The Relationship Among Microsomal Enzyme Induction, Liver Weight and Histological Change in Rat Toxicology Studies

Abstract

The purpose of this study was to determine what histological changes, if any, accompany liver enlargement and microsomal enzyme induction in rats administered high doses of therapeutic agents in preclinical toxicology studies. This was accomplished by evaluating a database derived from a series of 11 induction studies in rats with 10 novel compounds comprising five therapeutic classes. Results from serum enzyme chemistry analyses, gross organ weight changes, and histological analyses of the liver sections were evaluated and compared with the magnitude and extent of hepatic cytochrome P450 induction. All compounds were administrated via oral intubation once a day for the duration of the study using multiple doses, each proportionally based on body weight. During the course of these studies, serum clinical chemistry data and clinical observations were recorded. After necropsy, histopathology observations were made, and hepatic microsomes were assayed for cytochrome P450 content and associated drug-metabolizing enzymes. In some cases, cyanide-insensitive β-oxidation of palmitoyl CoA was also assayed. Liver weight increases of 20% or greater were associated with histological evidence of hypertrophy, but neither the severity of hypertrophy nor the magnitude of liver weight increase correlated with the magnitude of drug-metabolizing enzyme elevations. Hypertrophy alone was not associated with serum enzyme increases. While there was a correlation between the incidence of increased liver weights and microsomal enzyme induction, the magnitudes of these increases were not related. Decreased serum triglycerides were often associated with elevated β-oxidation attributed to hepatic peroxisome proliferation. It was concluded that, while slight ALT elevations occasionally were observed, hepatic microsomal enzyme induction was generally not accompanied by substantial morphological changes or elevated serum enzyme levels considered indicative of liver injury.

Introduction

A number of unrelated, structurally dissimilar, and typically lipophilic (at physiological pH) chemical compounds stimulate the microsomal induction of cytochrome P450 enzymes in the livers of rodents and other mammals when administered continuously over a period of days. In addition to components of the cytochrome monooxygenase system, NADPH-dependent cytochrome P450 reductase, epoxide hydrolase, and some conjugating enzymes may be induced as well. Morphologically, this may be accompanied by liver hypertrophy and the proliferation of smooth endoplasmic reticulum (Remmer and Merker, 1963), although hepatic hypertrophy is not an essential manifestation of P450 induction. While the most common cytochrome P450 enzyme induction mechanism involves increased gene transcription, inducers of at least one cytochrome P450 subfamily appears to act by multiple, post-translational mechanisms (Okey, 1990). In general, the induction response is rapid with significant increases in microsomal cytochrome P450 detected as early as 1 day after treatment (Davison and Wills, 1974). Peak induction responses for individual cytochromes may require 4–7 days or more, depending on the inducer, the dose, and the enzyme (Paolini et al., 1991).

In a study by Crampton et al. (1977), in which rats were fed phenobarbitone or butylated hydroxytoluene for up to 80 wk, liver enlargement and induction of drug-metabolizing enzymes were accompanied morphologically by centrilobular cell enlargement and hypertrophy of the smooth endoplasmic reticulum. These authors deemed this an adaptive response suggesting that the size of the liver, which is also the site of xenobiotic metabolism, increases in proportion to the increased functional load. This and similar studies (Simmons et al., 1991) have demonstrated the absence of overt hepatotoxicity at dose levels that nevertheless stimulate the drug-metabolizing enzyme system of the liver. Conversely, other studies have shown that the induction response can be accompanied by liver toxicity, as for example, the overt hepatotoxicity [i.e. decreased serum albumin, substantially increased serum alkaline phosphatase (AlkP) and 5′-nucleotidase (5′-NT) activities, cytoplasmic eosinophilia and some single-cell necrosis of hepatocytes] that accompanied a two- to threefold increase in cytochrome P450 content following 13-wk exposure to a novel lipid regulator in a study by Wolfgang et al. (1995). While examples can be found for both hepatotoxic and non-hepatotoxic induction responses, hepatotoxicity based on either liver function assays or histopathology is usually not assessed by investigators studying microsomal enzyme induction. Consequently, a comprehensive investigation of both phenomena using novel chemical agents with potential pharmacological application is warranted.

The purpose of this retrospective data evaluation was to explore the coincidence, if any, of possible hepatic injury, suggested by substantial clinical chemistry or histopathological changes, and cytochrome P450 induction by examining the compiled data from a series of 11 induction studies in rats with 10 novel compounds of therapeutic interest. In general, the duration of these studies and the dosing regimen were selected according to standardized protocols for regulatory submission prior to human clinical exposure. These compounds had been selected for cytochrome P450 analyses because either: (1) preliminary studies in the rat had produced increased liver weights after multiple dosing; or (2) chemical analogues or other members of the same chemical class increased liver weights in exposed rats. This basis for selection corresponds to studies of 1–3 months duration v. 2–3 wk duration, respectively. For each compound, dose levels were selected on the basis of preliminary toxicology studies and were expected to reveal target-organ toxicity at the highest dose. Owing to the proprietary nature of the studies, we are unable to reveal the chemical structures. Conclusions based on this analysis of 11 independent studies help clarify the hepatotoxicity/hypertrophy responses for chemicals with potential therapeutic use.