Biochemical effects of three carcinogenic chlorinated methanes in rat liver

Three chlorinated methanes, carbon tetrachloride, chloroform, and methylene chloride, known to cause liver tumors in rodents, were given by oral gavage to adult female rats both 21 h and 4 h before sacrifice. Then hepatic DNA damage, ornithine decarboxylase (ODC), cytochrome P-450, glutathione content, and serum alanine aminotransferase (SGPT) activity assays were performed. Carbon tetrachloride increased rat hepatic ODC activity and decreased cytochrome P-450 content at doses both below and above cytotoxicity (as measured by increased SGPT activity). At 54 and 160 mg/kg, chloroform increased hepatic ODC activity with minimal or no elevation in SGPT activity. At 480 mg/kg chloroform increased hepatic ODC and SGPT activity. A dose of 1,275 mg/kg methylene chloride caused a small, but significant amount of hepatic DNA damage. When these three compounds are compared on either an equimolar or equitoxic (1/5 LD50) basis, their ability to induce hepatic ODC or increase SGPT activity was carbon tetrachloride greater than chloroform greater than methylene chloride. The results of this biochemical study are interpreted with respect to the ability of chemicals to cause hepatic cancer by either genetic or epigenetic mechanisms.     

Effect of l-carnitine supplementation on xenobiotic-metabolizing hepatic enzymes exposed to methanol

The study aimed to evaluate the effect of L-carnitine on hepatic cytochrome P450-dependent monooxygenases exposed to methanol. Male Spraque-Dawley rats were given methanol (1/4 LD50 and 1/2 LD50) together with L-carnitine (1g/kg body weight). The parameters of microsome electron transport chains I and II and the levels of CYP2E1, CYP2B1/2 and CYP1A2 were measured 8, 12, 24, 48, 72 and 96 h after exposure. L-carnitine did not affect cytochrome P450 but it significantly increased at 72 and 96 h NADPH-cytochrome P450 reductase. It stimulated cytochrome b5 at 48 and 96 h and NADH-cytochrome b5 reductase activity at 12, 72 and 96 h. Methanol, especially the lower dose, inhibited cytochrome P450 after 48 h, but the higher methanol dose inhibited NADH-cytochrome b5 reductase activity in this time. L-carnitine, combined with the lower dose of methanol, stimulated NADPH-cytochrome P450 reductase after 48 h and cytochrome b5 and NADH-cytochrome b5 reductase over the whole period of observation. L-carnitine stimulated CYP2B1/2 but not CYP2E1 and CYP1A2. Methanol stimulated CYP2E1 at 24 h, but CYP1A2 at 96 h in the studied doses. CYP2B1/2 was induced by the lower dose of methanol at 24 h but by the higher one at 96 h. When given together, L-carnitine and methanol (1/2 LD50) significantly stimulated CYP2E1 up to 170% at 24 h and 145% at 96 h.     

Effects of inhibition and induction of cytochrome P-450 isozymes on hyperoxic lung injury in rats.

Pulmonary oxygen toxicity most likely results from excessive production of reactive oxygen species. The role of the cytochromes P-450 in this process is controversial because these enzymes have been reported both to enhance hyperoxic lung injury and to protect from the damaging effects of 100% oxygen. We sought to further determine the role of the cytochromes P-450 in hyperoxic lung injury by inhibiting and inducing pulmonary cytochrome P-450 isozymes in rats. Treatment with the cytochrome P-450 inhibitor cimetidine or 8-methoxypsoralen did not improve survival or reduce lung edema in rats exposed to 100% oxygen. The activity of cytochrome P-450IIB1, the major pulmonary cytochrome P-450 isozyme in rats, was clearly inhibited by 8-methoxypsoralen. beta-Naphthoflavone (beta NF), a selective inducer of cytochrome P-450IA1, was administered in two-dose and five-dose regimens. The two-dose regimen produced significant and sustained induction of cytochrome P-450IA1 activity, but survival in these rats was not improved when exposed to 100% oxygen. In rats treated with five doses of beta NF, a small increase in survival time was found from 71.1 +/- 8.7 to 88.0 +/- 20.2 h; however, there was no difference in the induction of cytochrome P-450IA1 activity between this five-dose regimen and the two-dose regimen. The small improvement in survival after five doses of beta NF is thus unrelated to cytochrome P-450IA1 induction. We conclude that neither inhibition of cytochrome P-450IIB1 activity nor induction of cytochrome P-450IA1 activity protects adult rats against hyperoxic lung injury.     

