Ethionine Diet Research Articles

Proglumide Reverses Nonalcoholic Steatohepatitis by Interaction with the Farnesoid X Receptor and Altering the Microbiome.

Nonalcoholic steatohepatitis (NASH) is associated with obesity, metabolic syndrome, and dysbiosis of the gut microbiome. Cholecystokinin (CCK) is released by saturated fats and plays an important role in bile acid secretion. CCK receptors are expressed on cholangiocytes, and CCK-B receptor expression increases in the livers of mice with NASH. The farnesoid X receptor (FXR) is involved in bile acid transport and is a target for novel therapeutics for NASH. The aim of this study was to examine the role of proglumide, a CCK receptor inhibitor, in a murine model of NASH and its interaction at FXR. Mice were fed a choline deficient ethionine (CDE) diet to induce NASH. Some CDE-fed mice received proglumide-treated drinking water. Blood was collected and liver tissues were examined histologically. Proglumide’s interaction at FXR was evaluated by computer modeling, a luciferase reporter assay, and tissue FXR expression. Stool microbiome was analyzed by RNA-Sequencing. CDE-fed mice developed NASH and the effect was prevented by proglumide. Computer modeling demonstrated specific binding of proglumide to FXR. Proglumide binding in the reporter assay was consistent with a partial agonist at the FXR with a mean binding affinity of 215 nM. FXR expression was significantly decreased in livers of CDE-fed mice compared to control livers, and proglumide restored FXR expression to normal levels. Proglumide therapy altered the microbiome signature by increasing beneficial and decreasing harmful bacteria. These data highlight the potential novel mechanisms by which proglumide therapy may improve NASH through interaction with the FXR and consequent alteration of the gut microbiome.

Cholecystokinin Receptor Antagonist Therapy Decreases Inflammation and Fibrosis in Chronic Pancreatitis.

Chronic pancreatitis is associated with recurrent inflammation, pain, fibrosis, and loss of exocrine and endocrine pancreatic function and risk of cancer. We hypothesized that activation of the CCK receptor contributes to pancreatitis and blockade of this pathway would improve chronic pancreatitis. Two murine models were used to determine whether CCK receptor blockade with proglumide could prevent and reverse histologic and biochemical features of chronic pancreatitis: the 6-week repetitive chronic cerulein injection model and the modified 75% choline-deficient ethionine (CDE) diet. In the CDE-fed model, half the mice received water supplemented with proglumide, for 18weeks. After chronic pancreatitis was established in the cerulein model, half the mice were treated with proglumide and half with water. Histology was scored in a blinded fashion for inflammation, fibrosis and acinar ductal metaplasia (ADM) and serum lipase levels were measured. RNA was extracted and examined for differentially expressed fibrosis genes. Proglumide therapy decreased pancreatic weight in the CDE diet study and the cerulein-induced chronic pancreatitis model. Fibrosis, inflammation, and ADM scores were significantly reduced in both models. Lipase values improved with proglumide but not in controls in both models. Proglumide decreased pancreas mRNA expression of amylase, collagen-4, and TGFβR2 gene expression by 44, 38, and 25%, respectively, compared to control mice. New strategies are needed to decreased inflammation and reduce fibrosis in chronic pancreatitis. CCK receptor antagonist therapy may improve chronic pancreatitis by reversing fibrosis and inflammation. The decrease in ADM may reduce the risk of the development of pancreatic cancer.

β-Arrestin1 alleviates acute pancreatitis via repression of NF-κBp65 activation.

