Dose Of Ethionine Research Articles

Cellular proliferation in the canine pancreas after d,l-ethionine dosage as detected by double immunohistochemical labelling

d,l-Ethionine produces pancreatic exocrine necrosis and islet proliferation in hamsters and dogs. As a first step in examining whether induction of islet proliferation has therapeutic applications in animals with exhausted or destroyed insulin-producing beta-cells, we studied pancreatic cellular proliferation after intravenous administration of d,l-ethionine in normal dogs. Double immunohistochemical labelling of pancreatic tissue was used to identify proliferating cells in three groups of six clinically normal crossbred dogs administered d,l-ethionine (100 mg/kg) intravenously three times a week for two weeks. Six additional dogs served as untreated controls. Group I was euthanased and necropsied on day 15 (72 hours after the final dose of ethionine). Groups II and III were euthanased on days 29 and 43 respectively. Utilising markers for proliferating nuclei, insulin and cytokeratin, proliferating cells were classified as acinar, endocrine (both intra or extra-islet), duct or 'other' (i.e. infiltrative or interstitial) and counted under the light microscope (40x magnification). Compared to controls, an increase in the number of proliferating cells was found in all categories except ducts. Acinar cells demonstrated statistically significant (p < 0.05) proliferation, greatest two weeks after ethionine cessation continuing over four weeks. The interstitial, infiltrative or 'other' group also showed proliferation, however this was a more immediate response, which substantially decreased two weeks after ethionine administration. Endocrine cells showed only minor and non-significant proliferative activity and were probably not responsible for a significant increase in apparent beta-cell mass. The number of proliferating duct cells was inconsequential and there appeared to be no specific relationship between any cell populations and duct cells.

Effect of d,l-ethionine administration on the histomorphology of canine pancreatic acinar and β-cells

d,l-Ethionine produces pancreatic exocrine necrosis and islet proliferation in hamsters and dogs. As a first step in examining whether induction of islet proliferation has therapeutic applications in animals with exhausted or destroyed insulin-producing beta-cells, we studied pancreatic pathological alterations after intravenous administration of d,l-ethionine in normal dogs. Histomorphological changes in pancreatic acinar cells and beta-cells were assessed in three groups of six clinically normal crossbred dogs administered d,l-ethionine (100 mg/ kg) intravenously three times a week for two weeks. Six additional dogs served as untreated controls. Group I was euthanased and necropsied on day 15 (72 hours after the final dose of ethionine). Groups II and III were euthanased on days 29 and 43 respectively. Severe acinar destruction occurred resulting in significant (p < 0.001) shrinkage of the pancreas in all groups. Although there was variability in histomorphology within groups, pancreases of group I generally exhibited widespread loss of pancreatic acinar structure. Remaining acinar cells were difficult to discern from other cell types within lobules and were surrounded by infiltrates, predominantly of lymphocytes. Partial acinar cellular regeneration had occurred by day 29, but was still incomplete at day 43. Immunohistochemistry suggested that the effect of d,l-ethionine administration on the histomorphology of beta-cells in the left lobe was minimal; however, morphometry demonstrated a significant (p < 0.05) increase in the number of individual beta-cells in groups II and III, and clusters of 2-10, 10-20 and 20+ cells in Group II. It is probable that the apparent increase in the number of individual and other beta-cell arrangements observed in some groups resulted primarily from alterations in the exocrine tissue, although some beta-cell hyperplasia cannot be excluded completely.

Biochemical alterations in hepatic tissue of ethionine and N-nitrosodimethylamine treated mice, and in a transplantable tumor of rat.