Effect of PSK, a protein-bound polysaccharide from Coriolus versicolor, on drug-metabolizing enzymes in sarcoma-180 bearing and normal mice

The effects of PSK and Propionibacterium acnes (anaerobic Corynebacterium) on hepatic drug-metabolizing enzymes were studied using sarcoma-180 bearing and non-tumor bearing mice. PSK had no influence on aminopyrine N-demethylase and aniline hydroxylase activities, cytochrome P-450 concentration in hepatic microsomes, and the reductase activity of cytochrome c in normal mice. The content of cytochrome P-450 was not significantly reduced in S-180 bearing mice. On the other hand, P. acnes administration significantly decreased the amount of cytochromes P-450 and b5 and aminopyrine N-demethylase activity. When FT-207 (Tegafur) was administered orally to S-180 bearing mice combined with the immunoadjuvants, only P. acnes significantly reduced the 5-FU levels in the serum and some organs.     

Induction of tolerance in mice and rats to the effect of endotoxin to decrease the hepatic microsomal mixed-function oxidase system. Evidence for a possible macrophage-derived factor in the endotoxin effect

Daily administration of low, non-lethal doses of bacterial endotoxin to mice and rats has been shown to induce tolerance to the effect of an acute challenge dose of endotoxin to decrease the hepatic microsomal drug metabolizing activity, the level of cytochrome P-450, and to increase heme oxygenase activity. The serum collected at various times after injection of endotoxin into control animals when injected into untreated animals markedly depressed aniline hydroxylase activity, ethylmorphine N-demethylase activity, and the level of cytochrome P-450. Tolerant animals were not affected by the post-endotoxin serum injection, suggesting the decreased activity caused by the serum in untreated animals was probably due to endotoxin contained in the serum. Injection of tolerant mice and rats with supernatant medium obtained from cultures of peritoneal macrophages incubated with 100 micrograms/ml of endotoxin caused a loss of hepatic microsomal drug-metabolizing activity, and a decrease in the level of cytochrome P-450. These results suggest that peritoneal macrophages release a factor in response to endotoxin that mediates the decreased hepatic mixed-function oxidase activity.     

Evaluation in a battery of in vivo assays of four in vitro genotoxins proved to be noncarcinogens in rodents.

2-Chlorethanol, 8-hydroxyquinoline, 2,6-toluenediamine, and eugenol, previously found to behave as genotoxins in in vitro systems and as noncarcinogens in rodents, were evaluated for their ability to induce genotoxic effects in vivo. Rats were given by gavage a single or two successive doses equal to one-half the corresponding LD50, killed at different times after treatment, and examined for the following end points: the frequency of both micronucleated polychromatic erythrocytes in the bone marrow and micronucleated hepatocytes (after partial hepatectomy); the in vivo-in vitro induction of DNA fragmentation, as measured by the alkaline elution technique, and of unscheduled DNA synthesis, as measured by autoradiography, in hepatocyte primary cultures. The two latter end points were also evaluated after in vitro exposure of hepatocytes to log-spaced subtoxic concentrations. 2-Chloroethanol, 8-hydroxyquinoline, and eugenol never produced effects indicative of genotoxic activity. The same happened with 2,6-toluenediamine, with the exception of a significant increase over controls in the amounts of DNA damage and repair displayed by hepatocyte cultures obtained from rats given two 1/2 LD50 separated by a 24 h interval. Our results, which, apart the above mentioned exception, are in concordance with the rodent carcinogenicity results, contribute to underline the role of in vivo short-term tests for the detection of potential genotoxic carcinogens.     

