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A. P. Hall, C. R. Elcombe, J. R. Foster, T. Harada, W. Kaufmann, A. Knippe, K. Ku¨ Ttler, D. E. Malarkey, R. R. Maronpot, A. Nishikawa, T. Nolte, A. Schulte, V. Strauss, and M. J. York
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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, frequently 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 PPARα. The generation of genetically engineered mice that express altered versions of these nuclear hormone receptors, together with other avenues of investigation, have now demonstrated that sensitivity to many of these effects is rodent-specific. These data are consistent with the available epidemiological 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, PPARα, weight, fasting, clinical pathology, omics.

Introduction

Drug and chemically induced liver enlargement in subchronic and chronic toxicology studies in rodents has, for many years, taxed the toxicology profession in terms of its perceived relevance to hepatotoxicity, to carcinogenicity in lifetime bioassays at similar dose levels, and in terms of its relevance to man (Cohen and Grasso 1981; Elcombe, Rose, and Pratt 1985; Grasso and Hinton 1991). The fact that after more than 50 years of debate (Gilbert and Golberg 1965; Rowe et al. 1959; Weil and McCollister 1963) the issue remains as contentious as ever prompted the European Society of Toxicologic Pathology (ESTP) to convene an expert opinion group to discuss the current state of the science. The purpose of the workshop was to discuss the significance of hepatocellular hypertrophy in rodents, define more clearly when adaptive responses become adverse, and understand the long-term consequences of hepatocellular hypertrophy in order to guide scientific opinion for risk assessment in man and dose setting for longer term animal studies. One critical aspect of hepatic hypertrophy which continues to pose serious questions for toxicologists is the definition of what constitutes an adverse hepatic effect, versus a nonadverse or adaptive effect. This was a major focus of discussion by the expert opinion group resulting in a consensus opinion detailed in this article.

Hepatic Hypertrophy—What Is It?

While to a histopathologist the term hepatic hypertrophy is well recognized and readily defined histologically, to a toxicologist, the term can have various connotations including an increase in the weight of the organ (liver hypertrophy), an increase in the average size of the hepatocytes (hepatocellular hypertrophy), and even hepatic enzyme induction (functionally sometimes referred to as ‘‘work’’ hypertrophy).

In practice, drug- or chemical-induced hepatic hypertrophy tends to be a combination of all of these parameters in addition to others including profound changes in the intracellular enzymes involved not only in phase 1 and 2 drug metabolism (Crampton et al. 1977b; Lake, Longland, et al. 1976; Maronpot et al. 2010) but also in other more fundamental cell processes such as altered oxidative status, fatty acid metabolism, energy production and utilization, cell turnover and altered hepatocellular cytoplasmic, and nuclear morphology (Cattley and Popp 1989; Crampton et al. 1977b; Grasso et al. 1974; Grasso and Hinton 1991).

Archetypical changes that often accompany this phenomenon are increases in hepatic-derived enzymes (transaminases, alkaline phosphatase, and γ-glutamyltransferase) that may appear in the plasma following liver enlargement (Ennulat, Magid-Slav, et al. 2010). Of prime importance for interpretation of these changes with regard to risk assessment are the significant species differences shown in response to chemicals that induce a classic hypertrophic response in the rodent liver (Lake 1995; Lake, Brantom, et al. 1976; Rhodes et al. 1986; Williams and Perrone 1996).

Liver Hypertrophy-table1

Liver Weight Increases

Increases in liver weight in rodents due to exposure to chemicals can be achieved through a number of mechanisms and can be accompanied by a range of differing histological appearances, some of which clearly show cytotoxicity and cell death, such as carbon tetrachloride and chloroform (Edwards and Dalton 1942; Eschenbrenner and Miller 1945; Grasso and Hinton 1991; Reuber and Glover 1968), while other chemicals such as sodium phenobarbitone, the PPARα (peroxisome activated receptor alpha) agonists, Nafenopin and Clofibrate, and trichloroethylene, induce an increase in liver weight without overt cell necrosis (Lake et al. 1989; Mitchell et al. 1985; Price et al. 1986). While there is an undoubted relationship between hepatic necrosis and its consequences on the eventual increased incidence of neoplasia of the liver (Eschenbrenner and Miller 1945; Grasso and Hinton 1991), this is less clear for chemicals, or rather for doses of chemicals, that induce liver weight increases in the absence of overt hepatocellular damage. The focus of the workshop was on those chemicals that are not traditionally associated with acute hepatic necrosis but rather exert their effect by increasing the weight of the liver through other means.

