Julian E.A. Leakey

Effects of aging and caloric restriction on hepatic drug metabolizing enzymes in the Fischer 344 rat. I: The cytochrome P-450 dependent monooxygenase system

The effects of long-term caloric restriction on the hepatic cytochrome P-450 dependent monooxygenase system were investigated in the 22-month-old Fischer 344 rat. Caloric restriction decreased the age-related changes in hepatic testosterone metabolism, which are associated with demasculinization of the liver. Caloric restriction also increased hepatic microsomal testosterone 6 beta-hydroxylase, lauric acid 12-hydroxylase and 4-nitrophenol hydroxylase activities over corresponding values in both ad libitum fed 22-month and 60-day-old control male rats. This suggests that cytochrome P-450 isozymes, P-450 pcn1&2, P-452 and P450j may be induced by caloric restriction. Such changes in cytochrome P-450 isozyme profiles could result in altered carcinogen activation, radical formation or drug detoxication in the calorically restricted rat.

Role of Glucocorticoids and “Caloric Stress” in Modulating the Effects of Caloric Restriction in Rodentsa

Caloric restriction (i.e., the balanced reduction of the protein, carbohydrate and fat content of the diet without reduction of its micronutrient content) has been shown, in rodents, to be an extremely powerful modulator of a broad spectrum of age-associated degenerative diseases as well as life span.’-5 Caloric restriction has been found to delay the occurrence of many age-associated neoplastic diseases or to slow their progression, often to such an extent that the clinical expression of the diseases is eliminated.* Since reducing individual dietary components such as protein or fat without reducing the overall caloric intake is less effective in increasing longevity or suppressing neoplasia than caloric restriction by itself, it is reasonable to assume that the observed effects of caloric restriction are primarily dependent upon a specific reduction in calories and not specific dietary compon e n t ~ . ~ ~ ’ The multiple actions of caloric restriction suggest that it may influence primary aging processes themselves; however, recent examination of pathological data suggests that caloric restriction delays different age-dependent pathological conditions at different rates.8 This observation would argue against the modulation by caloric restriction of a simple fundamental process of cellular aging, but rather, it implies that caloric restriction modifies the organism’s homeostatic set-point to one that is less susceptible to some but not all pathologies. Unfortunately, such theories on the nature of the caloric restriction effect will remain merely speculatory until the precise molecular and biochemical mechanisms by which caloric restriction influences the processes of aging and disease are fully elucidated.

Effects of caloric restriction on rodent drug and carcinogen metabolizing enzymes: implications for mutagenesis and cancer

Caloric restriction in rodents results in increased longevity and a decreased rate of spontaneous and chemically induced neoplasia. The low rates of spontaneous neoplasia and other pathologies have made calorically restricted rodents attractive for use in chronic bioassays. However, caloric restriction also alters hepatic drug metabolizing enzyme (DME) expression and so may also alter the biotransformation rates of test chemicals. These alterations in DME expression may be divided into two types: (1) those that are the direct result of caloric restriction itself and are detectable from shortly after the restriction is initiated; (2) those which are the result of pathological conditions that are delayed by caloric restriction. These latter alterations do not usually become apparent until late in the life of the organism. In rats, the largest direct effect of caloric restriction on liver DMEs is an apparent de-differentiation of sex-specific enzyme expression. This includes a 40-70% decrease in cytochrome P450 2C11 (CYP2C11) expression in males and a 20-30% reduction of corticosterone sulfotransferase activity in females. Changes in DME activities that occur late in life in calorically restricted rats include a stimulation of CYP2E1-dependent 4-nitrophenol hydroxylase activity and a delay in the disappearance of male-specific enzyme activities in senescent males. It is probable that altered DME expression is associated with altered metabolic activation of chemical carcinogens. For example the relative expression of hepatic CYP2C11 in ad libitum-fed or calorically restricted rats of different ages is closely correlated with the amount of genetic damage in 2-acetylaminofluorene- or aflatoxin B1-pretreated hepatocytes isolated from rats of the same age and caloric intake. This suggests that altered hepatic drug and carcinogen metabolism in calorically restricted rats can influence the carcinogenicity of test chemicals.

Differential stability of drug-metabolizing enzyme activities in primary rat hepatocytes, cultured in the absence or presence of dexamethasone