Carbon tetrachloride hepatotoxicity in endotoxin tolerant and polymyxin B-treated rats

Gut-derived endotoxin has been implicated in the hepatotoxic effects of CCl4. The present study has investigated whether two procedures known to block LPS effects would alter the action of CCl4 to decrease hepatic cytochrome P-450 and microsomal drug-metabolizing activity. Administration of polymyxin B or induction of LPS tolerance were shown to attenuate the effect of CCl4 administration to increase SGOT and SGPT levels, signs of hepatic damage. Polymyxin B administration but not LPS tolerance caused a slight decrease in cytochrome P-450. In pretreated animals given CCl4, only those which had received polymyxin B showed a reduced effect of CCl4 to alter cytochrome P-450 level and activity. However, the apparent protective effect was of the same magnitude as the loss of cytochrome P-450 caused by polymyxin B itself. These results suggest that the ability of polymyxin B to ablate the CCl4 loss of P-450 might be due to a reduced metabolic activation of CCl4 by P-450 and not due to any anti-LPS activity. The results suggest that gut-derived LPS does not participate in the effect of CCl4 decreasing cytochrome P-450-mediated reactions. However, participation of LPS in other hepatotoxic effects of CCl4 is not excluded.     

Effect of traditional Chinese herbal medicines on the pharmacokinetics of western drugs in Sprague-Dawley rats of different ages (II): Aminophylline-huan shao tan and aminophylline-pu chung yi chi tang.

The effect of Chinese herbal medicines (Huan Shao Tan and Pu Chung Yi Chi Tang) and western drugs (sodium phenobarbital and cimetidine) on the serum concentration and pharmacokinetic parameters of theophylline and cytochrome P-450 of Sprague-Dawley (SD) rats of three different ages were examined. The older rats without pretreatment with Chinese herbal medicines and western drugs exhibited higher serum theophylline concentration and lower pharmacokinetic parameters of theophylline than middle-aged and younger rats (P < 0.05), but there was no difference in cytochrome P-450 activity among the three different ages of rats. All rats when pretreated with sodium phenobarbital showed lower serum theophylline concentration and higher pharmacokinetics parameters of theophylline. Also, the activity of cytochrome P-450 was higher (P < 0.05). When cimetidine was pre-administered in SD rats of three age groups, all rats exhibited lower serum theophylline concentration and higher pharmacokinetics parameters (P < 0.05), but the activity of cytochrome P-450 remained unchanged (P > 0.05). The results were opposite to other studies, probably because the dose and dosing intervals were different. No single effect occurred on the younger and middle-aged rats after pretreatment with Huan Shao Tan and Pu Chung Yi Chi Tang: their serum theophylline concentration, pharmacokinetics parameters and cytochrome P-450 activity were the same as the control group. However, the older rats after pretreatment with Huan Shao Tan or Pu Chung Yi Chi Tang showed lower serum theophylline concentration and higher pharmacokinetics parameters than the younger and middle-aged rats pretreated with similar Chinese herbal medicines. This indicates that Huan Shao Tan and Pu Chung Yi Chi Tang may perhaps improve the elimination of theophylline in older rats. This might be attributed to the increase in hepatic blood flow or in liver volume, since the activity of cytochrome P-450 was not affected by the administration of Chinese herbal medicines.     

Aldehydic products of lipid peroxidation inactivate cytochrome P-450.

The effects of aldehydic products of lipid peroxidation, malondialdehyde (MDA) and 4-hydroxynonenal (HNE), on the structure of rat liver microsomal membrane and cytochrome P-450 was studied. MDA (15-30 microM) similarly to p-chlormercuribenzoate decreased the cytochrome P-450 content by 50 % and lowered microviscosity of lipid surrounding of the spin label OTMB bound to SH-groups of membrane proteins. OTMB was effectively reduced by K3Fe(CN)6 in microsomes preincubated with MDA (20 (M), but not in native microsomes. HNE (10 microM) decreased the cytochrome P-450 content by 90 %. Reduced glutathione and cysteine (5 mM) prevented the decrease of cytochrome P-450 under influence of both MDA or HNE, whereas cytochrome P-420 formation remains unchanged. MDA and HNE decreased activities of NADPH oxidase and NADPH cytochrome c reductase. HNE increased microviscosity of the OTMB lipid environment. The further increase of HNE concentration did not affect this parameter. Both MDA and HNE increased the absorbance at 420 nm, which indicated inactivation of cytochrome P-450 by changes in hydrophobicity of lipid surrounding. We suggest that HNE and aliphatic aldehydes at low concentrations can enter into hydrophobic environment of cytochrome P-450 binding to its SH-groups, which led to inactivation of cytochrome P-450. At the same time, the modification of membrane surface layer and subsequent decrease of hydrophobicity of cytochrome P-450 environment preceded the binding of MDA to SH-groups of cytochrome P-450 to develop its inactivating effect.     

