Invited Review

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:
[email protected].
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. TOXICOLOGIC PATHOLOGY
INTRODUCTION TABLE 1.—Dose response relationship for liver weight increase for the
PPARa agonist, Fenofibrate (adapted from Price et al. 1986).
Drug and chemically induced liver enlargement in subchro-
nic and chronic toxicology studies in rodents has, for many Dose (mg/kg/day) Liver weight (% of control)
years, taxed the toxicology profession in terms of its perceived
0 100
relevance to hepatotoxicity, to carcinogenicity in lifetime
13 125
bioassays at similar dose levels, and in terms of its relevance 60 157
to man (Cohen and Grasso 1981; Elcombe, Rose, and Pratt 200 187
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- Liver Weight Increases
tious as ever prompted the European Society of Toxicologic
Increases in liver weight in rodents due to exposure to
Pathology (ESTP) to convene an expert opinion group to
chemicals can be achieved through a number of mechanisms
discuss the current state of the science. The purpose of the
and can be accompanied by a range of differing histological
workshop was to discuss the significance of hepatocellular
appearances, some of which clearly show cytotoxicity and cell
hypertrophy in rodents, define more clearly when adaptive
death, such as carbon tetrachloride and chloroform (Edwards
responses become adverse, and understand the long-term
and Dalton 1942; Eschenbrenner and Miller 1945; Grasso and
consequences of hepatocellular hypertrophy in order to guide
Hinton 1991; Reuber and Glover 1968), while other chemicals
scientific opinion for risk assessment in man and dose setting
such as sodium phenobarbitone, the PPARa (peroxisome acti-
for longer term animal studies.
vated receptor alpha) agonists, Nafenopin and Clofibrate, and
One critical aspect of hepatic hypertrophy which continues
trichloroethylene, induce an increase in liver weight without
to pose serious questions for toxicologists is the definition of
overt cell necrosis (Lake et al. 1989; Mitchell et al. 1985; Price
what constitutes an adverse hepatic effect, versus a non-
et al. 1986). While there is an undoubted relationship between
adverse or adaptive effect. This was a major focus of discussion
hepatic necrosis and its consequences on the eventual increased
by the expert opinion group resulting in a consensus opinion
incidence of neoplasia of the liver (Eschenbrenner and Miller
detailed in this article.
1945; Grasso and Hinton 1991), this is less clear for chemicals,
or rather for doses of chemicals, that induce liver weight
HEPATIC HYPERTROPHY—WHAT IS IT?
increases in the absence of overt hepatocellular damage. The
While to a histopathologist the term hepatic hypertrophy is focus of the workshop was on those chemicals that are not tra-
well recognized and readily defined histologically, to a toxicol- ditionally associated with acute hepatic necrosis but rather
ogist, the term can have various connotations including an exert their effect by increasing the weight of the liver through
increase in the weight of the organ (liver hypertrophy), an other means.
increase in the average size of the hepatocytes (hepatocellular Organ weight data can provide sensitive indices of toxicolo-
hypertrophy), and even hepatic enzyme induction (functionally gic change where it can correlate and confirm changes seen
sometimes referred to as ‘‘work’’ hypertrophy). down the microscope. To this end, the STP have recently pub-
In practice, drug- or chemical-induced hepatic hypertrophy lished a position paper (Sellers et al. 2007) that gives careful
tends to be a combination of all of these parameters in addition guidance to the collection and weighing of organs in routine
to others including profound changes in the intracellular toxicology studies. The authors recommend that organ weight
enzymes involved not only in phase 1 and 2 drug metabolism data should be routinely expressed as an organ-to-body weight
(Crampton et al. 1977b; Lake, Longland, et al. 1976; Maronpot ratio to avoid large variations in body weight skewing organ
et al. 2010) but also in other more fundamental cell processes weight interpretation. In this way, increases in liver organ
such as altered oxidative status, fatty acid metabolism, energy weight can be accurately correlated with hepatocellular hyper-
production and utilization, cell turnover and altered hepatocel- trophy. In rodent studies, where routinely larger numbers of
lular cytoplasmic, and nuclear morphology (Cattley and Popp animals are used, these data can be compared with concurrent
1989; Crampton et al. 1977b; Grasso et al. 1974; Grasso and controls and statistically interrogated to derive p values that
Hinton 1991). may increase confidence through weight of evidence to allow
Archetypical changes that often accompany this phenom- a judgment of hepatocellular hypertrophy when the histological
enon are increases in hepatic-derived enzymes (transaminases, changes appear to be relatively small.
alkaline phosphatase, and g-glutamyltransferase) that may This is particularly important since the magnitude of the
appear in the plasma following liver enlargement (Ennulat, increased liver weight can vary considerably between different
Magid-Slav, et al. 2010). Of prime importance for interpreta- chemicals. Liver weight changes may also demonstrate a clear
tion of these changes with regard to risk assessment are the sig- dose relationship (Table 1; typically between 110% and 150%
nificant species differences shown in response to chemicals of control liver weight). However, it should be noted that
that induce a classic hypertrophic response in the rodent liver although liver weight increases are correlated with microsomal
(Lake 1995; Lake, Brantom, et al. 1976; Rhodes et al. 1986; enzyme induction, the degree of microsomal enzyme induction
Williams and Perrone 1996). 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 preference to other tissues of the body) and accounts for the
in rats, dogs, or monkeys (Amacher, Schomaker, and loss of liver weight seen under fasting conditions (Rothacker
Burkhardt 1998, 2001; Amacher et al. 2006; Ennulat, Walker, et al. 1988). It is currently unclear as to what percentage of
et al. 2010). laboratories utilizes fasting, but what was apparent from the
Increases in liver weights have been shown to be associated workshop was that there exists considerable inter-laboratory
with the induction of increased incidences of hepatocellular variation in the practice of overnight fasting of animals prior
neoplasia in 2-year carcinogenicity studies in rodents (Allen to necropsy.
et al. 2004; Carmichael et al. 1997) and while considered to Welfare reasons prohibit the overnight fasting of pregnant ani-
be of little relevance to man for some chemicals (Butler and mals but a systematic study looking into the differences between
Newberne 1975; Ito and Sugano 1991; McClain et al. 1995; fasted and non-fasted animals would seem a worthwhile exercise
Stevenson et al. 1990), dose levels of a chemical inducing this if the former leads to better discrimination of effects provided ani-
degree of liver weight increase would be considered to carry an mal welfare guidelines permit this (Animal and Plant Health
increased risk of inducing hepatic neoplasms in these types of Inspection Services [APHIS] 1997; Animal Welfare Information
study. In a survey of 138 chemicals used in the agrochemical Center 2005). However, it is clear that overnight fasting does have
industry, a relative increase in liver weight of 150% of con- some distinct advantages. First, overnight fasting decreases the var-
trol values after 1 year of treatment was positively correlated iation seen in some clinical pathology parameters and therefore
with the induction of liver tumors in mice (Carmichael et al. increases the probability of identifying statistically significant
1997). Similarly, in another survey of mouse NTP studies changes between control and treated animals (Matsuzawa and
where correlations between liver weight increases and histolo- Sakazume 1994). Second, animals exposed to high levels of a
gical parameters and carcinogenesis were assessed, the authors xenobiotic very frequently experience decreased food intake due
concluded that ‘‘the best single predictor of liver cancer in mice to inanition/toxicity which is often noted in toxicological studies
was hepatocellular hypertrophy’’ (Allen et al. 2004). This study as a treatment, and dose related, decrease in hepatocellular gly-
demonstrates a highly significant relationship between cogen storage (Agren, Wilander, and Jorpes 1931; Lockard et al.
increases in liver weight and the future outcome of hepatic neo- 1983). It is possible therefore that loss of glycogen in animals
plasia (p < .001; Table 2). In a similar review of the rat, a less exposed to a xenobiotic might mask statistically significant
statistically significant relationship (p ¼ .018) between liver increases in liver weight. Since liver weight increases are gener-
weight and hepatocarcinogenesis was also noted, whereby liver ally considered a sensitive measure of hepatocellular hypertro-
weight increases alone correctly predicted eight of the eleven phy, small changes in these parameters may not be visible
liver carcinogens (but overpredicted twenty-six false positives using H&E stained sections. This is especially so if animals are
and failed to predict three true positives; Allen et al. 2004). 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-
Effect of Fasting on Liver Weights
nosis of hypertrophy when comparing treated with concurrent
A potentially important confounder in terms of the evalua- control animals (Li et al. 2003). However, there are clearly
tion of liver weight changes following chemical treatment is well-described changes in gene expression in fasted animals
the practice of fasting animals overnight prior to sacrifice. It (Bauer et al. 2004; Lkhagvadorj et al. 2009) and further work
is likely that fasting alters the resulting organ weights in to assess the relative merits of overnight fasting for subsequent
rodents over those not fasted (Chatamra, Daniel, and Lam histopathology evaluation would help assess the importance or
1984; Rothacker et al. 1988). Studies have shown that the otherwise of this effect.
rodent liver weight increases in fed animals are maintained for The practice of fasting rodents before necropsy was one that
up to 8 hr after feeding. Water and glycogen account for the was debated at the workshop but because of the differing
major portion of this increase with the former comprising over experiences of the group a recommendation was made for a
66% of the increase (Leveille and Chakrabarty 1967). Over- comparison dose–response study with a known hepatotrophic
night fasting will rapidly deplete both components (in agent, such as sodium phenobarbitone, in the rat where
groups would be fasted and the results obtained compared to increases in humans (30- to 100-fold of the upper limit of nor-
non-fasted groups. The NOEL for the histopathology of mal) can also be accompanied by full recovery of hepatic func-
hepatocellular hypertrophy would then be compared between tion (Koch et al. 1997).
the fasted and non-fasted groups. Alternatively, small decreases in serum ALT might also be
associated with liver enzyme induction. In a rat study, where
CLINICAL PATHOLOGY sodium phenobarbitone was given at dose levels of 80 mg/kg,
and b-naphthoflavone (a CYP1A inducer) was given at 100
The following clinical pathology parameters can help in the
mg/kg, there were no recorded elevations in serum transami-
assessment of adverse effects on the liver evolving during
nases, but instead (statistically insignificant) decreases in ALT
enzyme induction:
or AST activities were recorded (Arvela, Reinila, and Pelkonen
1981). In these experiments, the only histopathology recorded
Alanine Aminotransferase and Aspartate
