Amino acid regulation of synthesis of ribonucleic acid and protein in the liver of rats

Weanling (23-day-old) rats were fed on either a low-protein diet (6% casein) or a diet containing an adequate amount of protein (18% casein) for 28 days. Hepatic cells from animals fed on the deficient diet were characterized by markedly lower concentrations of protein and RNA in all cellular fractions as compared with cells from control rats. The bound rRNA fraction was decreased to the greatest degree, whereas the free ribosomal concentrations were only slightly less than in control animals. A good correlation was observed between the rate of hepatic protein synthesis in vivo and the cellular protein content of the liver. Rates of protein synthesis both in vivo and in vitro were directly correlated with the hepatic concentration of individual free amino acids that are essential for protein synthesis. The decreased protein-synthetic ability of the ribosomes from the liver of protein-deprived rats was related to a decrease in the number of active ribosomes and heavy polyribosomes. The lower ribosomal content of the hepatocytes was correlated with the decreased concentration of essential free amino acids. In the protein-deprived rats, the rate of accumulation of newly synthesized cytoplasmic rRNA was markedly decreased compared with control animals. From these results it was concluded that amino acids regulate protein synthesis (1) by affecting the number of ribosomes that actively synthesize protein and (2) by inhibiting the rate of synthesis of new ribosomes. Both of these processes may involve the synthesis of proteins with a rapid rate of turnover.     

The regulation of protein synthesis in the liver of rats. Mechanisms of dietary amino acid control in the immature animal

Weanling (23-day-old) rats were fed either on an amino acid-deficient diet (6% of casein, which in effect represents an `amino acid-deficient' diet) or on a diet containing an adequate amount of protein (18% of casein) for 28 days. The hepatic cells from the animals fed on the low-protein diet were characterized by low amino acid content, almost complete inhibition of cell proliferation and a marked decrease in cell volume, protein content and concentration of cytoplasmic RNA compared with cells from control rats. The lower concentration of cytoplasmic RNA was correlated with a decreased ribosomal-RNA content, of which a larger proportion was in the form of free ribosomes. The protein-synthetic competence and messenger-RNA content of isolated ribosomes from liver cells of protein-deprived animals were 40–50% of those noted in controls. At 1hr. after an injection of radioactive uridine, the specific radioactivity of liver total RNA was greater in the group fed on the low-protein diet, but the amount of label that was associated with cytoplasmic RNA or ribosomes was significantly less than that noted in control animals. From these data it was concluded that dietary amino acids regulate hepatic protein synthesis (1) by affecting the ability of polyribosomes to synthesize protein and (2) by influencing the concentration of cytoplasmic ribosomes. It is also tentatively hypothesized that the former process may be directly related to the concentration of cellular free amino acids, whereas the latter could be correlated with the ability of newly synthesized ribosomal sub-units to leave the nucleus.     

Influence of the adrenal glucocorticoids on the stimulation of synthesis of hepatic ribonucleic acid and plasma acute-phase globulins by leucocytic endogenous mediator.

An injection of unpurified leucocytice endogenous mediator into rats results in an increased incorporation of [6(-14)C]orotate into hepatic RNA, an increase in the concentration of RNA associated with the bound ribosomal fraction of liver, and increases in the concentrations in serum of acute-phase proteins such as alpha2-macrofoetoprotein and haptoglobin. If given 3 days after adrenalectomy or 7 days after hypophysectomy,, leucocyte factor did not induce the increase in RNA synthesis or alpha2-macrofoetoprotein concentrations but did stimulate an increase in serum haptoglobin. When hypophysectomized or adrenalectomized rats received daily subcutaneous injections of 0.5mg of cortisol, leucocyte factor again induced a significant increase in the synthesis of hepatic RNA and an increase in the concentration of serum alpha2-macrofoetoprotein. These observations suggest that leucocyte factor can regulate acute-phase-protein synthesis at several different sites, one or more of which requires permissive action of the glucocorticoid hormones. Futher, leucocyte factor will stimulate an increase rate of incorporation of orotate into hepatic ribosomes when added in vitro in the presence of cortisol to a liver-perfusion system. Thus the stimulatory effect of leucocyte factor may be directy on liver but may require the presence of other hormones to stimulate the incorporation of orotate into RNA.     