β-Arrestins (β-arrs) are regulators and mediators of G protein-coupled receptor signaling that are functionally involved in inflammation. Nuclear factor-κB p65 (NF-κBp65) activation has been observed early in the onset of pancreatitis. However, the effect of β-arrs in acute pancreatitis (AP) is unclear. The aim of this study is to investigate whether β-arrs are involved in AP through activation of NF-κBp65. Acute pancreatitis was induced by either caerulein injection or choline-deficient supplemented with ethionine diet (CDE). β-arr1 wild-type and β-arr1 knockout mice were used in the experiment. The survival rate was calculated in the CDE model mice. Histological and western blot analyses were performed in the caerulein model. Inflammatory mediators were detected by real-time polymerase chain reaction in the caerulein-induced AP mice. Furthermore, AR42J and PANC-1 cell lines were used to further study the effects of β-arr1 in caerulein-induced pancreatic cells. β-Arr1 but not β-arr2 is significantly downregulated in caerulein-induced AP in mice. Targeted deletion of β-arr1 notably upregulated expression of the pancreatic inflammatory mediators including tumor necrosis factor α and interleukin 1β as well as interleukin 6 and aggravated AP in caerulein-induced mice. β-Arr1 deficiency increased mortality in mice with CDE-induced AP. Further, β-arr1 deficiency enhanced caerulein-induced phosphorylation of NF-κBp65 both in vivo and in vitro. β-Arr1 alleviates AP via repression of NF-κBp65 activation, and it is a potentially therapeutic target for AP.

Su1251 Overexpression of EGFR in the Exocrine Pancreas Induces a Chronic Pancreatitis

Background and Aim: Chronic pancreatitis and pancreatic ductal adenocarcinoma (PDA) frequently overexpress epidermal growth factor receptor (EGFR) and EGF-family ligands. Although chronic pancreatitis is a risk factor for PDA, the relationship between chronic pancreatitis and PDA remains obscure. A critical obstacle to understanding the role of chronic pancreatitis in development of PDA is the lack of appropriate animal models for chronic pancreatitis with similar clinical courses as in human patients. Serine protease inhibitor Kazal type 1 (SPINK1: mouse homolog Spink3), which is also known as tumor associated trypsin inhibitor (TATI), is mainly produced in the pancreatic acinar cells. Recently, we showed that SPINK1 binds to EGFR to activate its downstream signaling. The present study aims to generate chronic pancreatitis model mice and assess the molecular events from chronic pancreatitis to PDA. Methods: 1) C57BL/6 female mice were repeatedly induced acute pancreatitis by using choline-deficient with ethionine (CDE) diet for 54 weeks. 2) To assess the molecular events caused by long term CDE diet treatment using Western blotting. 3) To further analyze the clinical importance of EGFR and SPINK1 in the chronic pancreatitis and PDA, we examined the expression of SPINK1 and EGFR. Immunohistochemical analyses were performed using human chronic pancreatitis and PDA samples. Results: 1) Histological evaluation revealed that chronic pancreatitis, characterized by acinar atrophy, fibrosis and well-developed tubular complexes (TCs), was observed after 24 wk of CDE-diet treatment. In spite of the severe pancreatic degeneration, neoplastic changes were not observed. 2) Expression of EGFR and its ligands (Spink3 and TGF α) and activation of K-Ras (GTP-Ras formation), which are frequently observed in human PDA, were indeed observed in parallel with TCs formation. In addition, cyclooxygenase-2 which induced by stimuli involved in inflammatory responses was also overexpressed. 3) In the human chronic pancreatitis samples, TCs were positive EGFR immunostaining. Furthermore, PDA cases were positive for both SPINK1 and EGFR immunoreactivity. Conclusion: We have successfully developed a chronic pancreatitis model with CDE diet. We also showed expression of EGFR and its potent ligands, phosphorylation of EGFR, and activation of K-Ras as the step of chronic inflammation. It was suggested that EGFR signaling cascades were important for the development from chronic pancreatitis to PDA. Howevere, chronic pancreatitis itself is not sufficient for the development of PDA. Further combinations of CDE-diet treatment with the administration of other promotion insults or genetic alterations may result in PDA development.

