real-time PCR are now being used in conjunction
with more traditional approaches such as Western blotting,
immunocytochemistry, and electron microscopy to better eval-
uate the toxicological response to xenobiotics (Miyawaki et al.
2011). This has led to the evolution of the ‘‘omics’’ (metabo-
nomics, transcriptomics, and proteomics) technologies from
an observational science that essentially reported lists of differ-
entially expressed genes to one in which Principal Component
Analysis can now be used to better understand the effects of
drug treatment upon biologically relevant signaling networks.
The rodent liver transcriptome has been studied by numer-
ous researchers to gain mechanistic insights into the complex
biological processes involved in drug-induced liver injury,
repair, and hepatocarcinogenesis (Au, Navarro, and Rossi
2011). Combined toxicogenomic investigations now allow
investigators to study early changes in gene expression pro-
files. Since toxicogenomic profiling may precede clinical
chemistry, histopathology, clinical, or even ultrastructural
changes (Heinloth et al. 2007; Wang et al. 2011), it is possible
to gain insight into the early signaling perturbations that
presage toxicologic change (Ruepp et al. 2002).
The activation of nuclear hormone receptors and the induc-
tion of microsomal liver enzymes occur through well-
recognized mechanisms that are amenable to toxicogenomic
profiling. Since the majority of hepatotoxicants induce over-
whelming responses in gene expression related to cell injury,
degeneration, metabolism, DNA repair, and regeneration as the
liver begins the healing process (Gerrish and Malarkey 2007),
it seems likely that a toxicogenomic approach may provide a
useful tool to understand the mechanistic basis of these
changes. The challenge therefore now remains to find the key
genetic and molecular events that link drug induced liver dam-
age and, specifically, drug-induced enzyme induction with
986 HALL ET AL.
FIGURE 8.—Flow-chart diagram demonstrating the NTP strategy of
incorporating predictive hepatic transcriptomic data sets that may be
useful in classifying compounds as non-genotoxic carcinogens after
90 days exposure. Currently, the presence of hepatocyte hypertrophy
and increased liver weights have a low predictive value that will soon
in combination with gene array profiles of genotoxicity and carcino-
genicity make identifying and classifying carcinogenic agents more
sensitive and specific.
hepatocarcinogenesis in rodents and man (Figure 8 and
Table 4). To that end, there have been about a dozen studies
that profile known non-genotoxic or genotoxic carcinogens in
the rat or mouse in order to identify a common toxicogenomic
signature that may be used to classify chemicals with unknown
carcinogenic activity (Auerbach et al. 2010; Ellinger-Ziegelbauer
et al. 2005).
HEPATIC HYPERTROPHY: ADVERSE VERSUS NON-ADVERSE
An adverse change is one ‘‘that . . . affects the performance
of the whole organism’s ability to respond to an additional
environmental challenge’’ (Lewis et al. 2002). Another widely
accepted definition is that an adverse effect is ‘‘a change in
morphology, physiology, growth, reproduction, development,
or life span of an organism which results in impairment of func-
tional capacity or impairment of capacity to compensate for
additional stress or increased susceptibility to the harmful
effects of other environmental influences’’ (WHO/IPCS [Inter-
national Programme on Chemical Safety] 2004).
While such definitions may help summarize the broad cate-
gory of changes that might be considered adverse, it was clear
from the discussions arising from the expert panel that the
operational use of the term adverse in the context of liver
hypertrophy would benefit from some clarification. For exam-
ple, a xenobiotic that induces an increase in liver weight of
150% in a 3-month study might be considered ‘‘adverse’’ in the
context of dose setting for longer term studies but would not be
considered adverse in the context of safety evaluation. In this
situation, the increase in liver weight should be considered the
maximum tolerated dose (MTD) for longer term dose setting
TOXICOLOGIC PATHOLOGY
TABLE 4.—Proposed mechanisms involved in tumorigenesis in rat
liver according to observed gene expression changes.