Effect of dietary protein on cholesterol homeostasis in diabetic rats

Normal and streptozotocin (STZ)-diabetic rats were studied in order to examine the effects of altering the type of dietary protein on cholesterol homeostasis. Rats were fed a non-purified or a purified diet containing either casein or soybean protein. The results obtained on the specific aspects of lipid metabolism were remarkably similar in control rats fed the non-purified (Purina Lab Chow) diet or the purified diet with the soybean protein. However, most of the findings obtained with the above two groups were different from those obtained with rats fed the purified diet containing casein. In the latter group, plasma cholesterol was elevated following a 15-day feeding period as compared to the other two dietary groups. The excess plasma cholesterol in the casein-fed group was found in two lipoprotein fractions with densities of 1.023-1.045 g/ml and 1.045-1.086 g/ml, respectively. The latter lipoprotein fraction was also enriched with apolipoprotein E. The casein-fed animals also showed a lower fractional rate of plasma cholesterol esterification and an abnormal accumulation of cholesterol in the body despite inhibition of cholesterol synthesis in the liver and in the intestines. Twelve to 15 days after the induction of diabetes, plasma cholesterol increased to a similar extent in the rats on all three diets. However, the distribution of cholesterol among the lipoprotein fractions was markedly different. The percentage of cholesterol in fractions of d less than 1.086 g/ml was increased while that carried in the fraction of d 1.086-1.161 g/ml decreased in the rats fed the nonpurified diet and the casein diet. In contrast, there was no change in the distribution of lipoprotein cholesterol between the diabetic and the control rats fed the soybean protein diet. The hepatic synthesis of cholesterol was unaltered in diabetic rats fed the nonpurified diet and the purified diet with soybean protein, but was increased 2.4-fold in diabetic rats fed casein. Intestinal cholesterol synthesis was increased in all three dietary groups. The increase was highest in the rats fed casein and lowest in rats fed soybean protein. The rate of sterol synthesis in the kidneys was not significantly affected by the diet or diabetes. In all three dietary groups diabetes led to an abnormal accumulation of cholesterol in the body. This accumulation was highest in the casein-fed rats and lowest in those fed the soybean protein diet. The cholesterol content of the kidneys was markedly increased by dietary casein.(ABSTRACT TRUNCATED AT 400 WORDS)     

Regulation of hepatic synthesis of proteins by the chronology of protein ingestion

Circadian variations in liver protein synthesis were were assessed in control rats fed a mixed 10% protein diet and in rats fed proteins as a separate meal either at 09:00 (SF 09) or at 21:00 (SF 21) and provided with a protein-free diet ad libitum. Protein synthesis was measured by incorporation of labelled leucine over a short period of time (15 min) at time-points regularly spaced over 24 h. In controls, the circadian variations observed were of moderate amplitude (from 2.75 mg/h per g at 09:00 to 5.77 mg/h per g at 06:00) correlated with increased protein and RNA contents of the liver. In separately fed animals ingestion of the protein meal triggered a 300% increase in protein synthesis within 1 h while the feeding pattern was unaltered. In the SF 09 group, high synthetic activity was not followed by an increase of hepatic protein content while hepatic urea concentrations were sharply increased and glucogenic amino acid pools were greatly depleted. It is suggested that the high influx of amino acids consecutive to the absorption of the dietary proteins is the key factor stimulating protein synthesis, while synchronisation with the energetic metabolism controls the degree of degradation. The possible involvement of variations in the insulin to glucagon ratio is discussed.     