was centrilobular hepatocellular hypertrophy. Small decreases
Aminotransferase (ALT/AST)
in ALT/AST activity may usually be regarded as a ‘‘non-
Several studies in rats dosed with liver enzyme–inducing adverse’’ effect because they cannot be correlated with a toxico-
compounds have been published. In these studies ALT and AST logically relevant finding. Large changes (>50%), however, may
activity increases were below twofold of the controls: sodium result not only from induction of hepatic drug metabolism but
phenobarbitone (an archetypical mixed CYP 2B/3A inducer) from deficiencies in pyridoxal 50 -phosphate. This may be con-
given to rats at dose levels where centrilobular hypertrophy firmed or excluded by measuring enzyme activities with and
was present without accompanying degenerative changes without pyridoxal-phosphate addition as a cofactor to the reac-
induced small increases in both hepatic ALT (Boll et al. tion mixture (PSD Guidance Document 2007).
1998) and serum ALT and AST (Lake and Evans 1993).
In contrast, administration of the CYP 1A inducer,
Alkaline Phosphatase (ALP)
3-methylcholanthrene to rats, at dose levels that induced sig-
nificant levels of drug-metabolizing enzymes, failed to show Increases in ALP activity associated with hepatic microso-
any increase in serum ALT or AST (Lake and Evans 1993). mal enzyme induction, in the absence of accompanying degen-
Peroxisome proliferators (PPARa agonists and CYP4A indu- erative histopathological findings, have been reported in dog
cers), similarly show minimally increased levels of serum by a number of workers (Conning and Litchfield. 1971; Leeling
ALT and AST even when given at dose levels that induce et al. 1975; Robertson et al. 1993). These studies correlated an
significant increases in liver weight, provided that the only increase in ALP activity with increased microsomal enzyme
histological effect seen in the liver is hepatocellular hypertro- activity and demonstrated that the source of the ALP increase
phy (Eacho et al. 1985; Huang and Shaw 2002; Kramer et al. was of hepatic origin in the absence of histologically detectable
2003; Peterson et al. 2004). hepatobiliary injury. Increases in total serum ALP activity up to
Increases in serum ALT activity without increases in hepa- approximately 2.5-fold (Leeling et al. 1975), 3-fold (Conning
tic ALT activity are thought to be due to damage to, and leak- and Litchfield 1971), and 5-fold (Robertson et al. 1993) were
age from, hepatocyte cell membranes, resulting in a release of observed during the course of these studies, which were noted
enzymes from the cytosol into the blood (Amacher 1998). to recover after an 8-day treatment-free period in concordance
Alternatively, increased enzyme synthesis, as a consequence with a reduction in hepatic microsomal enzyme activity (Litch-
of liver enzyme induction, may also lead to higher ALT levels field and Conning 1972). Additionally, the ALP change reported
in both the liver and the serum. In this scenario, moderately by Robertson et al. (1993) was not associated with any other
higher serum ALT levels, especially in mice treated with liver changes in clinical pathology parameters. Leeling et al.
enzyme inducers, might occur without cell membrane damage (1975) concluded that marked changes in ALP levels during
(Strauss, pers. comm. 2011). Boone et al. (1985) concluded that drug treatment should not automatically be assumed to have
increases in serum ALT activity in the range of 2 to 4 or toxicological implications.
higher in individual or group mean data when compared with During the current ESTP-sponsored workshop, case study
concurrent controls should raise concern as an indicator of material was presented where a dose-related increase in plasma
potential hepatic injury unless a clear alternative explanation ALP activity (up to 10-fold) was shown to be of hepatic origin
is found. Based on the recommendations of regulatory author- in the dog and followed for up to 52 weeks. Increases in ALP
ities, (EMEA 2010; FDA 2009; HED 2002) increases in ALT activity were evident at 4 weeks (with maximal comparable
activity of two- to threefold should be considered as indicative levels observed at 13, 26, and 52 weeks). ALP activity corre-
of hepatocellular damage. Distinguishing irreversible and lated with hepatocyte hypertrophy and increases in liver weight
reversible liver injury is pragmatically dealt with by assessing up to 1.5-fold of control values. One individual animal showed
the magnitude of the transaminase elevation where minimal evidence of hepatocellular degeneration and atrophy at week
and reversible hepatic injury is commonly accompanied by 52, but this change was also associated with moderate increases
small increases in transaminase levels of less than twofold in AST (5-fold), ALT (2-fold), and GLDH (glutamate dehydro-
(Kramer et al. 2003; Lassen 2004; Peterson et al. 2004; Satoh genase; 15-fold). Therefore, it is considered that increases in
et al. 1982; Solter 2005). However, considerably larger ALT circulating ALP activity in the dog, with associated increased
liver weight and histological hepatocellular hypertrophy but normally not accompanied by histopathological evidence of
without hepatocellular degeneration could be interpreted as hepatic degeneration (Neely et al. 2010). When accompanied
an adaptive, rather than an adverse response to chemical by increased serum bile acids, increased bilirubin is a reliable
exposure. indicator of hepatic toxicity and loss of hepatic function (Levin
and Schwartz 1965).
g-Glutamyltransferase (gGT)
Induction of hepatocyte gGT has also been reported with Other Clinical Pathology Parameters
several chemicals, including sodium phenobarbitone which is
Global coagulation tests, such as prothrombin time and acti-
known to cause liver weight increases and induce hepatic meta-
vated partial thromboplastin time, can be shortened by drugs/
bolism as well as various CYPs (Ratanasavanh et al. 1979). In
chemicals that induce hepatic drug metabolism due to
case studies presented at the workshop, messenger RNA
increased synthesis of coagulation factors by liver cells
(mRNA) for hepatic gGT was shown to be induced in a number
(Niemegeers et al. 1981; Poulsen, Lerche, and Pedersen
of repeat dose (7 days) oral toxicity studies in the rat where
1985). Triglyceride levels can be decreased or increased during
centrilobular hypertrophy, increased liver weights, increased
enzyme induction depending on the administered compound
CYPs (predominantly 2B2 and 3A3), and phase 2 enzymes
(Amacher, Schomaker, and Burkhardt 1998). In the experience
activities (predominantly GSTA3 and UGT1A6) were
of the authors, administration of enzyme inducers may also
observed. However, this only translated into increases in circu-
alter protein levels—the change often being correlated with
lating gGT activity where marked induction (>50-fold) of
changes in lipid levels (Strauss, pers. comm. 2011).
hepatic gGT mRNA was observed. In rats treated with pheno-
In addition to the parameters discussed above, there are
barbital for 5 days, liver but not serum gGT activity was
several other clinical pathology changes that reflect alterations
increased, indicating the relative insensitivity of serum-
in the functional/anatomical status of the liver. In addition to
derived gGT as a marker of hepatobiliary change in laboratory
cholesterol, glucose, urea, and ammonia, many enzymes can
animals compared to measuring hepatic gGT (Goldberg et al.
be measured that are localized in different intracellular sites
1981). In a reported study where kava kava was administered
within the hepatocyte. Enzymes such as lactate dehydrogenase
to rats for 14 weeks, statistically significant increases in serum
(LDH), sorbitol dehydrogenase (SDH), and a-glutathione
gGT were observed in both males and particularly so in
S-transferase (aGST) are located in the cytosol; malate dehy-
females, which accompanied hepatocellular hypertrophy, liver
drogenase (MDH) is in the cytosol and mitochondria; ornithine
weight increases, and significant induction of CYPs (Clayton
carbamoyl transferase (OCT) and GLDH are in the mitochon-
et al. 2007). The authors concluded that the changes seen in the
dria and pseudocholinesterase (CHE) is in vesicles. These
liver, including the increases in serum gGT, were adaptive in
enzymes are not measured routinely, but they may be useful
nature.
on occasion to substitute for other more commonly used
However, cholestasis due to partial obstruction of intrahepa-
enzymes or to provide additional information—for example,
tic bile ducts may also result in similar changes in gGT enzyme
SDH may be used in the guinea pig because of its greater liver
activities. The decision therefore to ascribe increases in gGT to
specificity (Clampitt and Hart 1978). Additionally, GLDH and
enzyme induction should be made on a weight-of-evidence
OCT may be measured to estimate the grade of liver cell
approach with full consideration of increases in liver weight,
damage, while CHE can be used as an additional marker of the
the absence of biologically significant increases in transami-
synthetic capacity of the liver (Giffen et al. 2002; Kutty and
nase activity, as well as the absence of adverse histopathologi-
Payne 1994; Litchfield and Gartland 1974; O’Brien et al.
cal changes.
2002).
The measurement of more specific biomarkers of hepatic
Bilirubin/Bile Acids
damage in liver tissue, serum, or urine may help distinguish
Total bilirubin, as well as bile acid levels, can be reduced as adaptive enzyme induction from adverse liver cell toxicity.
a consequence of higher conjugation rates and excretion via Studies measuring transcriptome profiles in liver tissue
bile when hepatic enzyme–inducing chemicals are adminis- (Ellinger-Ziegelbauer et al. 2011; Kramer et al. 2003; Peterson
tered (Boiteux-antoine et al. 1989; Echchgadda et al. 2004; et al. 2004) as well as metabolome patterns in plasma (van
Gógl et al. 1979). An increase in plasma bilirubin levels is gen- Ravenzwaay et al. 2010) and urine (Robertson et al. 2000) are
erally not seen following liver enzyme induction, but is usually aimed at fulfilling this aspiration.
indicative of impaired hepatic bile flow, accelerated red blood If there are no accompanying histopathological changes,
cell destruction, or decreased bilirubin metabolism (Boone et then the default assumption is that any consistent, and signifi-
al. 2005; Jonker, Liddleb, and Downes 2011). Additionally, cant, adverse change in clinical pathology parameters can be
competitive inhibition of bilirubin conjugation by drugs (such regarded as an adverse effect with relevance to human health.
as Atazanavir that inhibits UDP-glucuronyltransferase isoen- However, small clinical chemistry changes beyond historical
zyme, 1A1) may lead to a dose-related, asymptomatic, uncon- control ranges can be regarded as adaptive, if sufficient evi-
jugated hyperbilirubinemia in humans and in rats (Neely et al. dence exists that these effects are due to altered metabolism,
2010; Zucker et al. 2001), which is clearly non-adverse and if the change by its own is doubtful or of minimal toxicological
importance, and the finding alone is not predictive for an enzyme inducers are administered without any histopathologi-
adverse liver effect (Regulation EC No. 1772/2008, Annex 1, cal indication of liver cell toxicity (Strauss, pers. comm. 2011).
chapter 3.9)—for example—decreases in total bilirubin levels When comparing the above with older published studies, it
without any sign of hypoproliferative anemia, accompanied is apparent that different methods can lead to widely different
by decreases in total bile acid concentrations, may be consid- results. There is general agreement that ALT and AST activities
ered adaptive provided that the synthetic pathway for these should be measured according to The International Federation
molecules remains unaffected (Kuipers et al. 1989) since these of Clinical Chemistry and Laboratory Medicine (IFCC) methods