Considerably lowere CO2-14C production from the degradative pathway of thymidine -14C utilization was observed in the hepatic tissues of mice (C3H) that recieved multiple intraperitoneal doses of ethionine (400 mg/kg on day 1 5, 9, 13, 17, 21 and 25). While the incorporation of thymidine -14C into DNA was unchanged throughout this period, the tRNA methylase activity of these mice receiving multiple doses of ethionine was significantly lowered at the final sacrifice. When a lower single dose of ethionine (80 mg/kg) was given to mice, the depression of CO2-14C produced from thymidine -14C utilization was less pronounced.Transplantable dimethylaminobenz(a)anthracene (DMBA) induced manunary tumor of rat showed both increased incorporation of thymidine -14C into DNA and t-RNA methylase activity. Also, transplantable Morris hepatoma, although not available for testing in this study, shares a similar pattern of thymidine utilization with transplantable mammary tumor and in general, all other transplantable tumors. In contrast to transplantable tumors N-nitrosodimethylamine (NDMA) induced hepatomas of mice showed no statistically significant alteration in their utilization of thymidine -14C.The known hepatotoxins, bromobenzene and carbon tetra-chloride, when administered to rats in a single dose all lowered the heptatic CO2-14C production from thymidine -14C utilization as compared to non-treated controls (p<0.001, p<0.01 respectively). The implication of these findings is discussed.

Fluorescence histochemical study of the pancreas in the cat.

The exocrine and endocrine pancreas was investigated according to the fluorescence histochemical method of Flack and Hillarp. 1) Green fluorescent adrenergic fibers were regularly seen associated with arteries and arterioles in the exocrine pancreas. 2) Cholinergic fibers as shown by cholinesterase activity, were also found in the parenchyma of pancreas. 3) Yellow fluorescent cells scattered in the exocrine parenchyma and localized to a population of pancreatic islet cells with a characteristic distribution at the islet periphery was found. 4) By the fluorescence microscopic observation, inter-or intralobular pancreatic ducts, involving the zymogen granules, can also be seen after treatment with HCL vapor. 5) Yellow fluorescent cells, beta-cells containing insulin, remained at the Islet periphery. At present, the above mentioned yellow fluorescent cells are identified as containing HPP (Human pancreatic polypeptide) according to the immunofluorescence technique. With the use of the Falck and Hillarp histochemical technique ethionine induced pancreatitis in cats has been investigated. 1) After seven days of ethionine (5 mg/kg BW oral ad.) treatment, pancreas showed histochemical changes such as hemorrhage, fat necrosis, destruction of acinar cells and degranulation of zymogen from the parenchyma of pancreas. 2) Oral administration of ethionine for ten days induced severe degranulation, rupture of vessels, especially of veins and venules and later influenced arteries or arterioles. 3) Necrosis and fibrosis began to appear in the spaces between the cellular debris and marked pancreatic atrophy could be found. 4) The destruction of Islets of Langerhans can be found in the ethionine induced pancreatic parenchyma. On the other hand, an increased number of Islets of Langerhans was also observed in the site of lobule. 5) The presented findings may also suggest that the duration of administration of ethionine is more important factor than graded doses of ethionine in the production of ethionine is more important factor than graded doses of ethionine in the production of ethionine induced pancreatitis in cats.

Prompt elevation of rat serum alpha-fetoprotein by acute liver injury following a single injection of ethionine.

The mechanism of increased alpha-fetoprotein (AFP) production following a single injection of ethionine was investigated by using rats aged 5 weeks at the time of killing. Marked elevations of serum AFP concentrations occurred within 4 days in both male and female rats after administration of DL-ethionine or L-ethionine, although the increased levels of serum AFP and liver triglyceride in the adults were less marked in the male than in the female. No apparent necrosis of liver cells was observed in ethionine-treated rats. Frequent administrations of adenosine triphosphate after a single dose of ethionine prevented the increases in liver triglyceride and serum AFP levels. The increased concentrations of serum AFP, reaching a maximum level within 4 days, occurred before a slight increase in incorporation of 3H-thymidine into liver DNA. The serum AFP from ethionine-treated rats was immunologically and electrophoretically indistinguishable from that of fetal, carbontetrachloride-treated or hepatoma-bearing rats. These observations suggest that the increased production of AFP in ethionine-treated rats is closely associated with hepatic injury and is not the consequence of liver cell regeneration.

Prompt elevation of rat serum α-fetoprotein by ethionine-induced acute liver injury and its electrophoretic properties.