Su1252 Factors Predictive of Relapse, Cancer and Cyst Formation in Autoimmune Pancreatitis Patients

Background and Aim: Chronic pancreatitis and pancreatic ductal adenocarcinoma (PDA) frequently overexpress epidermal growth factor receptor (EGFR) and EGF-family ligands. Although chronic pancreatitis is a risk factor for PDA, the relationship between chronic pancreatitis and PDA remains obscure. A critical obstacle to understanding the role of chronic pancreatitis in development of PDA is the lack of appropriate animal models for chronic pancreatitis with similar clinical courses as in human patients. Serine protease inhibitor Kazal type 1 (SPINK1: mouse homolog Spink3), which is also known as tumor associated trypsin inhibitor (TATI), is mainly produced in the pancreatic acinar cells. Recently, we showed that SPINK1 binds to EGFR to activate its downstream signaling. The present study aims to generate chronic pancreatitis model mice and assess the molecular events from chronic pancreatitis to PDA. Methods: 1) C57BL/6 female mice were repeatedly induced acute pancreatitis by using choline-deficient with ethionine (CDE) diet for 54 weeks. 2) To assess the molecular events caused by long term CDE diet treatment using Western blotting. 3) To further analyze the clinical importance of EGFR and SPINK1 in the chronic pancreatitis and PDA, we examined the expression of SPINK1 and EGFR. Immunohistochemical analyses were performed using human chronic pancreatitis and PDA samples. Results: 1) Histological evaluation revealed that chronic pancreatitis, characterized by acinar atrophy, fibrosis and well-developed tubular complexes (TCs), was observed after 24 wk of CDE-diet treatment. In spite of the severe pancreatic degeneration, neoplastic changes were not observed. 2) Expression of EGFR and its ligands (Spink3 and TGF α) and activation of K-Ras (GTP-Ras formation), which are frequently observed in human PDA, were indeed observed in parallel with TCs formation. In addition, cyclooxygenase-2 which induced by stimuli involved in inflammatory responses was also overexpressed. 3) In the human chronic pancreatitis samples, TCs were positive EGFR immunostaining. Furthermore, PDA cases were positive for both SPINK1 and EGFR immunoreactivity. Conclusion: We have successfully developed a chronic pancreatitis model with CDE diet. We also showed expression of EGFR and its potent ligands, phosphorylation of EGFR, and activation of K-Ras as the step of chronic inflammation. It was suggested that EGFR signaling cascades were important for the development from chronic pancreatitis to PDA. Howevere, chronic pancreatitis itself is not sufficient for the development of PDA. Further combinations of CDE-diet treatment with the administration of other promotion insults or genetic alterations may result in PDA development.

Deletion of toll-like receptor-4 downregulates protein kinase C-ζ and attenuates liver injury in experimental pancreatitis

Toll-like receptor-4 (TLR4) and protein kinase C-zeta (PKC-zeta) play a role in macrophage activation. We hypothesized that deletion of TLR4 downregulates PKC-zeta and attenuates liver cell apoptosis in experimental pancreatitis. Acute pancreatitis was induced by choline-deficient ethionine diet in C57/BL6 (TLR4+/+ and TLR4-/-) mice. During pancreatitis, staining for TLR4 and PKC-zeta, which colocalized in Kupffer cells but not in hepatocytes, increased in TLR4+/+ mice and decreased in TLR4-/- mice. In TLR4+/+ mice, pancreatitis increased TLR4 protein and mRNA and PKC-zeta protein and activity, nuclear factor (NF)-kappaB, ERK1/2, caspase-3 cleavage, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining; all P < .01 versus controls. In TLR4-/- mice with pancreatitis, PKC-zeta mRNA and activity were reduced, ERK1/2 and caspase-3 did not increase, and NF-kappaB and TUNEL (mostly in hepatocytes) increased mildly (all P < .01 vs control). PKC-zeta did not interact directly with NF-kappaB; however, during pancreatitis, coimmunoprecipitation of PKC-zeta with ERK1/2 was increased in TLR4+/+ mice and was attenuated in TLR4-/- mice (all P < .01 vs control), indicating that PKC-zeta interacts with ERK1/2. Acute pancreatitis upregulates TLR4, PKC-zeta, NF-kappaB, and ERK1/2, and increases apoptosis in mice livers. PKC-zeta induces nuclear translocation of NF-kappaB via ERK1/2-dependent mechanisms. Deletion of TLR4 downregulates PKC-zeta, NF-kappaB, and ERK1/2, and attenuates pancreatitis-induced liver cell apoptosis.