Effects of protein-deprivation on the regeneration of rat liver after partial hepatectomy.

Rats maintained on a protein-free diet for 3 days have an altered time course of hepatic DNA synthesis during liver regeneration. The delay in DNA synthesis is eliminated by the administration of casein hydrolysate (given as late as 6h after partial hepatectomy), but not by glucose or incomplete amino acid mixtures. Despite the change in the timing of DNA synthesis, the increases in hepatic amino acid pools, which take place at the earliest stages of the regenerative process, occur in a normal pattern in the regenerating liver of rats fed the protein-free diet. Protein-deprived rats have increased protein synthesis and decreased rates of protein degradation in the liver in response to partial hepatectomy, but these adaptations do not prevent a lag in protein accumulation and low protein/RNA ratios. The regenerating livers of these animals show a deficit in the accumulation of cytoplasmic polyadenylated mRNA as well as a smaller proportion of free polyribosomes. It is suggested that the deficit in free polyribosomes found in the regenerating liver of protein-deprived rats might be a consequence of the slow accumulation of mRNA species coding for intracellular proteins.     

Deficiency of dietary EAA preferentially inhibits mRNA translation of ribosomal proteins in liver of meal-fed rats

The goal of these studies was to investigate the mechanisms by which amino acid supply regulates global rates of protein synthesis as well as the translation of ribosomal protein (rp) mRNAs in liver. In the experiments conducted, male weanling rats were trained over a 2-wk period to consume their daily food intake within 3 h. On day 14, rats were fed the control diet or an isocaloric, isonitrogenous diet lacking glycine, tryptophan, leucine, or the branched-chain amino acids (BCAA) for 1 h. Feeding Trp-, Leu-, or BCAA-deficient diets resulted in significant reductions in serum insulin, hepatic protein synthesis, eukaryotic initiation factor 2B (eIF2B) activity, and phosphorylation of eIF4E-binding protein 1 (4E-BP1) and ribosomal protein S6 kinase (S6K1). Phosphorylation of eIF2alpha was inversely related to eIF2B activity under all conditions. Alterations in the hepatic synthesis of rp were assessed by changes in the distribution of rp (S4, S8, L26) mRNAs across sucrose density gradients and compared with non-rp (beta-actin, albumin) mRNAs. In all dietary treatments, non-rp mRNAs were mostly polysome associated. Conversely, the proportion of rp mRNAs residing in polysomes was two- to fivefold less in rats fed diets lacking tryptophan, leucine, or BCAA compared with rats fed the control diet. Total hepatic abundance of all mRNAs examined did not differ among treatment groups. For all parameters examined, there were no differences between rats fed the glycine-deficient diet and rats fed the control diet. The data suggest that essential amino acid (EAA) deficiency inhibits global rates of liver protein synthesis via a block in translation initiation. Additionally, the translation of rp mRNAs is preferentially repressed in association with decreased S6K1 phosphorylation.     

Effect of unbalanced diets on incorporation of delta-aminolevulinic acid into cytochrome P-450

The in vivo syntheses of two liver microsomal cytochromes P-450 PB3a, P-450 UT50 [(1987) Eur. J. Biochem., submitted] (Mr 50,000, 52,000) have been estimated by measuring the specific activity 2 h after i.p. administration of delta-[3H]aminolevulinic acid to male Sprague Dawley rats. The animals were fed either a standard rat chow (5% lard, 22% casein) or unbalanced diets (high lipid, 30% lard or low protein, 6% casein) with or without 50 ppm Phenoclor DP6. The high-lipid diet supported a more rapid body weight gain but had little impact on cytochrome P-450 content, expressed either per whole liver or per mg microsomal protein, and on the incorporation of the precursor into cytochrome P-450. The latter was determined by measuring the radioactivity incorporated into the cytochrome P-450 fraction, partially purified by affinity chromatography, as well as into two cytochrome P-450 isozymes (Mr 50,000 or 52,000) purified by DEAE-52 cellulose ion-exchange chromatography. The low-protein diet, on the other hand, severely depressed body weight gain and cytochrome P-450 content as well as incorporation of radioactivity, the lower-Mr cytochrome (Mr 50,000) being particularly affected. However, when a potent inducer, Phenoclor DP6, was added to the low-protein diet, cytochrome synthesis was restored indicating that the effect was reversible.     