changes may be due to an increase in the conjugation rate and without addition of the cofactor, pyridoxal 50 -phosphate. If
excretion via bile. Other confounding examples include reduc- decreased transaminase activities are observed, a repetition with
tions in global coagulation tests, which result from increased addition of the cofactor should be performed in order to exclude/
coagulation factor synthesis as a consequence of enzyme confirm alternative reasons for the decreased activity other than
induction (Poulsen, Lerche, and Pedersen 1985) but, alterna- enzyme induction (Evans and Whitehorn 1995).
tively, could result from an acute phase reaction or In addition, one should be aware that commercial gGT kits
thrombophilia. for human medicine have a limit of detection of about 3 U/L (at
A general rule of thumb is that changes affecting a single 37 C, i.e., 50 nkat/L, e.g., Roche instruction manual gGT Szasz
clinical chemistry parameter beyond historical control values mod., 2010-06, V7). Normal rat serum gGT activities are gen-
can normally be regarded as ‘‘non-adverse’’ when no histo- erally below this limit of detection, so that moderate gGT
pathological correlate can be found (ECETOC Technical increases cannot be measured with these tests. Also, total
Report No. 85, 2002). 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
Pitfalls When Interpreting Clinical Pathology
where decreases in bilirubin occur, unless reagents and proce-
Parameters
dures are appropriately modified.
Different results in studies with liver enzyme–inducing
compounds administered to various animal species as well as
Serum Enzyme Activity Elevation in Human Studies
literature studies of different facilities have to be cautiously
interpreted. Hepatic enzyme inducers, such as sodium phenobarbitone,
Although serum alanine aminotransferase (ALT) activity in have been reported to increase the serum enzyme levels in
the rat, mouse, dog, and humans mainly originates from liver human patient populations, and elevations in ALT, AST, and
tissue, species-specific differences exist regarding plasma gGT were seen in patients following treatment with several
half-life of the enzyme. In humans, the half-life of ALT in the anticonvulsant drugs (Aiges et al. 1980; Wall et al. 1992). In
plasma is 47 hr, in dogs it is reported to be 17 hr, while in rats it a study of epileptic patients receiving drug therapy, elevations
is approximately 3 to 4 hr (Boyd 1983). Depending on the in gGT were seen in almost all individuals leading the authors
enzyme kinetic ALT activity increases may be detectable in of the study to conclude that this did not indicate hepatic dam-
one species but not in the other in repeated dose studies. age, since none of the patients exhibited clinical symptoms.
g-glutamyltranspeptidase (gGT) and alkaline phosphatase Instead, these elevations in enzymes merely confirmed that the
(ALP) have different tissue distributions in the rat, mouse, and patients were receiving the drugs (Hirayanagi, Fujii, and
dog (Clampitt and Hart 1978; Keller 1981). For example, the Teshirogi 1991). Elevated gGT levels, in the absence of other
hepatic isoform is the main contribution to total circulating markers of hepatic damage, were reported to be commonly ele-
ALP activity in the dog, but has comparatively lower activity vated in patients taking rifampicin (Davis 1989), carbamaze-
in the rat and hence has lower diagnostic sensitivity in this pine, zarontin, phenobarbital, and phenytoin (Knight 2005;
species. Moreover, in young animals that are generally used Ohta and Toda 2001; Rosalki, Tarlow, and Rau 1971; Vanden-
in regulatory toxicology studies, serum ALP activity derives berghe 1996; Whitfield et al. 1973). Much of this elevated
mainly from the bone isoenzyme and not from the liver enzyme activity was considered due to hepatic induction of the
(Hoffmann et al. 1994). Therefore, in addition to liver enzyme enzyme, and its subsequent loss into the blood, rather than an
induction, changes in other organs may need to be assessed. indicator of frank toxicity to the liver (Ohta and Toda 2001).
Induction of the aforementioned enzymes by chemicals also Finally, anti-epileptic drugs in man have also been reported
appears to be species-specific: in dogs, corticoid-induced ALP to increase serum ALT up to 3-fold the upper limit of normality
isoenzyme activities are often increased (Gaskill et al. 2005), (ULN), and AST up to 2-fold the ULN in approximately 25%
whereas in mice, hepatic ALP is increased (Kawasaki, Mataki, of the patient cohort studied (Haidukewych and John 1986).
and.Takano 1994). In rats, hepatic gGT is induced in response Once again these were not considered to be indicative of hepa-
to liver enzyme inducers (Gallagher et al. 1998; Satoh et al. totoxicity but to be a consequence of hepatic enzyme induction
1982). In pregnant rats from gestation day 6 (GD6) until and hepatocellular hypertrophy. Indeed, liver biopsies taken
GD20, ALP, instead of gGT activity is increased when enzyme from patients on long-term therapy failed to show any histolo-
inducers are administered (Strauss, pers. comm. 2011). In mice, gical evidence of hepatotoxicity (Jacobsen et al. 1976). How-
in contrast to rats, ALT is more often increased when hepatic ever despite this, it should be noted that there is a correlation
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 storage of metabolism products, particles, or cleavage prod-
idiosyncratic hepatotoxicity in man (Li 2002). 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,
HISTOLOGICAL ASSESSMENT
necrosis, or degeneration, it is important to recognize that
The first indication of hepatomegaly is usually noted at increases in liver weight may be induced by hypertrophy,
necropsy by the measurement of an increase in organ weight, hyperplasia, or very frequently a combination of the two
and in extreme cases, by the concomitant visual observation (Maronpot et al. 2010).
of an increase in liver size (volume; Figure 1). In small animals An increase in liver cell size can result from accumula-
where experiments can be powered to gather statistically sig- tion of glycogen, lipid, and water (hydropic degeneration)
nificant data, the assessment of an increase in liver weight is or the proliferation of subcellular organelles (typically
relatively straightforward. However, in dogs and other larger smooth endoplasmic reticulum [sER] and/or peroxisomes).
animals, interindividual variation combined with smaller group Experiments in which hepatocellular hypertrophy was
sizes can make the assessment of meaningful increases in liver induced in mice with phenobarbital suggest that polyploi-
organ weight more uncertain. dization is an additional and inevitable component of liver
Clinical pathology can provide valuable ancillary data to cell hypertrophy caused by these types of drugs (possibly
indicate alterations in liver function; however, the diagnosis as an adaptive response to increased metabolic demands
of morphological or structural changes in the liver is ulti- —RNA and protein synthesis; Böhm and Noltemeyer
mately made by a trained pathologist assessing formalin fixed, 1981; Bursch et al. 2004). Morphologically in both rats
paraffin embedded, haematoxylin and eosin (H&E) stained and mice, hepatocellular hypertrophy may present as a dif-
sections. To this end, the National Toxicology Program (NTP) fuse change affecting all zones of the liver lobule, or a
for 2-year rat carcinogenicity studies advocates two liver sam- regional change usually affecting the centrilobular region,
ples taken at the widest part of the left lobe and right median but frequently extending to the mid-zonal region (and fur-
lobes (Maronpot et al. 1989; NTP 2006). The RITA trimming ther) with increasing exposure (Figure 2). Occasionally,
guide recommends to sample in addition the caudate lobe in periportal hypertrophy without evidence of centrilobular
rats and mice (Ruehl-Fehlert et al. 2003). hypertrophy is also seen—for example, both fenofibrate
Treatment-related increases in liver weight may result from and ciprofibrate induced periportal hypertrophy in the
a wide variety of causes such as hyperplasia (of any of the resi- cynomologus monkey as a result of both peroxisomal and
dent cell types), hypertrophy, inflammation, fibrosis, abnormal mitochondrial proliferation (Hoivik et al. 2004).
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
FIGURE 2.—(a) H&E stained low power photomicrograph of a male C57Bl mouse liver (right panel, 2c) on a 4-week feeding study show-
B6C3F1 mouse liver treated with 100 mg/kg/day of pentabromodiphe- ing diffuse macrovesicular fatty vacuolation (control) and diffuse
nyl oxide for 90 days showing centrilobular hepatocyte hypertrophy hepatocyte hypertrophy (treated) characterized by a diffuse enlarge-
with characteristic enlargement of hepatocytes noted by the decreased ment of hepatocytes. Note that diffuse changes tend to be more subtle
number of nuclei per unit area and pale staining centrilobular eosino- in appearance compared to regional changes and require careful com-
philic cytoplasm. (b and c). H&E stained low power photomicrograph parison to concurrent control samples and correlation with liver organ
of a control male C57Bl mouse liver (left panel, 2b) and a treated weight data to accurately identify.
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
FIGURE 3.—H&E stained high-power photomicrograph of a male
3A1, and CYP2B1 3A2) by Taqman quantitative RT-PCR.
B6C3F1 mouse liver chronically (2-year carcinogenicity study) treated
with chlordane showing the cellular features of extreme hypertrophy These data illustrate the utility of routine histological assess-
characterized by a notable increase in cellular size accompanied by ment, IHC and qRT-PCR to assess the nature and extent of
binucleate hepatocytes with enlarged hepatocyte nuclei (polyploidy). drug-metabolizing enzyme induction to help demonstrate the
adaptive nature of the toxicologic changes induced by
knowledge of ligand–receptor binding interactions. For exam- RO2910. Induction of hepatocyte drug-metabolizing enzymes
ple, in silico modeling of PXR–ligand crystal structure com- in this case resulted in increased turnover of T4 (thyroxine) and
plexes have elucidated the molecular basis for PXR receptor secondary thyroid hypertrophy/hyperplasia due to stimulation
promiscuity allowing it to interact with such diverse ligand of the pituitary–thyroid–endocrine axis. An ELISA method for
binding partners as phenobarbital and rifampicin (Watkins et total thyroxine (T4), total tri-iodothryonine (T3), and thyroid
al. 2001). However, these methods, although still widely used stimulating hormone (TSH) confirmed the increase in TSH and
and of general applicability, possess significant limitations— decrease in T4 (with no change in T3).
for example—immortalized cell lines tend to express the major
CYPs at lower levels than fresh hepatocyte preparations MECHANISMS OF HYPERTROPHY
(Fretland and Monshouwer 2010).
The use of newer techniques and, in particular, the use of Nuclear Hormone Receptors
cryopreserved human hepatocytes can now provide more The nuclear hormone receptors constitute a superfamily of
reliable data to build confidence for human risk assessment and xenosensing receptors that specifically interact with endogenous
to predict the effects of novel chemicals on drug-metabolizing and exogenous chemicals. Receptor–ligand binding results in the
enzyme induction in man (Fretland and Monshouwer 2010). activation of a battery of genes mediating oxidative drug metabo-
This type of information can also be supplemented by experi- lism, conjugation, and transport, which serves to eliminate the
ments generated in laboratory species including nonhuman pri- xenobiotic from the organism to maintain homeostasis. The
mates that show considerable sequence homology in their majority of these nuclear receptors are expressed on the liver