Suppression by phenobarbital of ethionine-induced hepatocellular carcinoma formation and hepatic S-adenosylethionine levels.

An 18-month carcinogenicity study was conducted in male weanling F344 rats (28/group) to examine the effects of the simultaneous feeding of selected concentrations of ethionine and 0.05% phenobarbital in a normal chow diet. The effects of a 1-6-week feeding of phenobarbital and ethionine on the hepatic levels of the related metabolites S-adenosylmethionine, S-adenosylhomocysteine and S-adenosylethionine were also examined. Ethionine at 0.3% or 0.1% induced hepatocellular carcinoma (HCCa) at incidences of 90% (19/21) and 89% (24/27), respectively. Adding phenobarbital to the 0.1% ethionine diet reduced the incidence of HCCa to 36% (10/28) and reduced the number of liver tumor-associated deaths occurring prior to terminal sacrifice from 10/27 to 1/28. No hepatic tumors were observed in rats fed 0, 0.003, 0.01, or 0.03% ethionine. Phenobarbital alone or combined with 0.03% ethionine produced no hepatic tumors. Dietary ethionine at 0.1% reduced the intracellular hepatic level of S-adenosylmethionine to <50% of that seen in control rats. Phenobarbital alone had little effect on either S-adenosylmethionine or S-adenosylhomocysteine levels. The combination of phenobarbital and 0.1% ethionine led to increases in the hepatic levels of S-adenosylmethionine of 40-60% after 3 and 6 weeks of feeding, compared to those seen in rats receiving 0.1% ethionine alone. Ethionine feeding resulted in high levels of S-adenosylethionine in the livers. Combining phenobarbital with ethionine in the diet led to 30-50% reductions in hepatic S-adenosylethionine content. The results indicate that phenobarbital inhibits hepatocarcinogenesis by ethionine, that ethionine may cause HCCa via methyl group insufficiency, and that at levels of < or =0.03% ethionine did not show evidence of tumorigenicity.

Effects of the cholecystokinin receptor antagonist L-364,718 on pancreatitis induced by a deficient in choline and supplemented with ethionine (CDE) diet in the rat.

The role of cholecystokinin (CCK) in the development of a necrotizing acute pancreatitis induced by a diet deficient in choline and supplemented with ethionine (CDE) has been evaluated in the rat by using a potent CCK receptor antagonist L-364,718. Acute pancreatitis was induced by administration of CDE diet for 14 days. L-364,718 administration was carried out by subcutaneous injections at dose of 0.1 mg/kg/day. Pancreatic exocrine secretion (flow, protein, amylase and trypsin outputs) in resting and under infusion of 1.25 microgram/kg/h of CCK-8 were used to evaluate the pancreatic functionality. Others parameters (serum amylase, percentage fluid in pancreas, haematocrit and mortality) evaluated the severity of pancreatitis. L-364,718 slightly reduced the mortality and the increases of percentage of fluid accumulated in pancreas in CDE diet acute pancreatitis. Basal and CCK stimulated pancreatic secretion was significantly depressed 36 hours after L-364,718 treatment. A slight response to CCK was observed. Nevertheless it was lower than usually observed in control rats. Our results demonstrate that in the rat, chronic L-364,718 treatment did not completely restore pancreatic activity in acute pancreatitis induced by CDE diet. Hence CCK cannot be considered as the main factor involved in the development of this pancreatitis model.

Effects of soybean trypsin inhibitors and dl-ethionine on growth and serum parameters in young rats