Organ weight data can provide sensitive indices of toxicologic change where it can correlate and confirm changes seen down the microscope. To this end, the STP have recently published a position paper (Sellers et al. 2007) that gives careful guidance to the collection and weighing of organs in routine toxicology studies. The authors recommend that organ weight data should be routinely expressed as an organ-to-body weight ratio to avoid large variations in body weight skewing organ weight interpretation. In this way, increases in liver organ weight can be accurately correlated with hepatocellular hypertrophy. In rodent studies, where routinely larger numbers of animals are used, these data can be compared with concurrent controls and statistically interrogated to derive p values that may increase confidence through weight of evidence to allow a judgment of hepatocellular hypertrophy when the histological changes appear to be relatively small.

This is particularly important since the magnitude of the increased liver weight can vary considerably between different chemicals. Liver weight changes may also demonstrate a clear dose relationship (Table 1; typically between 110% and 150% of control liver weight). However, it should be noted that although liver weight increases are correlated with microsomal enzyme induction, the degree of microsomal enzyme induction may not be closely correlated with either the magnitude of the liver weight increase or the degree of hepatocyte hypertrophy in rats, dogs, or monkeys (Amacher, Schomaker, and Burkhardt 1998, 2001; Amacher et al. 2006; Ennulat, Walker, et al. 2010).

Increases in liver weights have been shown to be associated with the induction of increased incidences of hepatocellular neoplasia in 2-year carcinogenicity studies in rodents (Allen et al. 2004; Carmichael et al. 1997) and while considered to be of little relevance to man for some chemicals (Butler and Newberne 1975; Ito and Sugano 1991; McClain et al. 1995; Stevenson et al. 1990), dose levels of a chemical inducing this degree of liver weight increase would be considered to carry an increased risk of inducing hepatic neoplasms in these types of study. In a survey of 138 chemicals used in the agrochemical industry, a relative increase in liver weight of ≥ 150% of control values after 1 year of treatment was positively correlated with the induction of liver tumors in mice (Carmichael et al. 1997). Similarly, in another survey of mouse NTP studies where correlations between liver weight increases and histological parameters and carcinogenesis were assessed, the authors concluded that ‘‘the best single predictor of liver cancer in mice was hepatocellular hypertrophy’’ (Allen et al. 2004). This study demonstrates a highly significant relationship between increases in liver weight and the future outcome of hepatic neoplasia (p < .001; Table 2). In a similar review of the rat, a less statistically significant relationship (p = .018) between liver weight and hepatocarcinogenesis was also noted, whereby liver weight increases alone correctly predicted eight of the eleven liver carcinogens (but overpredicted twenty-six false positives and failed to predict three true positives; Allen et al. 2004).

Liver Hypertrophy-table2

Effect of Fasting on Liver Weights

A potentially important confounder in terms of the evaluation of liver weight changes following chemical treatment is the practice of fasting animals overnight prior to sacrifice. It is likely that fasting alters the resulting organ weights in rodents over those not fasted (Chatamra, Daniel, and Lam 1984; Rothacker et al. 1988). Studies have shown that the rodent liver weight increases in fed animals are maintained for up to 8 hr after feeding. Water and glycogen account for the major portion of this increase with the former comprising over 66% of the increase (Leveille and Chakrabarty 1967). Overnight fasting will rapidly deplete both components (in preference to other tissues of the body) and accounts for the loss of liver weight seen under fasting conditions (Rothacker et al. 1988). It is currently unclear as to what percentage of laboratories utilizes fasting, but what was apparent from the workshop was that there exists considerable inter-laboratory variation in the practice of overnight fasting of animals prior to necropsy.