nuclear hormone receptors—for example, the ligand binding (Bookout et al. 2006) and can be functionally divided into those
domain of PXR in the rhesus monkey possesses 96% sequence that are generally associated with drug metabolism—the preg-
identity with the human receptor at the amino acid level nane X receptor (PXR) and the constitutive androstane receptor
(Moore et al. 2002). More recently, with the advent of huma- (CAR)—and those that are involved in the metabolic regulation
nized mice which express the human form of one or more of of endogenous compounds—principally glucocorticoids (gluco-
the nuclear receptors PXR (Gonzalez 2007), CAR (Ross corticoid receptor), lipid oxyseroids (liver X receptor (LXR)), bile
et al. 2010), or PPARa receptor (Morimura et al. 2006), inves- acids (farnesoid X receptor (FXR)), and lipid metabolism (peroxi-
tigators can compare the role of the human receptor with that of some proliferator-activated receptor (PPARa)) (reviewed in Plant
the rodent receptor to determine the species-specific responses and Aouabdi 2009; Table 3).
to prototypic and novel xenobiotics.
Peroxisome Proliferator-activated Receptor Alpha
Enzyme Induction in Safety Assessment
Currently, there are three peroxisome proliferators activated
The determination of enzyme induction is now carried out rou- receptors, termed peroxisome proliferator-activated receptor
tinely during the safety assessment of new chemical entities in the alpha (PPARa), PPARg and PPARb/d which heterodimerize
pharmaceutical sector. In one study where rats were dosed with with RXR to transactivate distinct but overlapping gene targets
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.
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 diverse range of xenobiotics from many classes and structures
liver, but is also distributed and highly expressed in the kidney and are also able to activate PPARa such as the fibrate hypolipidae-
intestine (Bookout et al. 2006) as well as heart and brown adipose mic agents (clofibrate, fenofibrate, gemfibrozil amongst oth-
tissue (Xu, Li, and Kong 2005). PPARg is expressed predomi- ers), methaphenilene, thromboxane synthetase inhibitors,
nantly in the adipose tissues but also in pancreatic beta-cells, dehydroepiandosterone, non-steroidal anti-oestrogens, ibupro-
intestines and spleen whereas PPARb/d is expressed nearly fen, Wy-14,643, diphenyl ether herbicides, and phenoxy herbi-
ubiquitously (Bookout et al. 2006; Greaves 2007). cides (Greaves 2007).
PPARs regulate lipid and cholesterol metabolism through
induction of (peroxisome proliferator response element
Peroxisome Proliferator-activated Receptor Alpha
(PPRE)) containing target genes resulting in increased beta-
Phenotype
oxidation of fatty acids (Xu, Li, and Kong 2005). Natural
ligands for PPARa include saturated and unsaturated fatty Activation of PPARa by hypolipidaemic agents results in
acids, eicosinoids, and linoleic acid metabolites. However, a liver enlargement through peroxisome proliferation (which
TABLE 3.—Summary of nuclear receptor actions.
Receptor CYP induction Inducer/Inducers Mechanism Histopathology
AhR CYP1A Dioxins, 3-methycholanthrene Arnt heterodimerization and Toxic hepatopathy, hepatocellular hypertrophy,
transcription of target genes multinucleated hepatocytes, fatty change, necrosis
CAR CYP2B Barbiturates RXR heterodimerization and Ground-glass hepatocyte hypertrophy
transcription of target genes
PXR CYP3A Rifampicin, dexamethasone, RXR heterodimerization and Ground-glass hepatocyte hypertrophy
pregnenolone 16 alpha-carbonitrile transcription of target genes
PPARa CYP4A Fibrates RXR heterodimerization and Granular eosinophilic hepatocyte hypertrophy
transcription of target genes
appears morphologically as an increase in peroxisomal volume such as the rat and mouse where liver expression is high
density) as well as proliferation of sER and increases in hepatic compared to non-responsive species such as the guinea pig,
cytochrome P450 enzyme activity (Cattley 2003; Walker et al. non-human primate, and human where expression (in the
1996). In rodents, these changes are highly correlated with human) is an order of magnitude lower (Choudhury et al.
liver tumors which has led to the general view that dose rates 2000; Palmer et al. 1998).
that induce >3 fold increases in peroxisomes and >1.5 fold The relatively recent development of PPARa gene knockout
increases in liver weight in short term studies are sufficient mice has now confirmed that the PPARa receptor is responsi-
to induce hepatocellular neoplasia in long term studies (Cattley ble for mediating the effects induced by the peroxisome prolif-
2004; Walker et al. 1996). erator–inducing agents clofibrate, WY-14,643 and DEHP (i.e.,
Increases in drug-metabolizing enzymes induced by PPARa hepatomegaly, increases in peroxisomes and enzyme induc-
activators are typically characterized by increased activity of tion) (Lee et al. 1995; Ward et al. 1998). It is also responsible
CYP4A and acyl CoA oxidase genes (Bell et al. 1991). How- for the transient increase in cell proliferation commonly
ever, more recent studies have demonstrated increased activity observed immediately after treatment as well as the more vari-
of other targets such as human SULT2A1 (Fang et al. 2005) able low level sustained increases in proliferation occasionally
and human/murine UGT2B4 (Barbier et al. 2003) as well as observed following chronic treatment (Cattley 2004).
decreased bile acid secretion due to decreased activity of cho- Not only do PPARa knockout mice not develop hepatocel-
lesterol 7a-hydroxylase (CYP7A1) and sterol 27-hydroxylase lular tumors in response to long-term treatment with peroxi-
(CYP27 ) (Li and Chiang 2009; Post et al. 2001). some proliferators, (Lake 1995; Peters, Cattley, and Gonzalez
1997), but PPARa humanized (PPARa knockout/knockin)
HEPATOCARCINOGENICITY mice also fail to develop tumors in response to these agents
suggesting that the human receptor is structurally different
Humans are regularly exposed to peroxisome proliferating from the murine receptor (Morimura et al. 2006). Since induc-
chemicals either in the form of environmental exposure, or tion of cell proliferation (with a concomitant decrease in apop-
intentionally through drug treatment. Given the clear associa- tosis) is thought to be the mechanistic basis for the induction of
tion between these agents and the induction of hepatocarcino- liver tumors by these non-genotoxic agents, it therefore prob-
genesis in long-term rodent studies, there is a clear concern able that humans would be resistant to this form of promotion.
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 Epidemiology and Risk to Man
induced upon treatment with peroxisomal proliferators result-
Studies using human hepatocytes (Bentley et al. 1993; Lake
ing in hepatocyte hyperplasia and fixation of spontaneous
2009) as well as human biopsy material exposed to gemfibrozil
mutations (Plant et al. 1998). Oxidative damage due to acyl
for up to 27 months (Greaves 2007) have generally concluded
CoA oxidase induction and escape of H2O2 from peroxisomes
that humans are insensitive and unresponsive to peroxisomal
has also been proposed as an indirect mechanism of genotoxi-
proliferators at therapeutic dose levels and that humans are not
city and carcinogencitiy (Conway et al. 1989). However it is
at an increased risk of developing liver tumors (Cattley et al.
uncertain whether this leads to DNA damage in hepatocytes
1998; International Agency for Research on Cancer [IARC]
and if it is causally related to the development of tumors
1995). In one study, humans were given oral fibrate therapy for
(Cattley 2004).
8 weeks and no differences in peroxisomal acyl CoA oxidase
mRNA transcripts were detected in groups receiving either
The Role of the PPARa Receptor
fenofibrate, bezafibrate, or gemfibrozil compared to placebo
The importance of the role of the PPARa receptor in controls (Roglans et al. 2002).
mediating hepatocarcinogenesis in response to peroxisomal These epidemiological data, however, have been collected
proliferators was initially raised by the observation that the from relatively small data sets and may therefore not be robust
relative expression level of the receptor in responder species enough to detect small increases in human liver carcinogenesis.
Further work over the past decade in nonhuman primates there- and appears morphologically as a temporal progression from
fore has been conducted and confirms that they are relatively centrilobular hepatocyte hypertrophy (with a concomitant
insensitive to the effects of PPARa agonists compared to induction of Cyp2b enzymes) to the development of altered
rodent species. Exposure to DEHP, diisononyl phthalate, or hepatic foci, and ultimately the appearance of adenomas and
clofibrate for 14 days failed to induce increases in peroxisomal carcinomas (Ross et al. 2010). Hand-in-hand with acute
fatty acid beta-oxidation or liver weight in the cynomolgus increases in cell proliferation is a CAR-dependent increase in
monkey (Pugh et al. 2000). In another study conducted in the endoreduplication in which Phenobarbitone and TCPOBOP
nonhuman primate, exposure to K-111, a potent PPARa stimulate an increase in the ratio of 8N:4N hepatocytes (Huang
agonist elicited only modest increases in lipid beta-oxidation et al. 2005), which appears morphologically as an increase in
(up to 3-fold) and peroxisome volume density (1.5- to 2-fold ploidy/nuclear size. Increases in functional drug-metabolizing
increase) compared to 50- to 100-fold increase in lipid beta- enzyme capacity through cell hypertrophy and hyperplasia act
oxidation and 10-fold increases in peroxisome volume density to promote clearance of xenobiotics and return the organism to
that can occur in rats and mice (Schäfer et al. 2004). Finally, homeostasis. In this respect, increases in hepatocyte ploidy
another recent report demonstrated that exposure to the potent have been correlated with increased cytochrome P450 activity
PPARa/g agonist, ciprofibrate, is capable of eliciting peroxi- (Rajvanshi et al. 1988).
some proliferation (Hoivik et al. 2004) in higher primates but Of note, recovery from Phenobarbitone or TCPOBOP treat-
that this modest induction was associated with transcript pro- ment results in complete reversal of changes marked by a return
files indicative of an antiproliferative and a pro-apoptotic effect to normal liver cell size and an associated increase in apoptosis
with no evidence of oxidative stress (Cariello et al. 2005). (Huang et al. 2005). Reversal may even extend to regression of
a proportion (30%) of liver tumors (adenomas and adenocarci-
Constitutive Androstane Receptor nomas) observed after the withdrawal of treatment with chlor-
dane in B6C3F1 mice (Malarkey et al. 1995).
CAR is an orphan nuclear receptor that forms heterodimers
with RXRa (retinoic X receptor) leading to nuclear transloca-
The Role of the CAR Receptor
tion and transactivation of target genes. This can occur even
in the absence of ligand (Honkakoski and Negishi 1998). CAR, It is now well established that the CAR, in a similar manner
however, is activated by a diverse range of ligands including to other nuclear receptors, mediates the acute and chronic
1,4-bis(2-(3,5-dichloropyridoxyloxy)) benzene (TCPOBOP) effects induced by Phenobarbitone and TCPOBOP. CAR
(murine CAR specific; Wei et al. 2000), CICO (human CAR knockout mice are resistant to the enlargement of liver cell size
specific, Maglich et al. 2003), phenobarbital, and clotrimazole and induction of drug-metabolizing enzymes/transporters as
(the latter two activators of both CAR and PXR, Moore et al. well as the acute and chronic increases in DNA synthesis (and
2000). Activated CAR heterodimers bind to a number of regu- reciprocal p53-mediated decreases in apoptosis) that leads to
latory regions of target genes eliciting transactivation of hepatocyte proliferation and ultimately hepatocarcinogenesis
CYP2B genes. (Huang et al. 2005).
CAR Phenotype Epidemiology and Risk to Man