The mechanism of increased α-fetoprotein (AFP) production following a single injection of ethionine was investigated by using 35 day-old rats at the time of sacrifice. After administration of DL-ethionine or L-ethionine, marked elevations of serum AFP concentrations occurred in 4 days either with male or female rats. D-Ethionine, however, did not cause the increase in serum AFP level. In adult rats, the increase in serum AFP level was less prominent in the male as compared with the female. No apparent necrosis of liver cells was observed in ethionine-treated rats. Frequent administration of ATP after the single dose of ethionine reduced the increases in liver triglyceride and serum AFP levels. The increased concentrations of serum AFP, reaching a maximum level in 4 days, occurred before a slight increase in hepatic DNA synthesis. The serum AFP from ethionine-treated rats was immunologically and electrophoretically indistinguishable from that in fetal, carbon tetrachloride (CCl4)-injured or hepatoma-bearing rat. These observations suggest that the increased production of AFP in ethionine-treated rats is closely associated with the hepatic injury and is not the consequence of liver cell regeneration.

Modification of toxic liver injury in the rat II. Protective effect of cycloheximide on ethionine-induced damage and autoprotective effects of high doses of ethionine, 3′-methyl-4-dimethylaminoazobenzene, and 2-acetylaminofluorene

Male and female rats were given various single oral doses of dl-ethionine, with and without accompanying doses of cycloheximide. It was found that a single dose of cycloheximide prevented the appearance of hepatic cell damage in dl-ethionine-treated female rats, up to 48 hr. In the male rat, two consecutive doses of cycloheximide, with an interval of 5.5 hr, were necessary to achieve protection. However, very large doses of dl-ethionine alone failed to produce the early liver injury which resulted from lower doses, although delayed damage did appear after several days. Similar results were obtained with high doses of 2-acetylamino-fluorene. Very high doses of 3′-methyl-4-dimethylaminoazobenzene failed entirely to induce the periportal hepatic cell lesion which is characteristic of this agent, instead giving rise to minimal centrilobular necrosis and leading to a massive proliferation of bile duct cells within a week.

Effects of glucose on the biochemical changes in rat liver in acute ethionine intoxication

When glucose is given to female rats 6 hours after a dose of ethionine, there is rapid reversal of the inhibition of RNA synthesis induced in the liver by the ethionine. Three hours after the administration of glucose, the level of RNA synthesis is 72% of normal, as compared with a level of 15% of normal in rats given ethionine but no glucose. The inhibition of protein synthesis is reversed by glucose more slowly, reaching 27% of normal in 3 hours, and 54% in 18 hours. In rats given ethionine, but not glucose, the level is 18% at 3 hours and 27% at 18 hours. Glucose failed to alter significantly the level of ATP in the liver, or to increase the rate of ATP turnover. The significance of these results is discussed.

Resistance of liver polysomes to ATP deficiency in the male rat

1. 1. Mature male and female rats respond in a similar fashion to a single dose of ethionine by the following changes in the liver: (a) marked depression of ATP concentration, (b) large accumulation of S- adenosylethionine and (c) severe inhibition of radioactive orotate incorporation into nuclear and microsomal RNA. 2. 2. However, a divergence in the response between the sexes is seen in hepatic protein synthesis. In the female rat, there is a rapid and marked inhibition of protein synthesis both in vivo and in vitro accompanied by a breakup of polysomes into monomers. In contrast, in male rats there is much less inhibition of protein synthesis in vivo, no inhibition in vitro and only a slight loss of polysomes. The unusual resistance of the polysomes from the male to the ATP deficiency lasts for at least 24 h. At that time, the male shows less depression of ATP concentration and a decrease in S- adenosylethionine content of the liver when compared to the female. The possible usefulness of this model for the analysis of some aspects of polysome metabolism in the intact cell is briefly discussed.

The use of chronically administered low doses of ethionine and puromycin to study increases in rat liver adenosine monophosphate deaminase activity caused by thioacetamide injections

Rat liver AMP deaminase activity was increased 2-fold 24 hr after three once-daily injections of thioacetamide at 50 mg kg body weight. Since higher doses of inhibitors, administered over several days, led to fatal toxicities, lower doses of ethionine (250 mg kg body weight, once daily) or puromycin (80 mg kg body weight day in three divided doses) were administered chronically throughout 4 or 5 days to test whether increased enzyme activity involved hepatic translation mechanisms. Ethionine inhibited thioacetamide-induced increases through 4 days in both male and female rats, but was no longer inhibitory after 5 days. Puromycin was not inhibitory. Studies on the incorporation of leucine-U- 14C into liver proteins and on cortisone induction of tryptophan oxygenase established that neither ethionine nor puromycin inhibited hepatic protein biosynthesis. Hepatic ATP levels were depleted by ethionine alone or in combination with thioacetamide, but it was considered unlikely that this change was linked with ethionine inhibition of increased AMP deaminase activity. The possibility that ethionine inhibition was related to allosteric modulation or to inhibition of cellular proliferation was discussed.