Dietary γ-aminobutyric acid affects the brain protein synthesis rate in young rats

Summary. The purpose of this study was to determine whether the γ-aminobutyric acid (GABA) affects the rate of brain protein synthesis in male rats. Two experiments were done on five or three groups of young rats (5 wk) given the diets containing 20% casein administrated 0 mg, 25 mg, 50 mg, 100 mg or 200 mg/100 g body weight GABA dissolved in saline by oral gavage for 1 day (d) (Experiment 1), and given the diets contained 0%, 0.25% or 0.5% GABA added to the 20% casein diet (Experiment 2) for 10 d. The plasma concentration of growth hormone (GH) was the highest in rats administrated 50 mg and 100 mg/100 g body weight GABA. The concentration of serum GABA increased significantly with the supplementation groups. The fractional (Ks) rates of protein synthesis in brain regions, liver and gastrocnemius muscle increased significantly with the 20% casein + 0.25% GABA diet and still more 20% casein + 0.5% GABA compared with the 20% casein diet. In brain regions, liver and gastrocnemius muscle, the RNA activity [g protein synthesized/(g RNA·d)] significantly correlated with the fractional rate of protein synthesis. The RNA concentration (mg RNA/g protein) was not related to the fractional rate of protein synthesis in any organ. Our results suggest that the treatment of GABA to young male rats are likely to increase the concentrations of plasma GH and the rate of protein synthesis in the brain, and that RNA activity is at least partly related to the fractional rate of brain protein synthesis.     

Nutritional control of branched chain alpha-ketoacid dehydrogenase in rat hepatocytes

Branched chain alpha-ketoacid dehydrogenase (EC 1.2.4.4) complex, the rate-limiting enzyme of branched chain amino acid catabolism in most tissues, is subject to regulation by covalent modification, with phosphorylation inactivating and dephosphorylation activating the complex. The enzyme complex from liver of chow-fed rats is mainly in the active form but that from liver of rats fed a low-protein diet is mainly in the inactive form. Isolated hepatocytes were used to identify factors that affect interconversion of branched chain alpha-ketoacid dehydrogenase. The enzyme present in hepatocytes of rats fed a low-protein diet appears much more responsive to regulation by covalent modification than the branched chain alpha-ketoacid dehydrogenase present in hepatocytes of normal chow-fed rats. alpha-Chloroisocaproate, a specific inhibitor of the kinase responsible for phosphorylation and inactivation of the complex, greatly stimulates oxidation of alpha-keto[1-14C]isovalerate by hepatocytes prepared from rats fed a low-protein diet but not from normal chow-fed rats. Oxidizable substrates are also much more effective inhibitors of branched chain alpha-ketoacid oxidation with hepatocytes from rats fed a low-protein diet than from normal chow-fed rats. Activity measurements with cell-free extracts suggest that changes in flux through the dehydrogenase with intact hepatocytes prepared from rats fed a low-protein diet are explained in large part by changes in the proportion of the enzyme in the active, dephosphorylated form. Regulation of liver branched chain alpha-ketoacid dehydrogenase by covalent modification functions to conserve branched chain amino acids for protein synthesis during periods of restricted dietary protein intake.     