Activation of CAR by potent drug-metabolizing enzyme Liver enlargement after 1 year of treatment with nongeno-
inducers such as Phenobarbitone causes hepatomegaly due to toxic xenobiotics in rodents correlates with an increase in liver
a profound induction of sER proliferation that occurs in com- tumors in chronic toxicology studies (Carmichael et al. 1997;
bination with an early and transient hepatocyte hyperplasia/ Ross et al. 2010). In a review of 138 pesticide carcinogenicity
suppression of apoptosis (Huang et al. 2005) and an enlarge- studies in mice, induction of liver tumors was very closely cor-
ment of the hepatic blood space (Massey and Butler 1979). related at 1 year with a mean relative liver weight of 150% and
Morphological changes are characterized by a centrilobular the presence of significant liver pathology (Carmichael et al.
hypertrophy that is apparent after 1 week of treatment and can 1997).
persist for 80 weeks without overt signs of liver cell damage In humans, liver enlargement associated with an increase in
(Crampton et al. 1977a). 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
HEPATOCARCINOGENESIS
(Pirttiaho et al. 1982). Therefore, clear concerns are raised for
Long-term treatment with CAR activators such as TCPO- the safety of human health and the risk of hepatocarcinogenesis
BOP and Phenobarbitone results in a strain- and species- in people exposed to these types of agents.
specific induction of liver tumors in rats and mice (Diwan Whereas treatment of CAR knockout mice clearly demon-
et al. 1992) that is dependent upon the fixation of somatic strated the dependence of hepatocarcinogenesis on the pres-
mutations due to the stimulation of S-phase DNA synthesis ence of the CAR receptor, it has only recently been
leading to hepatocyte proliferation and a decrease in apoptosis demonstrated that the human CAR receptor is relatively resis-
(Hasmall and Roberts 1999). This process is well characterized tant to the mitogenic effects of CAR pathway activation and,
therefore, less likely to induce liver tumors through this in Plant and Aouabdi 2009) to help integrate an organism’s
mechanism. This observation is based on the resistance of cul- response to xenobiotic exposure.
tured human hepatocytes to induction of replicative DNA This functional cross-talk is also reflected in the wide range
synthesis (Hirose et al. 2009; Lake 2009; Parzefall et al. of structurally diverse ligands that PXR can bind due to the
1991) as well as similar observations in huPXR/huCAR recep- markedly flexible pocket domain (Kliewer, Goodwin, and
tor humanized mice. Willson 2002). This results in activation by numerous xenobio-
In these later studies, treatment of humanized mice (huPXR/ tics from different chemical series such as steroids, antibiotics
huCAR) with either chlordane or Phenobarbitone resulted in (including the prototypic inducer rifampicin), antimycotics,
marked liver weight increases, hepatocellular hypertrophy, and bile acids, aflatoxins (Ratajewski et al. 2011), and the herbal
induction of cytochrome P450 in agreement with clinical data antidepressant St. John’s wort (Kliewer, Goodwin, and Willson
derived from human liver samples. However, no increases in 2002). As with other nuclear hormone receptors, species differ-
replicative DNA synthesis were observed either by direct mea- ences exist in sensitivity to different PXR activators. However,
surement of BrdU labeling index or by microarray analysis of since human and mouse PXR only share 80% amino acid
cell proliferation associated gene expression changes (Ross homology in their ligand-binding domain compared to 96%
et al. 2010). These experiments also demonstrated species differ- amino acid identity in the DNA-binding domain, mouse and
ences in response to Phenobarbitone and chlordane in that the human PXR differ markedly in their responses to PXR ligands
human receptor had greater sensitivity for Phenobarbitone- (Lehmann et al. 1998; Ma, Idle, and Gonzalez 2008). For
mediated increases in Cyp2b10 and Cyp3a11 compared to the example, human PXR is not activated by pregnenolone 16
mouse receptors, but the converse was true for chlordane. alpha-carbonitrile (PCN, a potent inducer of murine CYP3A
These data are in agreement with a number of epidemiolo- and PXR) whereas rifampicin is a selective activator of human
gical studies that demonstrate no linkage between hepatocarci- PXR but not murine PXR (Bertilsson et al. 1998).
nogenesis and chronic exposure to Phenobarbitone, phenytoin,
and other anticonvulsants at exposures similar to those that PXR Phenotype
produce liver tumors in rodents (IARC 2001; Olsen et al.
PXR is a master regulator of CYP3A transcription (Bertils-
1989; Whysner, Ross, and Williams 1996).
son et al. 1998; Gibson, El-sankary, and Plant 2002) as well as
The mechanistic basis underlying the differences in sensitiv-
other genes involved in phase I oxidation, phase II conjugation,
ity between the rodent and human receptor to induction of pro-
and phase III transport. The growing list of PXR target genes
liferation in response to CAR activation is still unclear.
include, in addition to CYP3A family members, CYP2B6
Microarray analysis of huPXR/huCAR mice has shown that
(Wang et al. 2003), Cyp7a1 (cholesterol 7 a-hydroxylase; Stau-
these mice are resistant to the upregulation of the polo-like
dinger et al. 2001), UDP-glucuronosyltransferase 1A1 (Hartley
kinase (Plk1)-mediated cell proliferation pathway induced by
et al. 2004), intestinal P-glycoprotein (Geick, Eichelbaum, and
Phenobarbitone treatment (Elcombe et al. 2010; Ross et al.
Burk 2001), OATP2 (Hartley et al. 2004), and MRP2 (Fromm
2010). Upregulation of this pathway in wild-type mice implies
et al. 2000).
that structural differences in the human receptor may account
Activation of PXR by PCN and cyproterone acetate (CPA),
for critical differences in the types of responder genes transac-
a synthetic steroid with anti-androgenic and contraceptive
tivated by CAR activation and induction of proliferation.
activity, results in liver hyperplasia and hypertrophy
(Japundzić et al. 1974) in line with the response seen with other
PREGNANE X RECEPTOR
nuclear hormone receptors. This effect is visible within 3 days
PXR Promiscuity and Cross-talk of treatment in rats (Schulte-Hermann et al. 1980) and is
accompanied by a decrease in the frequency of binucleated
PXR, like CAR, plays a central role in xenobiotic clearance.
hepatocytes and a concomitant increase in nuclear ploidy
CAR and PXR are structurally well conserved with a high
(Schulte-Hermann, Hoffmann, and Landgraf 1980). As with
degree of similarity in their DNA-binding domains but signif-
other nuclear hormone receptor agonists, long-term administra-
icant divergence in their ligand binding domains (Kliewer et al.
tion of CPA results in hepatocarcinogenesis in the mouse
2002). This enables considerable ‘‘cross-talk’’ between PXR
(Schuppler and Günzel 1979) and rat (Tucker, Kalinowski, and
and CAR due to the sharing of ligands, coregulators, or
Orton 1996).
DNA-binding elements resulting in a battery of genes coordi-
nately regulated by both nuclear receptors. To this end, Ueda
Role of the PXR Receptor
and Maglich identified sixty-nine genes regulated by CAR
(Ueda et al. 2002) and forty genes regulated by PXR (Maglich The generation of PXR knockout mice demonstrated that
et al. 2003) with many of them being coregulated by both PXR the PXR receptor is necessary for the induction of cellular
and CAR (Plant and Aouabdi 2009). hypertrophy and hyperplasia in response to PCN (as well as
Not only can PXR interact with other nuclear hormone other PXR dependent functions such as OATP2 upregulation
receptors to provide functional redundancy in xenobiotic meta- and bile acid excretion; Staudinger et al. 2001). This is consis-
bolism, but it is also regulated by a number of other receptors tent with similar observations made in CAR and PPARa
including PPARa, glucocorticoid receptor, and CAR (reviewed knockout mice and underlines the central coordination of
hepatocellular proliferation through the nuclear hormone Epidemiology and Risk to Man
receptor superfamily in response to a diverse range of
TCDD was first classified as a human carcinogen in 1997
xenobiotics.
(IARC 1997) and since then epidemiological studies have
linked dioxin exposure to cancers in man (Ma et al. 2006;
Epidemiology and Risk to Man
Schwarz and Appel 2005). However, the carcinogenic effect
The risk that PXR activators pose to the development of of TCDD and dioxin-like molecules is weak and there is some
liver tumors in man has not been extensively researched. Iso- uncertainty as to their true carcinogenic effect (Schwarz and
lated reports have noted that CPA is hepatotoxic in man (Savi- Appel 2005). This uncertainty is partly due to the binding char-
dou et al. 2006) but despite its possible mutagenic potential, acteristics of the human AhR receptor which shows approxi-
long-term follow-up of a group of fifty-three patients revealed mately a 10-fold lower affinity to TCDD than laboratory
no increase in liver cell carcinomas (Regidor et al. 2000). animal strains (Connor, and Aylward 2006). At this stage,
Instead, attention has focused on the role that PXR plays in therefore, it is not possible to definitively define what the
drug–drug interactions through enhanced PXR-mediated meta- adverse effects or risk is to human health, especially with
bolism resulting in either decreased exposure/efficacy or regard to hepatocarcinogenesis from exposure to xenobiotics
increased toxicity through metabolic activation (reviewed in that induce liver enlargement through this mechanism. This
Ma, Idle, and Gonzalez 2008). More recent research has also view is in line with that from a recent AhR Receptor expert
highlighted the role of PXR in bile acid homeostasis, PXR- panel workshop that concluded that the mode of action for AhR
mediated hepatic steatosis, steroid hormone homeostasis, and mediated carcinogenesis could not be excluded for humans
inflammatory bowel disease (Ma, Idle, and Gonzalez 2008). (Elcombe, pers. comm. 2011).
THE APPLICATION OF NEWER TOXICOGENOMIC TECHNOLOGIES