The biochemical pathology of ethionine-lnduced pancreatic damage: Ethionine incorporation into proteins, and ATP levels in pancreas of rats

Two biochemical mechanisms of possible pathogenetic significance in injury of cells by ethionine are synthesis of abnormal proteins by ethionine incorporation, and induction of ATP deficiency. These hypotheses were evaluated in relation to ethionine-in-duced pancreatic damage in rats. Ethionine incorporation into pancreatic protein was found to conform to the pattern which has been reported for several natural amino acids. Incorporation continued for at least 9 hours following a dose of 0.5 g per kg, apparently because a significant level of free ethionine persisted. The pancreas did not appear to have any unique tendency to incorporate ethionine into structural proteins when compared with liver and kidney, and therefore the results do not suggest that formation of abnormal proteins is of special significance in the pancreas. S-adenosylethionine accumulation and ATP deficit were demonstrated in pancreas 24 hours following single doses of ethionine (1 g per kg). However, at a lower dose which was adequate to produce severe pancreatic damage, there was no decrease in ATP in the pancreas. It therefore appears that the ethionine-induced pancreatic lesion cannot be attributed to ATP deficiency.

A potential method for increasing pancreatic accumulation of 75Se selenomethionine.

Since the introduction of 75Se selenomethionine as an agent for pancreatic scanning (1), numerous protocols designed to increase pancreatic uptake of this amino acid analogue have been proposed (1–4). For the most part, these methods have not been substantiated by animal investigation, and, where they have been tested experimentally, no significant increase in pancreatic radioactivity has been found (5). During the past year we have been engaged in determining the effects of numerous hormonal, pharmacologic, and biologic influences on pancreatic amino acid accumulation. Of the agents so far studied, only ethionine has been found to significantly increase pancreatic concentration of 75Se selenomethionine. Ethionine, the ethyl analogue of methionine, has been used, in doses of 1 g/kg body weight, to produce experimental pancreatitis and other cytopathogenic effects in animals (6, 7). When given in smaller daily doses over a period of months, it is carcinogenic (8). We have found in rats that when 1 mg/kg body weight (i.e., one one-thousandth the dose used to produce pancreatitis) of ethionine is given intraperitoneally and 75Se selenomethionine is administered subcutaneously thirty minutes later, a 63 per cent increase in pancreatic radioactivity (over control values) is produced. No significant increase in radioactivity of other organs results. The mechanism of ethionine's effect on accumulation of radioactivity in pancreas appears to reside in suppression of acinar cell enzyme release rather than in increased amino acid uptake or protein synthesis per se (7, 9, 10). It has been conclusively shown that when unlabeled methionine is given simultaneously with ethionine, all adverse cytopathological and physiological effects are prevented (6, 8, 9). We are currently investigating in animals: (a) whether the small dose of ethionine capable of increasing pancreatic amino acid concentration also produces adverse cytopathogenic and physiologic effects and (b) whether administration of unlabeled methionine thirty minutes after 75Se selenomethionine (and thus sixty minutes after ethionine) will prevent the adverse cytopathogenic and physiologic effects of ethionine. We have determined that administration of methionine in such a fashion does not affect the increased pancreatic accumulation of radioactivity. If any adverse effects of the single small dose of ethionine required to augment pancreatic amino acid accumulation can be prevented by subsequent administration of methionine, it should be possible to employ ethionine as an adjunct to clinical pancreatic photoscanning. Since, in practice, the value of pancreatic scanning is limited by relatively unfavorable statistics (i.e., low count rate and poor target: nontarget ratio), a 63 per cent increase in pancreatic radioactivity would represent a considerable advantage. Such an increase might be expected to transform a maximum count rate of, for example, 2500 c.p.m. to 4075 c.p.m.