Welfare reasons prohibit the overnight fasting of pregnant animals but a systematic study looking into the differences between fasted and non-fasted animals would seem a worthwhile exercise if the former leads to better discrimination of effects provided animal welfare guidelines permit this (Animal and Plant Health Inspection Services

[APHIS] 1997; Animal Welfare Information Center 2005). However, it is clear that overnight fasting does have some distinct advantages. First, overnight fasting decreases the variation seen in some clinical pathology parameters and therefore increases the probability of identifying statistically significant changes between control and treated animals (Matsuzawa and Sakazume 1994). Second, animals exposed to high levels of a xenobiotic very frequently experience decreased food intake due to inanition/toxicity which is often noted in toxicological studies as a treatment, and dose related, decrease in hepatocellular glycogen storage (Agren, Wilander, and Jorpes 1931; Lockard et al. 1983). It is possible therefore that loss of glycogen in animals exposed to a xenobiotic might mask statistically significant increases in liver weight. Since liver weight increases are generally considered a sensitive measure of hepatocellular hypertrophy, small changes in these parameters may not be visible using H&E stained sections. This is especially so if animals are not fasted, as a relative loss of glycogen due to toxicity in the high-dose groups is likely to reduce hepatocellular volume and obscure the change in dimensions which helps inform the diagnosis of hypertrophy when comparing treated with concurrent control animals (Li et al. 2003). However, there are clearly well-described changes in gene expression in fasted animals (Bauer et al. 2004; Lkhagvadorj et al. 2009) and further work to assess the relative merits of overnight fasting for subsequent histopathology evaluation would help assess the importance or otherwise of this effect.

The practice of fasting rodents before necropsy was one that was debated at the workshop but because of the differing experiences of the group a recommendation was made for a comparison dose–response study with a known hepatotrophic agent, such as sodium phenobarbitone, in the rat where groups would be fasted and the results obtained compared to non-fasted groups. The NOEL for the histopathology of hepatocellular hypertrophy would then be compared between the fasted and non-fasted groups.

Clinical Pathology

The following clinical pathology parameters can help in the assessment of adverse effects on the liver evolving during enzyme induction:

Alanine Aminotransferase and Aspartate Aminotransferase (ALT/AST)

Several studies in rats dosed with liver enzyme–inducing compounds have been published. In these studies ALT and AST activity increases were below twofold of the controls: sodium phenobarbitone (an archetypical mixed CYP 2B/3A inducer) given to rats at dose levels where centrilobular hypertrophy was present without accompanying degenerative changes induced small increases in both hepatic ALT (Boll et al. 1998) and serum ALT and AST (Lake and Evans 1993). In contrast, administration of the CYP 1A inducer, 3-methylcholanthrene to rats, at dose levels that induced significant levels of drug-metabolizing enzymes, failed to show any increase in serum ALT or AST (Lake and Evans 1993). Peroxisome proliferators (PPARα agonists and CYP4A inducers), similarly show minimally increased levels of serum ALT and AST even when given at dose levels that induce significant increases in liver weight, provided that the only histological effect seen in the liver is hepatocellular hypertrophy (Eacho et al. 1985; Huang and Shaw 2002; Kramer et al. 2003; Peterson et al. 2004).

Increases in serum ALT activity without increases in hepatic ALT activity are thought to be due to damage to, and leakage from, hepatocyte cell membranes, resulting in a release of enzymes from the cytosol into the blood (Amacher 1998). Alternatively, increased enzyme synthesis, as a consequence of liver enzyme induction, may also lead to higher ALT levels in both the liver and the serum. In this scenario, moderately higher serum ALT levels, especially in mice treated with liver enzyme inducers, might occur without cell membrane damage (Strauss, pers. comm. 2011). Boone et al. (1985) concluded that increases in serum ALT activity in the range of 2 to 4x or higher in individual or group mean data when compared with concurrent controls should raise concern as an indicator of potential hepatic injury unless a clear alternative explanation is found. Based on the recommendations of regulatory authorities, (EMEA 2010; FDA 2009; HED 2002) increases in ALT activity of two- to threefold should be considered as indicative of hepatocellular damage. Distinguishing irreversible and reversible liver injury is pragmatically dealt with by assessing the magnitude of the transaminase elevation where minimal and reversible hepatic injury is commonly accompanied by small increases in transaminase levels of less than twofold (Kramer et al. 2003; Lassen 2004; Peterson et al. 2004; Satoh et al. 1982; Solter 2005). However, considerably larger ALT increases in humans (30- to 100-fold of the upper limit of normal) can also be accompanied by full recovery of hepatic function (Koch et al. 1997).