ARYL HYDROCARBON RECEPTOR
Advanced technologies such as microarray analysis and
Aryl hydrocarbon receptor (AhR) is a member of the bHLH-
quantitative real-time PCR are now being used in conjunction
PAS (basic helix-loop-helix/Per-Arnt-Sim) gene family of
with more traditional approaches such as Western blotting,
transcription factors (reviewed in Schwarz and Appel 2005).
immunocytochemistry, and electron microscopy to better eval-
It is activated by polychlorinated dibenzo(p)dioxins (PCDD)
uate the toxicological response to xenobiotics (Miyawaki et al.
and coplanar polychlorinated biphenyls (PCBs) resulting in
2011). This has led to the evolution of the ‘‘omics’’ (metabo-
nuclear translocation, heterodimerization with its binding part-
nomics, transcriptomics, and proteomics) technologies from
ner Arnt (AhR nuclear translocator), and transcription of target
an observational science that essentially reported lists of differ-
genes (Landers and Bruce 1991).
entially expressed genes to one in which Principal Component
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a potent
Analysis can now be used to better understand the effects of
activator of AhR, and like other AhR receptor agonists, results
drug treatment upon biologically relevant signaling networks.
in induction of multiple drug-metabolizing enzymes such as
The rodent liver transcriptome has been studied by numer-
CYP1A1, CYP1A2, CYP2B1, and UGT1A6 (Schwarz and
ous researchers to gain mechanistic insights into the complex
Appel 2005). In parallel with hepatocyte drug-metabolizing
biological processes involved in drug-induced liver injury,
enzyme induction, there is liver enlargement due to hepatocyte
repair, and hepatocarcinogenesis (Au, Navarro, and Rossi
hypertrophy, multinucleated hepatocytes, fatty change, necro-
2011). Combined toxicogenomic investigations now allow
sis, and increased cellular replication with suppression of apop-
investigators to study early changes in gene expression pro-
tosis resulting in the eventual outgrowth of enzyme altered
files. Since toxicogenomic profiling may precede clinical
hepatic foci and hepatocarcinogenesis (Bock and Köhle
chemistry, histopathology, clinical, or even ultrastructural
2005; Kasai et al. 2009; Maronpot et al. 2010; Yoshizawa
changes (Heinloth et al. 2007; Wang et al. 2011), it is possible
et al. 2007).
to gain insight into the early signaling perturbations that
presage toxicologic change (Ruepp et al. 2002).
Role of AhR
The activation of nuclear hormone receptors and the induc-
TCDD and other polyhalogenated dibenzodioxins as well as tion of microsomal liver enzymes occur through well-
dibenzofuranes and dioxin-like PCBs are non-genotoxic. Stud- recognized mechanisms that are amenable to toxicogenomic
ies in AhR-receptor-deficient mice have demonstrated that the profiling. Since the majority of hepatotoxicants induce over-
acute toxic effects of TCDD are mediated via the AhR receptor whelming responses in gene expression related to cell injury,
(Fernandez-Salguero et al.1996). Similar work in mice mutated degeneration, metabolism, DNA repair, and regeneration as the
for the AhR receptor has also demonstrated that promotion of liver begins the healing process (Gerrish and Malarkey 2007),
hepatocarcinogenesis by TCDD is dependent upon sustained it seems likely that a toxicogenomic approach may provide a
activation of the AhR receptor (Beebe et al. 1995). In line with useful tool to understand the mechanistic basis of these
this, mice that have constitutively active AhR receptor show changes. The challenge therefore now remains to find the key
increased promotion and yield of liver tumors following genetic and molecular events that link drug induced liver dam-
N-nitrosodiethylamine initiation (Moennikes et al. 2004). age and, specifically, drug-induced enzyme induction with
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)
FIGURE 8.—Flow-chart diagram demonstrating the NTP strategy of Source: Ellinger-Ziegelbauer et al. (2005).
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 (Carmichael et al. 1997; Maronpot and Malarkey 2011). In
and increased liver weights have a low predictive value that will soon addition, while the initial effects of chemicals that induce hepa-
in combination with gene array profiles of genotoxicity and carcino- tic metabolism may be regarded as adaptive and noninjurious,
genicity make identifying and classifying carcinogenic agents more i.e., non-adverse (Greaves 2007; Schulte-Hermann 1974), it is
sensitive and specific. clear that at higher dose levels, or following prolonged expo-
sure, these adaptive responses can fail leading to degenerative
hepatocarcinogenesis in rodents and man (Figure 8 and hepatocellular changes including necrosis with additional
Table 4). To that end, there have been about a dozen studies involvement of the biliary systems as compensatory metabolic
that profile known non-genotoxic or genotoxic carcinogens in systems are overcome or where novel cytotoxic metabolites are
the rat or mouse in order to identify a common toxicogenomic generated (Klaunig et al. 1998; Williams and Iatropoulos
signature that may be used to classify chemicals with unknown 2002). In extreme cases, hepatocyte hypertrophy may lead to
carcinogenic activity (Auerbach et al. 2010; Ellinger-Ziegelbauer compression of the sinusoidal blood circulation and anoxic
et al. 2005). 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.
HEPATIC HYPERTROPHY: ADVERSE VERSUS NON-ADVERSE
Furthermore, the use of humanized mice has now shown that
An adverse change is one ‘‘that . . . affects the performance the rodent liver is primed toward proliferation in response to
of the whole organism’s ability to respond to an additional CAR/PXR/PPARa activation whereas the human liver shows
environmental challenge’’ (Lewis et al. 2002). Another widely considerable resistance to this mechanism of hepatocarcino-
accepted definition is that an adverse effect is ‘‘a change in genesis. Therefore, the induction of a proliferative or even neo-
morphology, physiology, growth, reproduction, development, plastic response in the rodent liver through enzyme induction
or life span of an organism which results in impairment of func- would be considered to have little relevance to man in the con-
tional capacity or impairment of capacity to compensate for text of estimating the risk of human hepatocarcinogenesis.
additional stress or increased susceptibility to the harmful The development of novel pharmaceuticals for the treatment of
effects of other environmental influences’’ (WHO/IPCS [Inter- human disease often results in adverse events (AEs) in man. How-
national Programme on Chemical Safety] 2004). ever, the identification of adverse effects in preclinical toxicity
While such definitions may help summarize the broad cate- studies or even in patients does not necessarily preclude the devel-
gory of changes that might be considered adverse, it was clear opment of these drugs into safe and effective treatments. Clearly,
from the discussions arising from the expert panel that the the risk evaluation of new therapeutic drugs requires careful con-
operational use of the term adverse in the context of liver sideration of the cost–benefit analysis. More tolerance to adverse
hypertrophy would benefit from some clarification. For exam- effects noted in preclinical toxicity studies is given to drugs that
ple, a xenobiotic that induces an increase in liver weight of treat serious disease and where the AEs are easily monitorable,
150% in a 3-month study might be considered ‘‘adverse’’ in the have a good therapeutic margin, or the effect is fully reversible
context of dose setting for longer term studies but would not be upon cessation of dosing without permanent loss of function.
considered adverse in the context of safety evaluation. In this Hence one of the underlying considerations when consider-
situation, the increase in liver weight should be considered the ing the adverse effects of a new chemical entity is that the
maximum tolerated dose (MTD) for longer term dose setting exposure dose and duration in the pivotal laboratory animal
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 FIGURE 9.—Flow-chart diagram showing a decision tree for adverse
dose setting in long-term toxicity studies, any change that versus non-adverse effects induced by compounds which increase
results in structural or biochemical alterations indicative of liver weight. (Modified from Andrew 2005.)
liver cell damage is likely to result in increased turnover of
hepatocytes, somatic mutation, and liver neoplasia in chronic et al. 2010). The exception to this rule is the dog where treat-
2-year rodent bioassays, and therefore exceed the MTD. In ment with phenobarbital or corticosteroids results in induction
addition, increases in liver weight in short-term experiments of liver ALP and gGT in the absence of liver injury and inde-
(3 months to 1 year) of approx. 150% (although considered pendently of enzyme induction (Ennulat, Walker, et al. 2010).
non-adverse for the purpose of risk assessment) even in the In terms of the hierarchy of effects on the liver, the general
absence of all other adverse findings is also likely to result in consensus of the panel discussion was that liver enlargement,
liver tumors in chronic carcinogencity studies in rats and mice as characterized by increases in liver weight, was the most sen-
(Carmichael et al. 1997; Maronpot and Malarkey 2011). There- sitive indicator of hypertrophy. This change precedes morpho-
fore, a dose level of a xenobiotic that in short-term tests logical effects, although measurable increases in mRNA
induced either structural or biochemical evidence of hepatocel- (indicative of enzyme induction) are likely to occur prior to
lular damage, or produced increases in liver weight of approx. increases in liver weight. Considerable discussion occurred
150% would be considered adverse in the context of dose set- regarding the extent of liver weight increase that could be
ting and exceed the MTD. expected to be non-adverse in the long term. While empirical
When considering dose levels below the MTD, various for- relationships of certain degrees of liver weight increase
mulae are applied but generally the bottom dose level needs to appeared to be correlated with the subsequent development
be a NOEL or NOAEL, as these will be used in estimating risk of irreversible toxicity, such as fibrosis, necrosis, vacuoliza-
assessment for occupational or long-term unintentional tion, fatty degeneration, and even neoplasia, it was considered
exposure (OECD Draft Guidance Document No. 116). The that a more scientific approach to the problem, taking into
dose levels are set on the basis of data derived from shorter account proposed mode of action analysis, would be of use to
term animal studies and those for the bottom dose level should guide the setting of better tolerated dose levels and a more sys-
either be completely clear of toxicity, induce effects that would tematic risk assessment process (Carmichael et al. 2011; Felter
be widely considered to be adaptive (i.e., non-adverse), or in et al. 2011). The general opinion of the group was that liver
the case of the pharmaceutical industry, produce manageable weight increase through hepatocyte enzyme induction, in the
or ‘‘desirable’’ toxicity that is considered a consequence of the absence of histopathologically demonstrated degenerative or
target (primary) pharmacology of the drug. necrotic changes and without significant changes in hepatic-
derived plasma enzymes, would not be considered adverse and
Non-adverse Effects in the Liver in Terms of the Hepatic would have little relevance to man in terms of risk assessment
Enzyme–inducing Agents and the development of liver tumors.
In the context of liver effects induced by hepatic enzyme–
inducing agents, a dose level at which a non-adverse toxicity
ASSESSMENT OF LIVER WEIGHT INCREASES
is present would, with every expectation, produce morphologi-
cal changes in the liver characterized by liver weight increases, When assessing a histological change caused by an increase
microscopic evidence of hepatocellular enlargement/hypertro- in liver weight, in order to conclude whether the change is
phy, and generally no increases in plasma enzymes such as adverse or not, a number of steps must be carefully considered,
alanine and aspartate aminotransferase (Ennulat, Walker, summarized below (and in Figure 9):
1. Is there histological evidence of structural degenera- SUMMARY AND CONCLUSIONS