Two three week feeding trials tested the effects of dietary soybean trypsin inhibitors (SBTI) and/or DL-ethionine (methionine antagonist) on growth, protein digestibility, and clinical parameters in young male and female Sprague Dawley and Wistar rats. SBTI-fed rats had decreased weight gain, feed efficiency, protein digestibility and increased serum urea nitrogen relative to casein-fed control animals. Ethionine without SBTI also had negative effects. Combination of the two factors resulted in greatest growth deficits and increased serum levels of urea nitrogen, triglycerides, alkaline phosphatase and glutamate pyruvate transaminase. Dietary inadequacy or unavailability of sulfur amino acids in rats fed SBTI and ethionine was suggested by poor growth and serum accumulation of total essential amino acids, methionine sulfoxide, and several amino acid precursors of the methionine transsulfuration pathway. DL-methionine supplementation of diets containing SBTI or SBTI + ethionine or use of a soy protein isolate + ethionine diet (low in SBTI) improved growth and feed efficiency and normalized clinical chemistry parameters and serum free amino acid levels further suggesting that a deficit in supply or availability of sulfur amino acids was a factor in the observed changes.

Acute hemorrhagic pancreatitis in mice: improved survival after indomethacin administration.

This study was designed to test whether indomethacin given in drinking water attenuates the severity of the acute hemorrhagic pancreatitis induced in mice fed a choline-deficient ethionine diet (CDE), and improves their survival. Three doses of indomethacin were tested, 20, 10 and 5 mg of indomethacin/l of drinking water, and their effects on the histopathology of the pancreas and liver, serum amylase activity levels, and mortality were determined. Even though 20 and 10 mg/l suppressed pancreatic injury, and the accompanying shock-like state, the mice died due to indomethacin hepatotoxicity. Hepatotoxicity was minimal at a dose of 5 mg/l, which resulted in preservation of significant amounts of normal pancreatic parenchyma, subsequent regeneration of acinar cells, and an increased survival of the mice. The results suggest that prostaglandins may play an important role in the pathogenesis of acute hemorrhagic pancreatitis, and that indomethacin attenuates the severity of the induced disease.

Altered insulin and glucagon secretion in perfused ethionine-treated rat pancreases.

The endocrine secretory function of rat pancreases in which pancreatitis had been induced by feeding rats a 0.5% ethionine diet was investigated. Despite loss of 50% of exocrine tissue and widespread destruction of acinar structure, pancreatic insulin and glucagon contents and 4-h fasting plasma insulin levels in vivo did not differ significantly from those of food-restricted, weight-matched controls. Plasma glucose concentrations (fasting and after oral glucose) were significantly lower than control. In isolated, perfused ethionine-treated pancreases secretin failed to stimulate insulin secretion, whereas basal insulin secretion and insulin responses to glucose, arginine, gastric inhibitory polypeptide, vasoactive intestinal peptide (VIP), and somatostatin were similar to those of controls. Basal glucagon secretion was elevated in ethionine-treated pancreases, and glucagon outputs in response to arginine, VIP, and somatostatin showed a consistent trend toward higher levels than those of controls. These findings demonstrate that ethionine-induced pancreatitis selectively impairs islet secretory function. These effects may be due to damage to islet cell membranes by exocrine enzymes and/or a direct pathogenic action of ethionine on the islets.

Theoretical mechanisms for synthesis of carcinogen-induced embryonic proteins: X correlation of the induceability of hepatomas and alpha-fetoprotein gene activity

We have observed a distinct difference in the alpha-fetoprotein production of Sprague-Dawley rats and Swiss white mice. When both animals are fed a 1% D L -ethionine diet, only the rat responds with increased levels of hepatic alpha-fetoprotein synthesis as shown by radioimmunoassay studies. Also, hybridization studies, with a complementary deoxyribonucleic acid for alpha-fetoprotein messenger ribonucleic acid, proved negative for the mouse, even after forty days on the 1% D L -ethionine diet. This evidence, combined with other information leads us to believe that the mouse has a genomic regulatory system that is resistant to carcinogenic change by certain chemicals. Furthermore it may eventually be proven that hepatic tissue that is competent to have certain embryonic genes activated may also be capable of becoming neoplastic.

Hepatic levels of S-adenosylethionine and S-adenosylmethionine in rats and hamsters during subchronic feeding of DL-ethionine.