Plasma high-density lipoproteins and liver lipids and proteins in man. Relation to hepatic histology and microsomal enzyme induction

The association of high-density lipoproteins (HDL) in plasma with liver lipids and proteins was investigated in 28 subjects with diagnostic liver biopsy. Lipids and proteins were evaluated in relation to hepatic histology and microsomal enzyme induction, assessed by liver cytochrome P-450. Moderate-severe hepatic parenchymal changes were associated with low liver phospholipids, protein and cytochrome P-450, low plasma HDL cholesterol (HDL-C), and high hepatic triglycerides. Liver microsomal induction accompanying anticonvulsant therapy was associated with high liver phospholipids and protein, high plasma HDL-C, apoproteins A-I and A-II, and high HDL-C/total cholesterol (T-C) ratio. HDL-C, A-I and the HDL-C/T-C ratio were directly proportional to liver phospholipids, protein and cytochrome P-450, inversely related to hepatic triglycerides. Increases in hepatic phospholipids and protein, characteristic of microsomal induction, may lead to the elevation of plasma HDL apoprotein and HDL-C levels and HDL-C/T-C ratios, and thus reduce the risk of coronary heart disease.     

The structure--activity relationships of flavonoids as inhibitors of cytochrome P-450 enzymes in rat liver microsomes and the mutagenicity of 2-amino-3-methyl-imidazo[4,5-f]quinoline

The antimutagenicity of 19 naturally occurring flavonoids andtheri derivatives including flavones, flavonols, flavanoes,inoflavones and flavanols were determined using Salmonella typhimuriumTA98 against 2-amino-3-methylimidazo[4,5-f] quinoline (IQ) inthe presence of Aroclor 1254-induced rat hepatic S9. In general,a relationship between the chemical structure of flavonoidsand their antimutagenicity was found for compounds containingone or more of the following featutes: (i) C4 keto group, (ii)aglycone, (iii double bond at positions C2 and C3, (iv) phenylgroup at position C2, and (v) three hydroxyl substituents atpositons C4', C5 and C7. The inhibitory effects of flavonoidson activities of 7-ethoxycoumarin deethylase (ECD) and 7-ethoxyresorufindeethylase (ESD) of Aroclor 12540-induced hepatic microsomeswere also examined. In addition, we studied the effects of flavonoidson the metabolism of IQ by Aroclor 1254-induced microsomes usinghigh-performance liquid chromatography. The antimutagenicitycorrelated with the inhibition of cytochrome P-450IA1-linkedESD and P-450IA2-linked ECD activity in hepatic microsomes,and with an inhibition of N-hydroxy-IQ fromation from IQ metabolismby hepatic microsomes. These reslute indicated that flavonesor flavonols that contasin C5, C7 and C4' hydroxyl groups arepotent inhibitors of P-450 enzyme activities induced by Aroclor1254 (P-450IA1 and P-450IA2), and masy potentially be usefulas chemopreventive agents against heterocyclic amine-inducedmutagenesis or carcinogenesis. ¹To whom correspondence sould be addressed     

The antitoxic function of the liver in D-galactosamine poisoning

Two intragastric administrations of 500 mg/kg of D-galactosamine reduce the RNA and the cytochrome P-45, and b5 content in the hepatic microsomes of rats; inhibit the activity of aminopyrine-N-demethylase, hexobarbital hydroxylase, aniline-p-hydroxylase, and glutathione-S-transferase; reduce the rate of NADP.H and NAD.H oxidation; accelerate inactivation of cytochrome P-450 to cytochrome P-420; reduce the number of points of hexobarbital binding with N-octilamine, though increase the hemoprotein affinity to these substrates. Destruction of the nucleus, endoplasmic reticulum, and mitochondria occurs in the hepatocytes of D-galactosamine poisoned rats.     

Cytochrome P-450 metabolic-intermediate complex formation with a series of diphenhydramine analogues

A series of diphenhydramine analogues have been studied with regard to their formation of a metabolic intermediate (MI) during their biotransformation in phenobarbital induced rat hepatic microsomes. The MI forms a complex with reduced cytochrome P-450. MI complexation of cytochrome P-450 may result in drug-drug interactions and/or in cumulation of the parent compound. The extent of MI complex formation could be correlated with the lipophilicity of the substrates in a parabolic manner. A hydrophobic pocket of limited dimensions in cytochrome P-450 for the N-alkyl substituent of the substrates can be assumed. Moreover our data indicate a role for the O-atom in the diphenhydramine analogues for the interaction with cytochrome P-450.     

