Programmed Effects in Neurobehavior and Antioxidative Physiology in Zebrafish Embryonically Exposed to Cadmium: Observations and Hypothesized Adverse Outcome Pathway Framework.

Non-communicable diseases (NCDs) are a major cause of premature mortality. Recent studies show that predispositions for NCDs may arise from early-life exposure to low concentrations of environmental contaminants. This developmental origins of health and disease (DOHaD) paradigm suggests that programming of an embryo can be disrupted, changing the homeostatic set point of biological functions. Epigenetic alterations are a possible underlying mechanism. Here, we investigated the DOHaD paradigm by exposing zebrafish to subtoxic concentrations of the ubiquitous contaminant cadmium during embryogenesis, followed by growth under normal conditions. Prolonged behavioral responses to physical stress and altered antioxidative physiology were observed approximately ten weeks after termination of embryonal exposure, at concentrations that were 50-3200-fold below the direct embryotoxic concentration, and interpreted as altered developmental programming. Literature was explored for possible mechanistic pathways that link embryonic subtoxic cadmium to the observed apical phenotypes, more specifically, the probability of molecular mechanisms induced by cadmium exposure leading to altered DNA methylation and subsequently to the observed apical phenotypes. This was done using the adverse outcome pathway model framework, and assessing key event relationship plausibility by tailored Bradford-Hill analysis. Thus, cadmium interaction with thiols appeared to be the major contributor to late-life effects. Cadmium-thiol interactions may lead to depletion of the methyl donor S-adenosyl-methionine, resulting in methylome alterations, and may, additionally, result in oxidative stress, which may lead to DNA oxidation, and subsequently altered DNA methyltransferase activity. In this way, DNA methylation may be affected at a critical developmental stage, causing the observed apical phenotypes.

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.

[The effect of amino acids in the asparagine family on the aspartate kinase and homoserine dehydrogenase of ethionine-resistant mutants of Pseudomonas putida]. / Vliianie aminokislot asparaginovogo semeistva na aktivnost' aspartatkinazy i gomoserindegidrogenazy étioninrezistentnykh mutantov Pseudomonas putida.

The activity of aspartate kinase and homoserin dehydrogenase from ethionine resistant mutants Pseudomonas putida 25 and 6 have been studied as affected by amino acids from the family of asparagine. They are characterized by a capacity to the surplus synthesis of methionine. It is shown that mutants have negative regulation of the level of activity of the studied enzymes. It is supposed that the mutations (or mutation) could take place which affected properties of enzymes, which participated directly in the biosynthesis of methionine, in the analogue resistant clones 25 and 6.

Enhancement and attenuation of cytogenetic damage by vitamin C in cultured human lymphocytes exposed to thiotepa or L-ethionine.

Vitamin C (vit C) at 2 mM enhanced sister chromatid exchange (SCE) frequencies induced by Thiotepa (THIO) or L-ethionine (L-ETH) in cultured human lymphocytes. However, when vit C was tested at 0.02 mM and 0.2 mM a rather protective effect on SCE rates induced by THIO or L-ETH was identified. Vit C (2 mM) caused a cell division delay in cultures treated with THIO or L-ETH. Division delays caused by THIO or L-ETH were reversed in the presence of 0.02 mM or 0.2 mM vit C. Mitotic indices (MIs) in cultures treated with THIO or L-ETH continued to be suppressed in the presence of 2 mM vit C. However, vit C at 0.02 mM reversed suppression of MIs caused by L-ETH or THIO. These findings illustrate the complexity of the interactions of vit C in biological systems and indicate that with different concentrations vit C can cause or prevent genetic toxicity.

[Effects of gomisin A, a lignan component of Schizandra fruits, on experimental liver injuries and liver microsomal drug-metabolizing enzymes].

Effects of oral administration of gomisin A, one of the components isolated from Schizandra fruits, on liver injuries induced by CCl4, d-galactosamine and dl-ethionine and on liver microsomal drug-metabolizing enzyme activities were investigated. Gomisin A suppressed the increase of serum transaminase activities and the appearances of histological changes such as degeneration and necrosis of hepatocyte, inflammatory cell infiltration and fatty deposition in each type of liver injury. The repeated administration of gomisin A (30 or 100 mg/kg, p.o., daily for 4 days) induced an apparent increase of liver weight in liver-injured and normal rats. Gomisin A decreased serum triglyceride and lipid contents of the liver in biochemical studies. Increases of microsomal cytochrome b5 and P-450, elevations of NADPH cytochrome C reductase, aminopyrine N-demethylase and 7-ethoxycoumarin O-deethylase activities and decrease of 3,4-benzo(a)pyrene hydroxylase activity per cytochrome P-450 were observed after the administration of gomisin A. In addition, gomisin A was found to enhance the incorporation of 14C-phenylalanine into liver protein and to shorten the hexobarbital-induced sleeping time. These changes caused by gomisin A were similar to those by phenobarbital. However, gomisin A is distinctly different from phenobarbital in the finding that phenobarbital lessened the survival ratio of CCl4-intoxicated mice, but gomisin A did not. Our observation suggest that gomisin A shows an antihepatotoxic action by oral application and also has hypolipidemic (mainly triglyceridemic) and liver protein synthesis-facilitating actions and that the enlargement of the liver seen with gomisin A is the adaptive hypertrophy which is due to the induction of drug-metabolizing enzymes.