The levels of S-adenosylethionine (AdoEt) and of S-adenosylmethionine (AdoMet) in the livers of weanling male rats and male and female hamsters fed ethionine for 1-6 weeks were determined. Ethionine was fed at levels of 0, 0.1, and 0.3% in the diet, and the animals were sacrificed after 0, 1, 3 and 6 weeks of treatment. In both species the hepatic contents of AdoEt were dependent upon the level of ethionine in the diet. For the 6-week experimental period the hepatic levels of AdoEt average 81 microgram/g liver in male hamsters fed 0.1% ethionine in the diet and 160 microgram/g in those fed 0.3% ethionine; the corresponding AdoEt levels in female hamsters were 104 and 191 microgram/g liver, respectively. No marked shifts in hepatic AdoEt levels were seen in either male or female hamsters although a gradual rise in hepatic AdoEt from 145 to 233 microgram/g was noted in the female hamsters receiving 0.3% ethionine in the diet for 1-6 weeks. AdoEt levels in the livers of rats fed 0.3% ethionine were quite variable with values of 123, 305 and 127 microgram/g liver noted at weeks 1, 3 and 6 respectively. In rats fed the 0.1% ethionine diet the liver AdoEt levels dropped from 103 to 61 microgram/g from weeks 1 to 6, In animals fed the ethionine-free diet, the hepatic contents of AdoMet were relatively constant throughout the 6-week experimental period, with average values of 25, 17, and 29 microgram/g liver respectively in the male rats, male hamsters and female hamsters. Chronic ethionine administration always suppressed hepatic AdoMet levels. This suppression was generally greater in animals fed the 0.1% ethionine than in those fed the 0.3% ethionine diet. Thus, the average hepatic AdoMet level in rats, male hamsters and female hamsters receiving the 0.1% ethionine diet for 3-6 weeks were 32, 18, and 45% respectively, of the corresponding AdoMet levels in control animals: however, the corresponding AdoMet levels in animals receiving the 0.3% ethionine diet were 66, 42, and 62% of the respective control values. Feeding 0.1% ethionine to male hamsters led to exceedingly low levels of liver AdoMet (1.4-2.9 microgram/g). No direct correlations could be made between the effects of ethionine feeding on the hepatic AdoEt and AdoMet levels in rats and hamsters and the previously reported differences in carcinogenicity by ethionine in these species.

Enhancement of DL-Ethionine-Induced Liver Carcinogenesis in Rats Fed a Choline-Devoid Diet&lt;xref ref-type="fn" rid="FN2"&gt;2&lt;/xref&gt;

The effects of a choline-devoid (CD) or a choline-supplemented (CS) diet on the induction of liver tumors in rats by DL-ethionine were investigated. Groups of male outbred Sprague-Dawley rats were fed a plain CD or a plain CS diet, or the same diets containing 0.05% DL-ethionine. Hepatocellular carcinomas developed in 50% of the rats fed the CD+ethionine diet for 14 weeks and in about 80% of the rats fed the same diet for 22-30 weeks. No hepatocellular carcinomas developed in rats fed the CS+ethionine diet, the plain CD diet, or the plain CS diet up to 30 weeks. The findings suggest that a CD diet alters the response of rat liver to DL-ethionine and leads to an early and enhanced induction of hepatocellular carcinoma.

Effect of dietary methionine level on anserine and carnosine contents in the gastrocnemius muscle of rat.

Anserine and carnosine in the muscle of growing rats were measured to explore the effect of dietary methionine. Carnosine was reduced in the gastrocnemius muscle of rats fed on a methionine-free diet. After refeeding on methionine for one week, carnosine in the muscle approached to the normal level. In contrast, anserine increased in the muscle of rats fed on a methionine-free diet. Anserine also increased in the gastrocnemius muscle of rats fed on a methionine-excess diet, whereas carnosine did not vary. Npi-Methylhistidine was detected in the muscle of rats fed on a methionine-excess diet. Moreover, Npi-methylhistindine and Ntau-methylhistine in the urine increased in the rats fed on both methionine-excess and methionine-free diets. Carnosine in the gastrocnemius muscle was not affected by a ethionine diet nor by a orotic acid diet which induced fatty liver. However, anserine increased in the muscle of rats fed on a ethionine diet.