Genotoxic carcinogens
Direct DNA damage response
Fixation of DNA damage
" of p53, p21
" of MGMT (O-6-methylguanine-DNA methyltransferase)
Regenerative hyperplasia after cytotoxicity
" of genes mediating cell cycle progression (PCNA, cdc2, cyclin B1, mitotic
spindle)
Nongenotoxic carcinogens
Oxidative stress and secondary responses
" Proteasome, " of HSPs , " of ARE target genes
" Cell proliferation, cell survival
# Apoptosis
Directly mitogenic/receptor-mediated
" of survival promoting proteins (GDF15, NRG1, SCYB10,LOC60380,
TIMP, LGALS3)
Source: Ellinger-Ziegelbauer et al. (2005).
(Carmichael et al. 1997; Maronpot and Malarkey 2011). In
addition, while the initial effects of chemicals that induce hepa-
tic metabolism may be regarded as adaptive and noninjurious,
i.e., non-adverse (Greaves 2007; Schulte-Hermann 1974), it is
clear that at higher dose levels, or following prolonged expo-
sure, these adaptive responses can fail leading to degenerative
hepatocellular changes including necrosis with additional
involvement of the biliary systems as compensatory metabolic
systems are overcome or where novel cytotoxic metabolites are
generated (Klaunig et al. 1998; Williams and Iatropoulos
2002). In extreme cases, hepatocyte hypertrophy may lead to
compression of the sinusoidal blood circulation and anoxic
necrosis (Farber 1980). In these circumstances, the use of the
term non-adverse is only valid for the dose and duration of
exposure of that chemical as defined by the study in question.
Furthermore, the use of humanized mice has now shown that
the rodent liver is primed toward proliferation in response to
CAR/PXR/PPARa activation whereas the human liver shows
considerable resistance to this mechanism of hepatocarcino-
genesis. Therefore, the induction of a proliferative or even neo-
plastic response in the rodent liver through enzyme induction
would be considered to have little relevance to man in the con-
text of estimating the risk of human hepatocarcinogenesis.
The development of novel pharmaceuticals for the treatment of
human disease often results in adverse events (AEs) in man. How-
ever, the identification of adverse effects in preclinical toxicity
studies or even in patients does not necessarily preclude the devel-
opment of these drugs into safe and effective treatments. Clearly,
the risk evaluation of new therapeutic drugs requires careful con-
sideration of the cost–benefit analysis. More tolerance to adverse
effects noted in preclinical toxicity studies is given to drugs that
treat serious disease and where the AEs are easily monitorable,
have a good therapeutic margin, or the effect is fully reversible
upon cessation of dosing without permanent loss of function.
Hence one of the underlying considerations when consider-
ing the adverse effects of a new chemical entity is that the
exposure dose and duration in the pivotal laboratory animal
Vol. 40, No. 7, 2012 LIVER HYPERTROPHY—ADVERSE OR NON-ADVERSE?
toxicology studies should equal or, ideally, exceed that in the
intended human population (IHC Guidance for Industry
M3(R2), 2010).
Conversely, agrochemicals are exposed to healthy humans
and therefore the risk-benefit assessment is different to drugs
exposed to patients.
Non-adverse Effects in the Liver for Setting Dose Levels
for Longer Term Studies
The highest dose level used on long-term animal studies
generally requires that it satisfies the regulatory conditions of
achieving a MTD level that is high enough to adequately assess
the toxicity while preventing unnecessarily high dose levels
that would induce chronic physiological dysfunction or reduce
overall survival (OECD Guidance Notes 2002; ICH Guidance
for Industry S1C(R2) 2008).
These requirements therefore dictate that for the purposes of
dose setting in long-term toxicity studies, any change that
results in structural or biochemical alterations indicative of
liver cell damage is likely to result in increased turnover of
hepatocytes, somatic mutation, and liver neoplasia in chronic
2-year rodent bioassays, and therefore exceed the MTD. In
addition, increases in liver weight in short-term experiments
(3 months to 1 year) of approx. 150% (although considered
non-adverse for the purpose of risk assessment) even in the
absence of all other adverse findings is also likely to result in
liver tumors in chronic carcinogencity studies in rats and mice
(Carmichael et al. 1997; Maronpot and Malarkey 2011). There-
fore, a dose level of a xenobiotic that in short-term tests
induced either structural or biochemical evidence of hepatocel-
lular damage, or produced increases in liver weight of approx.
150% would be considered adverse in the context of dose set-
ting and exceed the MTD.