Alternatively, small decreases in serum ALT might also be associated with liver enzyme induction. In a rat study, where sodium phenobarbitone was given at dose levels of 80 mg/kg, and b-naphthoflavone (a CYP1A inducer) was given at 100 mg/kg, there were no recorded elevations in serum transaminases, but instead (statistically insignificant) decreases in ALT or AST activities were recorded (Arvela, Reinila, and Pelkonen 1981). In these experiments, the only histopathology recorded was centrilobular hepatocellular hypertrophy. Small decreases in ALT/AST activity may usually be regarded as a ‘‘nonadverse’’ effect because they cannot be correlated with a toxicologically relevant finding. Large changes (>50%), however, may result not only from induction of hepatic drug metabolism but from deficiencies in pyridoxal 5′ -phosphate. This may be confirmed or excluded by measuring enzyme activities with and without pyridoxal-phosphate addition as a cofactor to the reaction mixture (PSD Guidance Document 2007).

Alkaline Phosphatase (ALP)

Increases in ALP activity associated with hepatic microsomal enzyme induction, in the absence of accompanying degenerative histopathological findings, have been reported in dog by a number of workers (Conning and Litchfield. 1971; Leeling et al. 1975; Robertson et al. 1993). These studies correlated an increase in ALP activity with increased microsomal enzyme activity and demonstrated that the source of the ALP increase was of hepatic origin in the absence of histologically detectable hepatobiliary injury. Increases in total serum ALP activity up to approximately 2.5-fold (Leeling et al. 1975), 3-fold (Conning and Litchfield 1971), and 5-fold (Robertson et al. 1993) were observed during the course of these studies, which were noted to recover after an 8-day treatment-free period in concordance with a reduction in hepatic microsomal enzyme activity (Litchfield and Conning 1972). Additionally, the ALP change reported by Robertson et al. (1993) was not associated with any other changes in clinical pathology parameters. Leeling et al. (1975) concluded that marked changes in ALP levels during drug treatment should not automatically be assumed to have toxicological implications.

During the current ESTP-sponsored workshop, case study material was presented where a dose-related increase in plasma ALP activity (up to 10-fold) was shown to be of hepatic origin in the dog and followed for up to 52 weeks. Increases in ALP activity were evident at 4 weeks (with maximal comparable levels observed at 13, 26, and 52 weeks). ALP activity correlated with hepatocyte hypertrophy and increases in liver weight up to 1.5-fold of control values. One individual animal showed evidence of hepatocellular degeneration and atrophy at week 52, but this change was also associated with moderate increases in AST (5-fold), ALT (2-fold), and GLDH (glutamate dehydrogenase; 15-fold). Therefore, it is considered that increases in circulating ALP activity in the dog, with associated increased liver weight and histological hepatocellular hypertrophy but without hepatocellular degeneration could be interpreted as an adaptive, rather than an adverse response to chemical exposure.

γ-Glutamyltransferase (γGT)

Induction of hepatocyte γGT has also been reported with several chemicals, including sodium phenobarbitone which is known to cause liver weight increases and induce hepatic metabolism as well as various CYPs (Ratanasavanh et al. 1979). In case studies presented at the workshop, messenger RNA (mRNA) for hepatic γGT was shown to be induced in a number of repeat dose (7 days) oral toxicity studies in the rat where centrilobular hypertrophy, increased liver weights, increased CYPs (predominantly 2B2 and 3A3), and phase 2 enzymes activities (predominantly GSTA3 and UGT1A6) were observed. However, this only translated into increases in circulating γGT activity where marked induction (>50-fold) of hepatic γGT mRNA was observed. In rats treated with phenobarbital for 5 days, liver but not serum γGT activity was increased, indicating the relative insensitivity of serumderived γGT as a marker of hepatobiliary change in laboratory animals compared to measuring hepatic γGT (Goldberg et al. 1981). In a reported study where kava kava was administered to rats for 14 weeks, statistically significant increases in serum γGT were observed in both males and particularly so in females, which accompanied hepatocellular hypertrophy, liver weight increases,