Hyperglucagonemia and altered responsiveness of hepatic adenylate cyclase-adenosine 3′,3′-monophosphate system to hormonal stimulation during chronic ingestion of dl-ethionine

Basal activity and hormonal responsiveness of the adenylate cyclase-adenosine 3′,5′-monophosphate system were examined in premalignant liver from rat chronically fed the hepatic carcinogen DL-ethionine, and these data were correlated with endogenous levels of plasma glucagon. By 2 weeks basal hepatic cyclic AMP levels, determined in tissue quick-frozen in situ, were 2-fold higher in rats ingesting ethionine than in the pair-fed control. Enhanced tissue cyclic AMP content was associated with an increase in the adenylate cyclase activity of whole homogenates of fresh liver from rats fed ethionine (68 ± 5 pmol cyclic AMP/10 min per mg protein) compared to control (48 ± 4). Cyclic AMP-dependent protein kinase activity ratios were also significantly higher (control, 0.38 ± 0.04; ethionine 0.55 ± 0.05) and the percent glycogen synthetase activity in the glucose 6-phosphate-independent form was markedly reduced (control, 52 ± 7%; ethionine, 15 ± 1.5 %) in the livers of ethionine-fed rats compared to the controls, suggesting that the high total hepatic cyclic AMP which accompanied ethione ingestion was biologically effective. These changes persisted throughout the 38 weeks of drug ingestion. Immunoreactive glucagon levels, determined in portal venous plasma, were 8-fold higher than control after 2 weeks of the ethionine diet (contro, 185 ± 24 pg/ml; ethionine, 1532 ± 195). Analogous to the changes in hepatic parameters, plasma glucagon levels remained elevated during the entire period of drug ingestion until the development of hepatomas. The hepatic cyclic AMP response to a maximal stimulatory dose of injected glucagon was blunted in vivo in ethionine-fed rats (control, 14-fold increase over basal, to 8.63 ± 1.1 pmol/mg wet weight; ethionine, 4.6-fold rise over basal, to 5.42 ± 0.9). Reduced cyclic AMP responses to both maximal and submaximal glucagon stimulation were also evident in vitro in hepatic slices prepared from rats fed the drug, and the reduction was specific to glucagon. Absolute or relative hepatic cyclic AMP responses to maximally effective concentrations of prostaglandin E 1 or isoproterenol in hepatic slices from ethionine-fed rats were greater than or equal to those observed in control slices. Parallel alterations in hormonal responsiveness were observed in adenylate cyclase activity of whole homogenates of these livers, implying that the changes in cyclic AMP accumulation following hormone stimulation were related to an alteration in cyclic AMP generation in the premalignant tissue. In view of the recognized hepatic actions of glucagon and the desensitization of adenylate cyclase which can occur during sustained stimulation of the liver with this hormone, the endogenous hyperglucagonemia that accompanies ethionine ingestion could play a role in the pathogenesis of both the basal alterations in hepatic cyclic AMP metabolism and the reduced responsiveness to glucagon observed in liver from rats fed this carcinogen.

Effect of ethionine on hepatic polyribosomes and protein synthesis. Studies dealing with the acute administration of ethionine to fasted or fed mice or to rats fed a diet containing ethionine

In female mice, the administration intraperitoneally of ethionine ( 16 or 25 mg 18–25 gm of body weight) to overnight fasted animals (up to 3 hours before killing) caused polyribosomal disaggregation, decreased in vitro protein synthesis, and a marked decrease of ATP of the livers. In contrast, the administration of ethionine to nonfasted animals led to greater polyribosomal aggregation, increased in vitro protein synthesis, decreased in vivo protein synthesis and a moderate decrease of ATP of the livers. In female rats, the effects of the administration intraperitoneally of ethionine ( 100 mg 100 gm of body weight) to overnight fasted animals (4 hours before killing) that had been fed ad libitum a basal diet or an ethionine diet (basal diet plus 0.25% ethionine) for 1 to 5 weeks or force-fed a basal diet or an ethionine diet for 3 days were investigated. Following ethionine administration, the animals fed the basal diet responded with polyribosomal disaggregation, decreased in vitro protein synthesis and decreased ATP levels in the livers while, in contrast, the animals fed the ethionine diet showed no change in polyribosomes along with little or no change in in vitro protein synthesis and in ATP levels of the livers. This adaptive response of the livers of rats fed the ethionine diet could be reversed, since rats fed ad libitum the ethionine diet for 21–31 weeks and then switched to the basal diet for 5–12 weeks reacted to the ethionine administration as did control animals.