When considering dose levels below the MTD, various for-
mulae are applied but generally the bottom dose level needs to
be a NOEL or NOAEL, as these will be used in estimating risk
assessment for occupational or long-term unintentional
exposure (OECD Draft Guidance Document No. 116). The
dose levels are set on the basis of data derived from shorter
term animal studies and those for the bottom dose level should
either be completely clear of toxicity, induce effects that would
be widely considered to be adaptive (i.e., non-adverse), or in
the case of the pharmaceutical industry, produce manageable
or ‘‘desirable’’ toxicity that is considered a consequence of the
target (primary) pharmacology of the drug.
Non-adverse Effects in the Liver in Terms of the Hepatic
Enzyme–inducing Agents
In the context of liver effects induced by hepatic enzyme–
inducing agents, a dose level at which a non-adverse toxicity
is present would, with every expectation, produce morphologi-
cal changes in the liver characterized by liver weight increases,
microscopic evidence of hepatocellular enlargement/hypertro-
phy, and generally no increases in plasma enzymes such as
alanine and aspartate aminotransferase (Ennulat, Walker,
987
FIGURE 9.—Flow-chart diagram showing a decision tree for adverse
versus non-adverse effects induced by compounds which increase
liver weight. (Modified from Andrew 2005.)
et al. 2010). The exception to this rule is the dog where treat-
ment with phenobarbital or corticosteroids results in induction
of liver ALP and gGT in the absence of liver injury and inde-
pendently of enzyme induction (Ennulat, Walker, et al. 2010).
In terms of the hierarchy of effects on the liver, the general
consensus of the panel discussion was that liver enlargement,
as characterized by increases in liver weight, was the most sen-
sitive indicator of hypertrophy. This change precedes morpho-
logical effects, although measurable increases in mRNA
(indicative of enzyme induction) are likely to occur prior to
increases in liver weight. Considerable discussion occurred
regarding the extent of liver weight increase that could be
expected to be non-adverse in the long term. While empirical
relationships of certain degrees of liver weight increase
appeared to be correlated with the subsequent development
of irreversible toxicity, such as fibrosis, necrosis, vacuoliza-
tion, fatty degeneration, and even neoplasia, it was considered
that a more scientific approach to the problem, taking into
account proposed mode of action analysis, would be of use to
guide the setting of better tolerated dose levels and a more sys-
tematic risk assessment process (Carmichael et al. 2011; Felter
et al. 2011). The general opinion of the group was that liver
weight increase through hepatocyte enzyme induction, in the
absence of histopathologically demonstrated degenerative or
necrotic changes and without significant changes in hepatic-
derived plasma enzymes, would not be considered adverse and
would have little relevance to man in terms of risk assessment
and the development of liver tumors.
ASSESSMENT OF LIVER WEIGHT INCREASES
When assessing a histological change caused by an increase
in liver weight, in order to conclude whether the change is
adverse or not, a number of steps must be carefully considered,
summarized below (and in Figure 9):
988 HALL ET AL.
1. Is there histological evidence of structural degenera-
tive or necrotic changes such as:
 hepatocyte necrosis, fibrosis, inflammation, and
*steatotic vacuolar degeneration
 biliary/oval cell proliferation, degeneration,
fibrosis, and cholestasis
 necrosis and degeneration of other resident cells
within the liver
(*Minimal to mild increases in steatotic macro-vesicular
vacuolation without other changes indicating cellular damage
should be distinguished from micro-vesicular vacuolation and
considered non-adverse since this is a common change induced
by feeding high-fat diets.)
(Of note, transient increases in proliferative indices together
with changes in hepatocyte ploidy, if induced through CAR/
PXR/PPARa activation, is likely to be a rodent-specific
phenomenon and therefore of little relevance to man even if
this results in altered hepatic foci and/or primary liver tumors
in chronic studies.)
2. In the absence of histological changes, using a
weight-of-evidence approach, is there clinical
pathology evidence of hepatocyte damage character-
ized by a dose dependent and biologically significant
and consistent increase in at least two liver
parameters:
 at least 2 to 3 increase in ALT (EMEA 2010,
FDA 2009; HED Guidance Document 2002) or
 a biologically significant change in other bio-
markers of hepatobiliary damage (ALP, AST,
gGT, GLDH, etc.)
 a biologically significant change in another clin-
ical pathology marker indicating liver dysfunc-
tion (albumin, bilirubin, bile acids, coagulation
factors, cholesterol, triglycerides etc.).