On the Mechanism of Induction of Hepatic Adenosine Triphosphate Deficiency by Ethionine

Abstract A method is described for the quantitative determination of S-adenosylethionine (AEt) in perchloric acid extracts of liver. The administration of ethionine to female rats is followed by the rapid accumulation of AEt in the liver. The concentration of AEt increases with increasing doses of ethionine up to about 0.25 to 0.35 mg per g of body weight. The AEt concentration remains at a maximum level for at least 24 hours after a single injection of ethionine. The concentration of free ethionine in the liver and blood is sufficiently high to account for this persistence of maximal AEt levels over this period of time. A close inverse relationship has been found between the concentration of adenosine triphosphate and AEt when measured as a function of time after injection of ethionine. However, a discrepancy exists between the concentrations of ATP and AEt when measured as a function of the dose of ethionine. A maximum liver concentration of AEt is obtained at a dosage level of ethionine which induces only a moderate decrease in ATP concentration. The ATP level continues to fall as the amount of ethionine administered is increased above this level. The basis for this discrepancy has been studied and found to be a progressive inhibition of synthesis of adenine nucleotides de novo with large doses of ethionine associated with the decreasing levels of ATP. The data concerning the various effects of ethionine upon the concentrations of hepatic AEt and ATP and in the induction of various biochemical lesions are consistent with the hypothesis that the primary effect of ethionine is to trap adenosine as AEt by reacting with ATP. With low doses of ethionine, the trapping effect is largely compensated for by a large increase in the rate of synthesis of adenosine monophosphate de novo. With increasingly larger doses of ethionine, the rate of trapping exceeds the rate of synthesis de novo with a resultant decrease in the concentration of adenine nucleotides including ATP. This, in turn, further compromises the new formation of AMP, leading to a progressive decrease in ATP concentration. A new steady state is established at a low level of ATP. During this steady state, AEt is not stable but is continuously broken down and resynthesized. The administration of methionine several hours after ethionine inhibits the further synthesis of AEt, thus allowing the partial resynthesis of ATP from adenosine liberated during transethylation from AEt. This accounts for the partial reversal of those biochemical lesions resulting from the decrease in ATP such as inhibition of protein and ribonucleic acid synthesis and accumulation of excess triglyceride in the liver (fatty liver). The biochemical and structural changes in the liver induced by ethionine offer an interesting example of biochemical lesions induced primarily by lethal synthesis, as enunciated by Peters.

The Inhibition of Liver Ribonucleic Acid Synthesis by Ethionine

SUMMARY The administration of DL-ethionine to female rats inhibits liver ribonucleic acid synthesis up to 90 to 95%. The inhibition of RNA synthesis follows in time the decrease in adenosine triphosphate concentration and precedes the inhibition of protein synthesis. The degree of inhibition of RNA synthesis is a function of the dose of ethionine. The curve for this inhibition parallels closely the curves for the decrease in ATP concentration and for the inhibition of protein synthesis with varying dosages of ethionine. The simultaneous administration of methionine reverses to a considerable extent the inhibition of RNA synthesis by ethionine.

Nicotinamide-adenine dinucleotides in acute liver injury induced by ethionine, and a comparison with the effects of salicylate.

1. The effects of single doses of ethionine or sodium salicylate on the nicotinamide-adenine dinucleotide content of rat liver have been studied. 2. There was no significant change in the sum of NAD+NADH(2) during the early period (0-2hr.) of the liver injury induced by ethionine but there was a decrease in this value of approx. 30% by 4hr. after administration. 3. Ethionine had no significant effect on the NADP+NADPH(2) during the first 2hr. period after administration. The sum then decreased to a value approx. 70% of the control by 3hr. after dosing but showed a partial recovery at the 4hr. period before decreasing again in later stages of the poisoning. 4. Salicylate produced a very rapid decrease in the NADP+NADPH(2) in the liver after intraperitoneal injection. After 1hr. the decrease was approx. 30% of the initial value; the sum slowly returned towards the normal range during the following 4hr. 5. A high parenteral dose of salicylate was found to cause only a small depression in the concentration of ATP in rat liver in contrast with the rapid depletion produced by ethionine. 6. These results are discussed in terms of the liver disturbances produced by ethionine and salicylate.