Effects of dietary soybean peptides on hepatic production of ketone bodies and secretion of triglyceride by perfused rat liver

Soybean protein isolate (SPI) was digested with protease to produce a peptides containing the low-molecular fraction (LD3) or a mixture of high- and low-molecular fractions (HD1). Rats were fed a diets containing SPI, LD3, or HD1 at a protein level equivalent to the 20% casein diet for 4 weeks. The serum triglyceride concentration was lower in rats fed SPI, LD3, and HD1 diets than in rats fed the casein diet, and the differences were significant for the cholesterol-enriched diet. The value for the LD3 group was the lowest among all groups for both the cholesterol-free and -enriched diets. The level of triglyceride in the post-perfused liver was significantly lower in the LD3 and HD1 groups and the SPI group than in the casein group irrespective of the presence of cholesterol in the diet. In the cholesterol-free diet, LD3 feeding as compared to casein feeding caused a reduction in triglyceride secretion from the liver to perfusate and an increment of hepatic ketone body production. The addition of cholesterol to the diets somewhat attenuated these effects of LD3. These results suggest that the low-molecular fraction in soybean peptides causes triglyceride-lowering activity through a reduction in triglyceride secretion from the liver to the blood circulation and the stimulation of fatty acid oxidation in the liver. There is a possibility that soybean peptides modulate triglyceride metabolism by changes in the hepatic contribution.     

Dietary orotic acid affects antioxidant enzyme mRNA levels and oxidative damage to lipids and proteins in rat liver

We investigated the effects of the dietary addition of orotic acid on liver antioxidant enzymes, mRNA levels of these enzymes, and peroxidative products by comparing casein with soy protein as the source of dietary protein. Rats fed the casein diet accumulated more liver lipids than those fed the soy protein diet when orotic acid was added. The addition of orotic acid lowered both the activity of liver Cu, Zn-superoxide dismutase and the level of Cu, Zn-superoxide dismutase mRNA. The addition of orotic acid led to a significant increase in the contents of conjugated dienes and protein carbonyls in the liver. In addition, dietary soy protein protected the increase in the levels of lipids and proteins peroxide induced by orotic acid. The addition of orotic acid to the casein diet increased the activities of both serum ornithine carbamoyltransferase and alanine aminotransferase. Thus, liver damage might result from the increased superoxide anion due to the decrease in the activity of hepatic superoxide dismutase, as well as increase in the production of hepatic peroxidative products in rats fed the casein diet with orotic acid.     

Adaptive changes in translation initiation activities for rat pancreatic protein synthesis with feeding of a high-protein diet

We have previously demonstrated that dietary protein induced pancreatic hypergrowth in pancreaticobiliary diverted (PBD) rats. Dietary protein and dietary amino acids stimulate protein synthesis by regulating translation initiation in the rat skeletal muscle and liver. The aim of the present study was to determine whether feeding a high-protein diet induces activation of translation initiation for protein synthesis in the rat pancreas. In PBD rats in which the bile-pancreatic juice was surgically diverted to the upper ileum for 11-13 days, pancreatic dry weight and protein content were doubled compared with those in sham rats and further increased with feeding of a high-protein diet (60% casein diet) for 2 days. These pancreatic growth parameters were maintained at high levels for the next 5 days and were much higher than those of sham rats fed a high-protein diet. In both sham and PBD rats, feeding of a high-protein diet for 2 days induced phosphorylation of eukaryotic initiation factor 4E-binding protein 1 and 70-kDa ribosomal protein S6 kinase, indicating the activation of the initiation phase of translation for pancreatic protein synthesis. However, this increased phosphorylation returned to normal levels on Day 7 in PBD but not in sham rats. We concluded that feeding a high-protein diet induced pancreatic growth with increases in the translation initiation activities for pancreatic protein synthesis within 2 days and that prolonged feeding of a high-protein diet changed the initiation activities differently in sham and PBD rats.     