[Pharmacological studies on schizandra fruits. III. Effects of wuweizisu C, a lignan component of schizandra fruits, on experimental liver injuries in rats].

The effects of wuweizisu C, a lignan component of schizandra fruits, on liver injuries induced by carbon tetrachloride (CCl4), d-galactosamine and dl-ethionine were investigated by means of serum-biochemical and histopathological examinations in rats. Pretreatment or combined administration of wuweizisu C dose-dependently reduced the elevation of serum transaminase activity and histological changes such as fatty degeneration, cell necrosis, inflammatory cell infiltration, etc., which were caused by the single administration of 1 ml/kg, p.o., or the repeated administration of 0.2 ml/kg, s.c., daily for 4 days of CCl4, respectively. The effects of wuweizisu C on the liver injuries induced by a low dose (200 mg/kg, i.p.) and a high dose (400 mg/kg, i.p.) of d-galactosamine were compared with those of uridine. Wuweizisu C significantly lowered the rise of serum transaminase activity after the administration of a low dose of d-galactosamine in the serum-biochemical analysis. A tendency was also shown to inhibit cell necrosis and inflammatory cell infiltration caused by both doses of d-galactosamine in the histopathological examination. On the other hand, uridine markedly repaired the serum-biochemical and histopathological changes after the administrations of both doses of d-galactosamine. Also wuweizisu C cured the liver injury by the repeated administration of 150 mg/kg, i.p., daily for 4 days of d-galactosamine. After the repeated administration of 250 mg/kg, s.c., daily for 4 days of ethionine, liver cell atrophy, diffuse fatty degeneration and decrease of serum triglyceride were observed, but not cell necrosis. Wuweizisu C dose-dependently inhibited fatty degeneration and decrease of serum triglyceride. These findings suggest that wuweizisu C can be protective and/or therapeutic on hepatocellular phenomena such as cell necrosis, fatty degeneration, inflammatory cell infiltration, etc., in human hepatitis.

Beneficial effect of pancreatic polypeptide in experimental pancreatitis.

Two animal models have been employed to examine the role of pancreatic polypeptide, a potent and selective inhibitor of pancreatic exocrine secretion, in the treatment of acute pancreatitis. In one model pancreatitis was induced by feeding young female Swiss Webster mice an ethionine-supplemented, choline-deficient diet for 48 hr. Animals (N = 30 per group) were injected subcutaneously every 8 hr for 7 days with pancreatic polypeptide (0, 2, 20, and 200 micrograms/kg/day). Treatment with 20 and 200 micrograms/kg/day pancreatic polypeptide significantly (P less than 0.05) reduced mortality from a control rate of 70% to 42% and 33%, respectively. Treated animals also exhibited significant (P less than 0.05) decreases in pancreatic content of activated chymotrypsin and an improvement in pancreatic histology. Pancreatic polypeptide was effective whether treatment was started before or at the same time the test diet was introduced. In contrast, pancreatic polypeptide failed to protect dogs with acute pancreatitis induced by retrograde injection of the pancreas with bile, which may reflect the rapid and mechanical nature of pancreatic damage in this animal model.

Inhibition by ethionine and beta-diethylaminoethyl diphenylpropylacetate of RNA and protein synthesis in the rat liver, and its reversal by pregnenolone-16 alpha-carbonitrile.

The effect of pregnenolone-16 alpha-carbonitrile (PCN) on female Sprague-Dawley rats was studied in conjunction with ethionine and beta-diethylaminoethyl diphenylpropylacetate (DADP). The cyanosteroid virtually abolished in inhibitory action of ethionine on orotic acid and leucine uptake by liver microsomes. When PCN was given conjointly with DADP, it completely overshadowed the latter's effect on leucine uptake. DADP was found to act as an inhibitor of the translational mechanism 5 h after its administration, while it exerted a stimulatory influence on the transcriptional mechanism after the same time period.

[Effect of thiol compounds on experimental liver damage (IV). Detoxicating effect of tiopronin, glutathione and cysteine on ethionine induced lived damage (author's transl)].