Collagen biosynthesis in normal and cirrhotic rat liver slices.

The fibrosis that accompanies hepatic cirrhosis, either that produced in animals experimentally (for example, by CCI4, ethionine, or choline-free diet) or that occurring in man (viral, alcoholic-nutritional or biliary) is one of the main features of the diseas.Two mechanisms have been proposed for the development of fibrosis: (a) condensation or collapse of preexisting hepatic stroma (2), and (b) induction of fibroblastic proliferation and de novo collagen deposition (5). The latter mechanism is supported by evidence showing a net increase of total collagen content or an increase of hydroxyproline content per unit weight of liver (3, 7). The present experimens were designed to explore the capacities of normal and cirrhotic rat liver slices to synthesize protein containing radioactive hydroxyproline in the course of incubation with labeled proline.Materials and Methods. Male Wistar albino rats weighing from 60 to 70 g were fed ad libitum with Purina chow. Cirrhosis was produced by intraperitoneal injection...

Ethionine induced sRNA neomethylase activity

Recent studies in this laboratory have been directed towards the methylation of sRNA using adult, embryonic, and neoplastic mouse liver cell preparations (Hancock, 1966; Hancock, 1967a; Hancock, 1967b; Hancock, et al. , 1967) . After characterizing the sRNA ethylase activity of mouse liver (manuscript submitted) investigations were initiated to detect any changes in sRNA ethylase activity of ethionine treated mice. Although the sRNA ethylase activities thus far are not remarkable, it was discovered during experiments analyzing rates of methylation that sRNA methylase activity had definitely increased in livers from ethionine treated mice. This paper will present evidence that new cytidylate, adenylate, and uridylate sRNA methylase activities arise in the liver cells of ethionine treated mice. Only data concerned with mice fed ethionine for six months will be presented here and a full report including three month data will appear elsewhere after the analysis of mice fed for nine months with an ethionine diet and completion of sRNA ethylase experiments.

Effect of Feeding DL-Ethionine on the Polyunsaturated Fatty Acids of Rat Liver Phospholipids

An experiment was carried out to investigate the effect of dietary ethionine on the essential fatty acid composition of rat liver phospholipids. Male and female rats were fed, for 14 days, a 9% casein diet supplemented with either 0.25% DL-ethionine, 0.5% DL-ethionine or 0.25% DL-ethionine plus 0.5% DL-methionine. At the end of the feeding period, livers were removed, their lipids were extracted and separated into triglycerides and phospholipid fractions, and their fatty acids were determined. While ethionine caused variable concentrations of triglyceride in both sexes, it had very little effect on the fatty acid composition of that fraction. Hepatic phospholipid concentrations, however, were much more consistent and similar for both sexes. The percentage of arachidonic acid in this lipid was significantly reduced in both sexes by ethionine, whereas the proportions of linoleic and oleic acids were increased. In both sexes, the largest phospholipid fraction, the phosphatidyl cholines showed the same changes as did the whole phospholipid, but they were more pronounced. The ethionine also decreased the percentage of stearic acid in this fraction, but the change was only significant in females. Only arachidonic acid and a pentaenoic acid were reduced in the phosphatidyl ethanolamines of both sexes by ethionine. Supplementation of the ethionine diet with methionine prevented most of the fatty acid changes in the phospholipid fractions. The results showed, therefore, that dietary ethionine exerts an influence on the metabolism of the phospholipid polyunsaturated fatty acids of both sexes by decreasing the relative amounts of arachidonic acid at the same time that linoleic acid is being increased. Possible reasons for the observed fatty acid changes are discussed.