The effects of primary hepatocyte culture on the rat cytochrome P450-dependent monooxygenase system and several conjugating enzyme activities were examined using a culture system similar to those used for evaluation of chemicals as potential genotoxins. Cytochrome P450 and cytochrome b5 contents progressively decreased throughout the 72-h culture period to less than 25% of initial values, whereas cytochrome P450 reductase rapidly decreased by 50% during attachment, but then remained stable. Cytochrome P450-dependent testosterone hydroxylase activities decreased more rapidly in culture than did cytochrome P450 content reaching less than 50% of attachment levels by 24 h. Cytochrome P450IIIA immunoreactive protein decreased at a similar rate to testosterone-6 beta-hydroxylase. Activated UDP-glucuronyltransferase activities towards 1-naphthol and testosterone declined more slowly over the 72 h than cytochrome P450 and remained at 50-60% of initial values at 72 h. UDP-glucuronyltransferase activity towards digitoxigenin monodigitoxoside (DIG) did not decrease during culture. Glutathione-S-transferase and sulfotransferase activities also declined during the 72 h at rates which appeared to be isozyme-dependent. Addition of 1 microM dexamethasone (DEX) to the culture medium increased UDP-glucuronyltransferase activity towards DIG, cytochrome P450 reductase and testosterone-6 beta-hydroxylase activities up to 2.5-, 2.0- and 7-fold, respectively and induced cytochrome P450IIIA immunoreactive protein(s) in the hepatocytes after 24 and 48 h of culture; DEX was less effective at the 72 h time-point. DEX treatment also significantly accelerated the decreases in glutathione-S-transferase activities and in sulfotransferase activities towards 1-naphthol and estrone. Thus, it appears that primary rat hepatocytes cultured under standard conditions, not only rapidly lose their monooxygenase capabilities, but also some of their capacity for conjugation. Furthermore, the use of DEX in cell culture medium to enhance cell survival does not maintain total drug-metabolizing enzyme capability, but appears to transiently and selectively increase expression of certain isozymes at the expense of others.

FDA Points-to-Consider Documents: The Need for Dietary Control for the Reduction of Experimental Variability within Animal Assays and the Use of Dietary Restriction to Achieve Dietary Control

Standard protocols for conducting chronic toxicity and carcinogenicity studies have been refined over the years to carefully control for many variables. Nevertheless, over the last 2 decades, there has been a steady increase in variability, a decrease in survival, an increase in tumor incidence rates, and an increase in the average body weight of control animals among the various rodent species and strains used for toxicity testing. These observations have prompted an evaluation of chronic study designs to determine what factor(s) may be responsible for such confounding changes. Ad libitum feeding and the selection of successful breeders with rapid offspring growth is believed to be at least partially responsible for the heavier, obese rodents with which many laboratories are coping today. As a result of these changes, some studies used for the evaluation of safety have been deemed inconclusive or inadequate for regulatory purposes and either additional supportive studies have been requested and/or studies per se have been repeated. Research on the molecular mechanisms of caloric restriction and agent-induced toxicity at the Food and Drug Administration (FDA) National Center for Toxicological Research stimulated the first international conference on the biological effects of dietary restriction in 1989; this was followed in 1993 by an FDA workshop exploring the utility of dietary restriction in controlling reduced survival in chronic tests and an international conference in 1994 exploring the implications for the regulatory community of using dietary restriction in toxicity and carcinogenicity studies used in support of a sponsors submission or in risk assessments. The outcome of that conference was the FDAs commitment to develop Points-to-Consider documents that address the issue of dietary control in chronic toxicity and carcinogenicity studies.

Metabolism of the arylamide herbicide propanil: II. Effects of propanil and its derivatives on hepatic microsomal drug-metabolizing enzymes in the rat☆

Propanil (3,4-dichloropropionanilide) is an arylamide herbicide that has been reported to be contaminated with the cytochrome P450 enzyme inducers 3,3,4,4-tetrachloroazobenzene (TCAB) and 3,3,4,4-tetrachloroazoxybenzene (TCAOB), which are structural analogs of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). We determined if treatment of rats with TCAB, TCAOB, propanil, 3,4-dichloroaniline, TCDD, or phenobarbital induced the hepatic microsomal metabolism of propanil and 3,4-dichloroaniline. Acylamidase-catalyzed hydrolysis of propanil to 3,4-dichloroaniline was not induced by any of the pretreatments; however, hydroxylation of propanil at the 2-position was induced by TCDD, TCAB, TCAOB, propanil, and 3,4-dichloroaniline pretreatments. Ring- and N-hydroxylations of 3,4-dichloroaniline were induced by TCDD, TCAB, TCAOB, and 3,4-dichloroaniline pretreatments. Microsomal 7-ethoxyresorufin-O-deethylase (EROD) and 7-benzoxyresorufin-O-dealkylase (BROD) activities and electrophoretic mobility of microsomal proteins suggested that cytochromes P450c and P450d were induced by TCAB and TCAOB pretreatment. EROD, BROD, and 7-pentoxyresorufin-O-dealkylase activities were slightly increased in microsomes from propanil- and 3,4-dichloroaniline-pretreated rats, which suggests that these compounds may be weak inducers of cytochrome P450 isozymes.

Effects of Long-Term Caloric Restriction on Hepatic Drug-Metabolizing Enzyme Activities in the Fischer 344 Rat

It is well established that caloric restriction (CR) in rodents can result in an increased maximally achievable life span and a decreased incidence and proliferative rate of neoplasia (Maeda et al. 1985; Masoro 1985, 1988). Although the precise molecular mechanisms underlying these effects have not been satisfactorily elucidated, CR has been associated with a wide range of biochemical and endocrinological alterations that occur in a spectrum of tissues (Weindruch and Walford 1988).