Immunohistochemical localization of cytochrome P-450 in rat brain

The presence of cytochrome P-450 in rat brain was studied by immunohistochemistry, using antibodies to cytochrome P-450 purified from livers of phenobarbital- or 3-methylcholanthrene-treated rats. Immunoreactive nerves were observed only in brain sections incubated with immunoglobulin-G to 3-methylcholanthrene-induced cytochrome P-450. This immunoreactivity was abolished by preabsorption of the antibody with highly purified rat liver cytochrome P-450c, the major cytochrome P-450 isozyme induced by 3-methylcholanthrene, but was not affected by other cytochrome P-450 isozymes induced by phenobarbital, isosafrole or pregnenolone-16-carbonitrile.

The most abundant concentration of nerve fibers with cytochrome P-450 immunoreactivity was observed in the globus pallidus. Immunoreactive fibers were also observed in the caudate putamen, amygdala, septum, ventromedial nucleus of the hypothalamus, medial forebrain bundle, ansa lenticularis, and ventromedial portion of the internal capsule and crus cerebri. Cell bodies with cytochrome P-450 immunoreactivity were observed in the caudate putamen and in the perifornical area of the hypothalamus. The cytochrome P-450 immunoreactive fibers in the globus pallidus and caudate putamen do not appear to emanate from cell bodies in the substantia nigra, since there was no reduction in the density of these fibers after unilateral stereotaxic electrolytic destruction of the substantia nigra (zona compacta and reticulata). Our data suggest that these striatal nerve processes are derived from cell bodies within the caudate putamen itself.

The present results indicate that rat brain contains a form of cytochrome P-450 with antigenic relatedness to the hepatic 3-methylcholanthrene-inducible cytochrome P-450c. This cytochrome P-450 isozyme was detected in brain areas which metabolize morphine and convert estradiol and estrone into catecholestrogens, which suggests an important role for this enzyme in the metabolism of both ex´ogenous and endogenous compounds in brain.     

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.     

In vivo and in vitro inhibition of hepatic microsomal drug metabolism by ketoconazole.

Ketoconazole (KC), a broad spectrum antifungal drug, has been recognized recently as a cause of hepatic injury. The mechanism of the adverse reaction remains unclear: a metabolic idiosincrasy has been suggested. However as a substituted imidazole, KC might be expected to interfere with the hepatic microsomal mixed function oxidases. Ethylmorphine N-demethylase (E-DM) and aniline hydroxylase (A-OH) activities were determined in rat liver microsomes in the presence of increasing amounts of KC. Both were inhibited in an exponential fashion. The E-DM inhibition was almost complete at concentrations greater than 250 microM and was of the mixed type. A much weaker effect was observed for A-OH. A significant inhibition of E-DM was also observed when KC was administered in vivo to rats either orally for 7 days at the dose of 100 mg/kg/day (P less than 0.02) or intraperitoneally for 4 days at the dose of 50 or 100 mg/kg day (P less than 0.01 or P less than 0.001 respectively). A-OH activity was significantly reduced (P less than 0.01) only after ip administration of 100 mg/kg/day of the drug for 4 days. Neither the amount of cytochrome P-450 nor NADPH cytochrome c reductase activity were affected at the doses considered. These data show that KC interferes with hepatic oxidative drug metabolism and suggest that this mechanism might be involved in the unwanted side effects of therapy with KC.     

In vivo and in vitro inhibition of hepatic microsomal drug metabolism by ketoconazole

Ketoconazole (KC), a broad spectrum antifungal drug, has been recognized recently as a cause of hepatic injury. The mechanism of the adverse reaction remains unclear: a metabolic idiosincrasy has been suggested. However as a substituted imidazole, KC might be expected to interfere with the hepatic microsomal mixed function oxidases. Ethylmorphine N-demethylase (E-DM) and aniline hydroxylase (A-OH) activities were determined in rat liver microsomes in the presence of increasing amounts of KC. Both were inhibited in an exponential fashion. The E-DM inhibition was almost complete at concentrations greater than 250 microM and was of the mixed type. A much weaker effect was observed for A-OH. A significant inhibition of E-DM was also observed when KC was administered in vivo to rats either orally for 7 days at the dose of 100 mg/kg/day (P less than 0.02) or intraperitoneally for 4 days at the dose of 50 or 100 mg/kg day (P less than 0.01 or P less than 0.001 respectively). A-OH activity was significantly reduced (P less than 0.01) only after ip administration of 100 mg/kg/day of the drug for 4 days. Neither the amount of cytochrome P-450 nor NADPH cytochrome c reductase activity were affected at the doses considered. These data show that KC interferes with hepatic oxidative drug metabolism and suggest that this mechanism might be involved in the unwanted side effects of therapy with KC.     