tive or necrotic changes such as:
The purpose of the 3rd International ESTP workshop was to
discuss the significance of hepatocellular hypertrophy in
hepatocyte necrosis, fibrosis, inflammation, and
*steatotic vacuolar degeneration rodents and define more clearly which responses were consid-
ered to be adaptive in nature so as to help guide scientific opin-
biliary/oval cell proliferation, degeneration,
fibrosis, and cholestasis ion. The consensus of opinion was that hepatomegaly as a
consequence of hepatocellular hypertrophy without histologic
necrosis and degeneration of other resident cells
within the liver or clinical pathology alterations indicative of liver toxicity was
considered to be an adaptive non-adverse change. This conclu-
(*Minimal to mild increases in steatotic macro-vesicular sion should normally be reached by an integrative weight of
vacuolation without other changes indicating cellular damage evidence approach. Under these circumstances, hepatocellular
should be distinguished from micro-vesicular vacuolation and hypertrophy through enzyme induction may therefore be
considered non-adverse since this is a common change induced considered a fully reversible change that does not compromise
by feeding high-fat diets.) viability or functional integrity and potentially benefits the
organism through increased metabolic capability.
(Of note, transient increases in proliferative indices together
During the assessment of increased liver weight due to
with changes in hepatocyte ploidy, if induced through CAR/
enzyme induction, one should consider for long-term dose
PXR/PPARa activation, is likely to be a rodent-specific
setting that non-adverse adaptive changes at low exposures
phenomenon and therefore of little relevance to man even if
may very well become adverse at higher/longer dosages. In
this results in altered hepatic foci and/or primary liver tumors
addition, increases in liver organ weight 150% in 3-month
in chronic studies.)
to 1-year studies may very well lead to hepatocarcinogenesis
and/or endocrine tumors (though increased degradation of thyr-
2. In the absence of histological changes, using a
oid and possibly reproductive hormones) in longer term studies
weight-of-evidence approach, is there clinical
and appropriate measures should be put in place to ensure ade-
pathology evidence of hepatocyte damage character-
quate survival of the top dose animals on the study.
ized by a dose dependent and biologically significant
Finally, the development of newer technologies and the use
and consistent increase in at least two liver
of genetically modified mice have considerably advanced our
parameters:
understanding of the underlying mechanisms involved in
at least 2 to 3 increase in ALT (EMEA 2010, nuclear hormone receptor (CAR, PXR, and PPARa) induced
FDA 2009; HED Guidance Document 2002) or hepatocellular hypertrophy. The imminent generation of triple
a biologically significant change in other bio- humanized mice are likely to further increase our understand-
markers of hepatobiliary damage (ALP, AST, ing of the response of the humanized receptor to these types
gGT, GLDH, etc.) of activators by removing the potentially confounding effects
a biologically significant change in another clin- of ‘‘off-target’’ nuclear hormone receptor signaling by ‘‘pro-
ical pathology marker indicating liver dysfunc- miscuous’’ activators and thus increase the confidence that
tion (albumin, bilirubin, bile acids, coagulation hepatocarcinogenesis in response to these types of compounds
factors, cholesterol, triglycerides etc.). is a rodent-specific phenomenon.
Clinical pathology changes should corroborate each other, be AUTHORS’ NOTE