Restoration by dietary glutamine of reduced tumor necrosis factor production in a low-protein-diet-fed rat model

Tumor necrosis factor-alpha (TNF) production by peritoneal macrophages and its dietary modification were investigated by using rats fed on a low-protein diet. The rats were given a 20% casein (control) diet or a 3% casein diet for 21 days, and TNF production was measured in activated macrophages of these animals. TNF production was significantly lower in macrophages from rats fed on the low-protein diet than that in macrophages from rats fed on the control diet. Oral administration of a cabbage extract, a known modulator of TNF production, to the low-protein-diet-fed rats significantly enhanced TNF production by macrophages. Glutamine supplementation to the low-protein diet significantly enhanced TNF production as well as TNF mRNA expression. These results indicate that the 3%-casein-diet-fed rat would be useful as a model for reduced TNF production in protein malnutrition. These results also suggest that glutamine administration restored the reduced TNF production associated with protein malnutrition.     

Effect of the Supplementation of Sulfur Amino Acids to a Diet Containing Soy Protein Isolate on Lipid Metabolism in Rats

The effect of the supplementation of sulfur amino acids to a low casein or soy protein isolate diet on tissue lipid metabolism was investigated. Supplementation of methionine to a 8% casein diet produced a fatty liver in rats, however, supplementation of methionine to a 8.8% soy protein diet (corresponding to a 8% casein diet as to sulfur amino acids content) did not produce a fatty liver. At the level of 8% or less of soy protein in the diet, the accumulation of liver lipids and serum triglyceride was observed. An amino acid mixture simulating the composition of soy protein isolate caused significant accumulation of liver lipids, but serum triglyceride was not changed. Serum cholesterol in rats fed the soy protein diet was lower than that in rats fed the casein diet, but on feeding the amino acid mixtures simulating these protein diets, there was no difference between the two groups. The small differences between soy protein isolate and casein as to lipid metabolism might be due to the small differences in the contents of sulfur amino acids or the specific nature of the soy protein or casein. The supplemental effect of methionine and cystine was also studied. About 60% of total sulfur amino acids could be substituted by cystine for maximum growth.     

Effect of food deprivation and hypophysectomy on in vitro protein synthesis by membrain-bound and free hepatic ribosomes.

The protein synthetic activities of membrane-bound and free hepatic ribosomes isolated from intact rats fed ad libitum, and normal rats subjected to food restriction to match that of hypophysectomised (Hx) rats were compared to the in vitro protein synthetic capacity of hepatic ribosomes isolated from Hx rats. Hypophysectomy resulted in decreased protein synthetic ability of bound ribosomes, whether protein synthesis was directed by endogenous messenger RNA (mRNA) (p less than 0.05) or by polyuridylic acid (polyU) (p less than 0.01). In contrast, the protein synthetic activity of free hepatic ribosomes from Hx rats was reduced when protein synthesis was directed by endogenous mRNA (p less than 0.05) but, when polyU was substituted as the messenger, the protein synthetic activity of these free ribosomes was equal to that of control rats. On the other hand the effects of food restriction on hepatic ribosomal function could be clearly differentiated from the effects observed following hypophysectomy. Thus, the reduced protein synthetic activity of hepatic bound ribosomes isolated from food restricted normal rats was not demonstrable, when polyU was used to direct protein synthesis. Further, food restriction had no effect on the protein synthetic activity of free hepatic ribosomes, and this was true when protein synthesis was directed by either endogenous or artificial messenger. It is concluded that hypophysectomy reduces the protein synthetic ability of both bound and free hepatic ribosomes, and this change of ribosomal function of Hx rats cannot be attributed to their decreased food intake.     

Increased Plasma Homocysteine Concentration in Rats from a Low Casein Diet

Rats were fed on a 10% casein (10C) diet, 30% casein (30C) diet, 10C+0.5% methionine diet, or 30C+0.5% methionine diet for 14 d to investigate the relationship between the dietary protein level and plasma homocysteine concentration. The plasma homocysteine concentration was significantly higher in the rats fed on the 10C diet than in the rats fed on the 30C diet, and this phenomenon persisted even under the condition of methionine supplementation. The activity of hepatic cystathionine β-synthase (CBS) was significantly lower in the rats fed on the 10% casein diets than in the rats fed on the 30% casein diets, irrespective of methionine supplementation. This is the first demonstration of a low-protein diet increasing the plasma homocysteine concentration in experimental animals. It is suggested that the decreased CBS activity might be associated, at least in part, with the hyperhomocysteinemia caused by the low-casein diet.     