Premature birth in rats induced by ethionine injection.

THE injection of pregnant rats with certain antimeta-bolites, such as ethionine, in sub-lethal doses, may result in resorption or stillbirth1–4. We wish to report the effect of smaller, repeated doses of ethionine on the course and outcome of pregnancy in rats. Daily subcutaneous injections, in the doses listed in Table 1, were given from the twelfth day of pregnancy until delivery. Control animals received saline injections. All injections were in a volume of 1 ml. The date of conception was established by the first appearance of sperm on vaginal smears.

Induction of Premature Birth in Rats by a Methionine Antagonist

Pregnant rats injected with ethionine have shown a high incidence of resorption and stillbirth and pups born alive have been smaller than normal. Without precise knowledge of the time of conception it has not been known to what extent the low birth weight could be attributed to prematurity. The present study was designed to shed light on this question. Pregnant rats were injected with graded doses of ethionine up to and including the amount known to cause resorption or stillbirth. The date of conception was inferred from the first appearance of sperm in the vagina. Relatively small doses of ethionine caused premature birth with only a low incidence of resorption and stillbirth. The incidence of prematurity, and its degree, were directly related to the dose of ethionine. The premature pups grew faster than the control pups, thus correcting an initial weight deficit by the age of 3 months. Sub-lethal doses of ethionine given to non-pregnant animals did not adversely affect subsequent conception or gestation.

PANCREATIC LESIONS BY N-ACETYLMETHIONINE.

IN THE COURSE of the last few yearsDLethionine, an analogue of methionine, has been shown to have effects on the pancreas and some other organs.* One of its principal effects on the pancreas is to diminish the number of zymogenic granules and also the amount of ribonucleic acid in the cytoplasm of the acinary cells. The drug administered to rats for more than 15 consecutive days leads to pancreatic atrophy,3,4a fact that aroused suspicion that it might suppress enzyme secretion, as Kaiser and Grossman13and Almeida and Grossman demonstrated in dogs after giving themDLethionine for several consecutive days. To be useful in the treatment of acute pancreatitis, a drug that inhibits pancreatic secretion would have to act rapidly, in a few hours. With this in mind, Alvizouri et al2tried giving a single dose of ethionine, with unsatisfactory results: pancreatic secretion was

IMMUNE MECHANISMS IN CHRONIC PANCREATIC DISEASE. I. OCCURRENCE OF PRECIPITINS TO PANCREATIC HOMOGENATES FOLLOWING CERTAIN FORMS OF PANCREATIC DAMAGE: AN EXPERIMENTAL STUDY.

The presence of precipitins to pancreatic antigens was investigated by gel diffusion in two experimental conditions in dogs: pancreatic-duct ligation, and ethionine-induced pancreatitis. Precipitins to the pancreas were not demonstrable after pancreatic-duct ligation, but were demonstrable in other dogs, after small doses of ethionine had been administered for a short time, and in the absence of clinical or other laboratory evidence of pancreatic damage.

Plasma and liver lipids of ethionine-treated rats.

AbstractThe changes in liver and plasma lipid concentrations caused by ethionine have been studied in the rat. A dose‐response curve was obtained for one time interval. For a constant dosage the time response curve was measured. The response of rats of different sex and nutritional state was also studied. Ethionine caused a rise in liver lipids, essentially confined to the glyceride fraction, a fall of plasma phospholipids, cholesterol esters, free cholesterol and glycerides and a rise of plasma free fatty acids (FFA). The liver lipids rose to a higher level in fasted female than in fasted male rats. Male rats also required a higher dose of ethionine for maximal response. Carbohydrate feeding to otherwise fasted female rats diminished, but did not abolish, the liver fat accumulation. The changes in plasma and liver lipid levels were maximal 24 hours after the ethionine administration. At 72 hours the liver lipids had fallen again almost to the zero time value. During the recovery phase the plasma glycerides rose above the zero time value and at 72 hours were 3 times higher.