Protection from N-nitrosodimethylamine-mediated liver damage by indole-3-carbinol

Indole-3-carbinol (I-3-C) was examined for its ability to protect mice against 24-hr N-nitrosodimethylamine (NDMA)-mediated hepatotoxicity. NDMA (20 mg/kg body weight) alone produced extensive hemorrhagic and centrolobular necrotic lesions, with a necrotic severity index of 3.0 +/- 0.4 (scale of 0-5). Treatment with 50 mg/kg body weight of I-3-C by gavage, 1 hr prior to NDMA, substantially protected against hemorrhagic lesions. Furthermore, I-3-C lowered the NDMA-mediated tissue necrotic index to 1.5 +/- 0.3, by reducing the extent of tissue necrosis rather than the severity in the necrotic region. Release of liver enzymes into the blood correlated with the histopathology; I-3-C reduced NDMA-mediated elevated activities of plasma alanine transaminase and ornithine transcarbamylase by 84 and 51.3%, respectively. Although no changes in nonprotein sulfhydryls were evident at 24-hr after NDMA, ascorbate levels were reduced to 40% of control values. However, treatment with I-3-C prior to NDMA prevented the decline in tissue ascorbate concentrations. In vitro, I-3-C was found to be a type II ligand for cytochrome P-450, with a Ks value of 237 microM. However, if such binding occurs in vivo, it does not protect against the approximately 60% decrease in hepatic cytochrome P-450 or the 80% decrease in NDMA demethylase I activity produced by NDMA. Since I-3-C slightly enhances cytochrome P-450 content and NDMA demethylase activity, the histopathologic protection by I-3-C must be due to factors other than inhibiting metabolic activation of NDMA.     

Impairment of hepatic cytochrome P-450-dependent monooxygenases by the malaria parasite Plasmodium berghei

The effect of Plasmodium berghei infection on hepatic monooxygenase activities and cytochrome P-450 contents was investigated in mice. NIH/NMRI or A/J mice infected with active P. berghei showed 30-40% decreases in hepatic cytochrome P-450 contents and the ability to metabolize the test substrates, ethylmorphine and benzo(a)pyrene. These decreases were observed during the erythrocytic stage of the infection, but not during the initial exoerythrocytic stage, or after heat-inactivated sporozoites were injected. These results strongly suggest that malaria infections may significantly impair the capacity of the liver to metabolize drugs, carcinogens, and other foreign compounds.     

Hepatic ornithine decarboxylase activity in the rat as influenced by retinyl acetate and ethanol or tryptophan

Ethanol and tryptophan have been demonstrated earlier to induce a rapid stimulation of hepatic ornithine decarboxylase (ODC) activity in overnight-fasted rats. In this study the effect of the administration of retinyl acetate prior to administering ethanol or tryptophan was investigated. The levels of ODC activity in the livers of control and experimental rats were assayed in vitro by measuring the release of 14CO2 from DL-[1-14C]ornithine. Intraperitoneal administration of retinyl acetate (1 microgram/100 g body wt) 1 hr before tube feeding ethanol (0.75 g as a 50% solution/100 g body wt) or L-tryptophan (30 mg in 3 ml water/100 g body wt) and 3 hr before killing caused an enhanced stimulation of hepatic ODC activity compared to that when each agent was administered alone. In vitro [14C]leucine incorporation into protein using hepatic microsomes of tryptophan-treated rats with or without retinyl acetate was increased in comparison with that of controls while decreases were observed when using microsomes of ethanol-treated rats with or without retinyl acetate. Although retinyl acetate has been reported earlier to inhibit the stimulation of hepatic ODC activity due to a variety of agents, including some agents known as carcinogens or promoters, it did not act in this manner against the acute administration of ethanol or tryptophan.