Effect of maternal ethanol consumption on foetal and neonatal rat hepatic protein synthesis.

Effects of maternal ethanol consumption were investigated on the rates of protein synthehsis by livers of foetal and neonatal rats both in vivo and in vitro, and on the activities of enzymes involved in protein synthesis and degradation. The rates of general protein synthesis by ribosomes in vitro studied by measuring the incorporation of [14C]leucine into ribosomal protein showed that maternal ethanol consumption resulted in an inhibition of the rates of protein synthesis by both foetal and neonatal livers from the ethanol-fed group. The rates of incorporation of intravenously injected [14C]leucine into hepatic proteins were also significantly lower in the foetal, neonatal and adult livers from the ethanol-fed group. Incubation of adult-rat liver slices with ethanol resulted in an inhibition of the incorporation of [14C]leucine into hepatic proteins; however, this effect was not observed in the foetal liver slices. This effect of externally added ethanol was at least partially prevented by the addition of pyrazole to the adult liver slices. Pyrazole addition to foetal liver slices was without significant effect on the rates of protein synthesis. Cross-mixing experiments showed that the capacity of both hepatic ribosomes and pH5 enzyme fractions to synthesize proteins was decreased in the foetal liver from the ethanol-fed group. Maternal ethanol consumption resulted in a decrease in hepatic total RNA content, RNA/DNA ratio and ribosomal protein content in the foetal liver. Foetal hepatic DNA content was not significantly affected. Ethanol consumption resulted in a significant decrease in proteolytic activity and the activity of tryptophan oxygenase in the foetal, neonatal and adult livers. It is possible that the mechanisms of inhibition of protein synthesis observed here in the foetal liver after maternal ethanol consumption may be responsible for at least some of the changes observed in 'foetal alcohol syndrome'.     

The acute effects of N-hydroxy-acetylaminofluorene on rat liver protein synthesis

A single intraperitoneal injection of N-hydroxy-acetylaminofluorene (N-hydroxy-AAF) at a dosage of 30 mg/kg significantly inhibited rat liver protein synthesis within 15 min. Marked alterations in the subcellular distribution of hepatic RNA accompanied the decline in protein synthesis in treated rats. These changes included decreases in nuclear and bound polysomal RNA and increases in free polysomal and non-sedimentable RNA. Heavy polysomal aggregates, both free and bound, were almost completely degraded to monomers and dimers during this period. Sedimentation profiles of total cytoplasmic RNA revealed no evidence of gross RNA breakdown in N-hydroxy-AAF-treated animals. To determine the mechanisms responsible for the inhibition of protein synthesis by N-hydroxy-AAF, cellular components involved in protein synthesis were purified from control and treated animals and examined in two cell-free systems. In a system which measures polypeptide chain elongation and release, the incorporation of amino acids into protein was reduced by 35% using polysomes from N-hydroxy-AAF treated animals compared with controls. By contrast, the function of the pH 5 fraction (containing aminoacylating enzymes and tRNA) from the carcinogen-treated animals was unimpaired. A wheat germ lysate system was used to determine the ability of mRNA to program polypeptide chain initiation and elongation. Cytoplasmic poly(A)⁺ RNA from N-hydroxy-AAF treated rats showed reduced capacity to stimulate protein synthesis in wheat germ lysates compared with similar preparations from DMSO-injected control rats. The rapid inhibition of protein synthesis by N-hydroxy-AAF may be an important contributing factor to other toxic effects of the carcinogen, including the inhibition of rRNA synthesis.