consistent with the expected species-specific patterns of Workshop Participants—Clifford Elcombe, CXRBios-
change resulting from hepatobiliary injury, and take into ciences, Great Britain, BSTP; John Foster, AstraZeneca, Great
account target pharmacology that may non-adversely alter one Britain, BSTP; Peter Hall, AstraZeneca, Great Britain, BSTP;
or more of these biomarkers. It should also be noted that statis- Takanori Harada, IET (Institute of Environmental Toxicology),
tical significance alone is not a reliable indicator of hepatic Japan, JSTP; Wolfgang Kaufmann, Merck KGaA, Germany,
toxicity (HED guidance document 2002). ESTP (organizer of the 3rd International ESTP expert work-
If the above mentioned adverse criteria are not observed, shop); Anja Knippel, Merck KGaA, Germany, ESTP; Karin
then increases in liver organ weight and liver cell hypertrophy Küttler, BASF SE, Germany, ESTP; David E Malarkey,
due to enzyme induction can be considered as an adaptive NIEHS (National Institute for Environmental Health Science),
response to a xenobiotic and of little relevance to man. USA, STP; Robert R. Maronpot, Consultant, USA, STP;
However, prolonged exposure to a xenobiotic at levels that Akiyoshi Nishikawa, NIHS (National Institute of Health
have previously been shown to be adaptive may eventually Sciences), Japan, JSTP; Thomas Nolte, Boehringer Ingelheim
result in liver cell injury due to a failure of adaptive mechan- Pharma GmbH & Co. KG, Germany, ESTP; Agnes Schulte,
isms. In this case, the combination of dose level and duration BfR (German Federal Institute for Risk Assessment),
of exposure to the xenobiotic under the terms and conditions Germany, ESTP; Volker Strauss, BASF SE, Germany,
of the new experiment would now be considered adverse. ESVCP/ESTP; Malcolm York, GSK, Great Britain, ESVCP.
ACKNOWLEDGMENTS Bertilsson, G., Heidrich, J., Svensson, K., Asman, M., Jendeberg, L., Sydow-
bäckman, M., Ohlsson, R., Postlind, H., Blomquist, P., and Berkenstam,
The authors wish to thank ESTP for organizing the workshop A. (1998). Identification of a human nuclear receptor defines a new signal-
and MerckSerono for giving logistical support at the venue. ing pathway for CYP3a induction. Proc Natl Acad Sci USA 95, 12208–13.
Bock, K. W., and Köhle, C. (2005). Ah receptor- and TCDD-mediated liver
tumor promotion: Clonal selection and expansion of cells evading growth
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Toxicologic Pathology, 40: 971-994, 2012

Liver Hypertrophy: A Review of Adaptive (Adverse and

At the ESTP liver hypertrophy expert group, it was

MOLECULAR METHODS TO ASSESS LIVER ENZYME INDUCTION

RO9210, a non-nucleoside reverse transcriptase inhibitor for the

TABLE 3.—Summary of nuclear receptor actions.

Receptor CYP induction Inducer/Inducers Mechanism Histopathology

CAR Phenotype Epidemiology and Risk to Man

THE APPLICATION OF NEWER TOXICOGENOMIC TECHNOLOGIES

TABLE 4.—Proposed mechanisms involved in tumorigenesis in rat

toxicology studies should equal or, ideally, exceed that in the

Non-adverse Effects in the Liver for Setting Dose Levels

1. Is there histological evidence of structural degenera- SUMMARY AND CONCLUSIONS

Clinical pathology changes should corroborate each other, be AUTHORS’ NOTE

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