Clinical pathology changes should corroborate each other, be
consistent with the expected species-specific patterns of
change resulting from hepatobiliary injury, and take into
account target pharmacology that may non-adversely alter one
or more of these biomarkers. It should also be noted that statis-
tical significance alone is not a reliable indicator of hepatic
toxicity (HED guidance document 2002).
If the above mentioned adverse criteria are not observed,
then increases in liver organ weight and liver cell hypertrophy
due to enzyme induction can be considered as an adaptive
response to a xenobiotic and of little relevance to man.
However, prolonged exposure to a xenobiotic at levels that
have previously been shown to be adaptive may eventually
result in liver cell injury due to a failure of adaptive mechan-
isms. In this case, the combination of dose level and duration
of exposure to the xenobiotic under the terms and conditions
of the new experiment would now be considered adverse.
TOXICOLOGIC PATHOLOGY
SUMMARY AND CONCLUSIONS
The purpose of the 3rd International ESTP workshop was to
discuss the significance of hepatocellular hypertrophy in
rodents and define more clearly which responses were consid-
ered to be adaptive in nature so as to help guide scientific opin-
ion. The consensus of opinion was that hepatomegaly as a
consequence of hepatocellular hypertrophy without histologic
or clinical pathology alterations indicative of liver toxicity was
considered to be an adaptive non-adverse change. This conclu-
sion should normally be reached by an integrative weight of
evidence approach. Under these circumstances, hepatocellular
hypertrophy through enzyme induction may therefore be
considered a fully reversible change that does not compromise
viability or functional integrity and potentially benefits the
organism through increased metabolic capability.
During the assessment of increased liver weight due to
enzyme induction, one should consider for long-term dose
setting that non-adverse adaptive changes at low exposures
may very well become adverse at higher/longer dosages. In
addition, increases in liver organ weight 150% in 3-month
to 1-year studies may very well lead to hepatocarcinogenesis
and/or endocrine tumors (though increased degradation of thyr-
oid and possibly reproductive hormones) in longer term studies
and appropriate measures should be put in place to ensure ade-
quate survival of the top dose animals on the study.
Finally, the development of newer technologies and the use
of genetically modified mice have considerably advanced our
understanding of the underlying mechanisms involved in
nuclear hormone receptor (CAR, PXR, and PPARa) induced
hepatocellular hypertrophy. The imminent generation of triple
humanized mice are likely to further increase our understand-
ing of the response of the humanized receptor to these types
of activators by removing the potentially confounding effects
of ‘‘off-target’’ nuclear hormone receptor signaling by ‘‘pro-
miscuous’’ activators and thus increase the confidence that
hepatocarcinogenesis in response to these types of compounds
is a rodent-specific phenomenon.
AUTHORS’ NOTE
Workshop Participants—Clifford Elcombe, CXRBios-
ciences, Great Britain, BSTP; John Foster, AstraZeneca, Great
Britain, BSTP; Peter Hall, AstraZeneca, Great Britain, BSTP;
Takanori Harada, IET (Institute of Environmental Toxicology),
Japan, JSTP; Wolfgang Kaufmann, Merck KGaA, Germany,
ESTP (organizer of the 3rd International ESTP expert work-
shop); Anja Knippel, Merck KGaA, Germany, ESTP; Karin
Küttler, BASF SE, Germany, ESTP; David E Malarkey,
NIEHS (National Institute for Environmental Health Science),
USA, STP; Robert R. Maronpot, Consultant, USA, STP;
Akiyoshi Nishikawa, NIHS (National Institute of Health
Sciences), Japan, JSTP; Thomas Nolte, Boehringer Ingelheim
Pharma GmbH & Co. KG, Germany, ESTP; Agnes Schulte,
BfR (German Federal Institute for Risk Assessment),
Germany, ESTP; Volker Strauss, BASF SE, Germany,
ESVCP/ESTP; Malcolm York, GSK, Great Britain, ESVCP.
Vol. 40, No. 7, 2012 LIVER HYPERTROPHY—ADVERSE OR NON-ADVERSE?
ACKNOWLEDGMENTS
The authors wish to thank ESTP for organizing the workshop
and MerckSerono for giving logistical support at the venue.
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