S-GPT elevated due to ethionine (Eth) administration was suppressed by thiol compounds such as tiopronin (2-mercaptopropionylglycine), glutathione, cysteine, in which tiopronin proved to be more effective than glutathione or cysteine. In thin-layer chromatography of urinary metabolites, Eth and ethionine sulfoxide were detected with administration of Eth, and S-ethyltiopronin plus Eth and ethionine sulfoxide by the administrations of Eth and tiopronin. These S-ethyl derivatives were not detected in the urine with administration of Eth and glutathione or cysteine. In the analysis of Eth and its metabolites by gas chromatography, cumulative urinary excretion of Eth within 72 hr after Eth administration was 40.7% in the Eth administered group, 23.6% in the Eth-tiopronin administered group and 38.2% in the Eth-glutathione administered group, respectively. In the urine of the Eth-tiopronin administered group, S-ethyltiopronin was excreted by 13.6%. Detoxicating effect of tiopronin on Eth induced liver damage was considered to involve the following mechanism. Tiopronin is considered to excrete part of Eth as S-ethyltiopronin by being an acceptor of transfer reaction of the ethyl group of Eth. Neither glutathione nor cysteine was an acceptor of the ethyl group and a detoxicating effect on Eth was not observed.

Acute lethality of D- and L-ethionine in Swiss mice.

The acute, 7-day LD50 in Swiss mice of D-ethionine administered intraperitoneally, was determined as 185 mg/kg with 95% confidence limits of 163 and 210 mg/kg. L-ethionine, the stereoisomer, which is more potent in inhibiting liver RNA synthesis, was not lethal at 2500 mg/kg. This acute toxicity of D-ethionine was suppressed by L-methionine and adenine. Blood accumulation in the thoracic cavity and pericardium was the probable cause of death.

Plasma and tissue iron changes in the rat after acute administration of ethionine.

1. Ethionine administered acutely to the adult female rat markedly elevates and then lowers plasma iron concentration over several days. Liver iron undergoes a reverse cycle. 2. Ethionine does not cause changes in the blood parameters, including total plasma iron-binding capacity and plasma iron clearance. Erythrocytes of rats injected with ethionine show altered responses to hypertonicity. 3. Increased reticulo-endothelial activity of the spleen, indicated by increased uptake of 59Fe-labelled erythrocytes by liver and spleen, apparently contributes to plasma iron elevation. Also the liver releases iron which further raises plasma iron.

The effect of cupric acetate on ethionine metabolism.

The addition of cupric acetate, a potent inhibitor of ethionine carcinogenesis, to a diet containing ethionine increased the ethionine toxicity. The concentration of S-adenosylethionine in liver was found to be significantly higher when compared to animals fed only ethionine in the diet. Ethionine forms a complex(es) with cupric acetate that is insoluble at a pH higher than 4; however, this complex can be solubilized at a low pH. Ethionine, if administered p.o. in the form of this complex, was absorbed from the intestinal lumen in the same order of magnitude as when administered alone; however, as the body weight increased over 200 g, the portion of absorbed ethionine decreased. The absorption of ethionine bound in the complex was completed within 16 hr compared to 2 hr for free ethionine. This time delay was accompanied by a shift in the concentration maximum of ethionine metabolities in the liver form 8 to 24 hr. When ethionine was administered alone, it was metabolized in the intestinal lumen as demonstrated by the analysis of the soluble intestinal contents; the presence of cupric acetate inhibited this process. The chromatographic analysis of ethionine metabolites in urine of rats treated by the complex revealed an increased excretion of ethionine sulfoxide and other ethionine metabolities at the expense of N-acetylethionine sulfoxide. The increased concentration of S-adenosylethionine in the liver in chronic experiments may be, at least partly, a result of a diminished capacity of the rat to detoxify (acetylate) ethionine sulfoxide, which is considered the main reserve pool of ethionine for the maintenance of a high level of S-adenosylethionine.

[Immunosuppressive effect of ethionine in rats. Resistance of this effect to methionine, tryptophan, ATP, adenosine and uridine]. / Effet immunodépresseur de l'éthionine chez le rat. Résistance de cet effet a l'administration de méthionine, de tryptophane, d'ATP, d'adénosine et d'uridine

One hundred eleven male adult rats of a pathogen-free Sherman or Charles River strain were divided into 3 series. Each of them contained non-treated controls and rats treated with intraperitoneal (i.p.) injections of DL-ethionine (E) [75 mg/d p. 100 g] for 6 weeks (series 1) or for only 2 weeks (series 2 and 3). Series 1 and 2 included also rats injected simultaneously with E and DL-methionine (68.4 mg/d p. 100 g). In the series 2 there were also groups receiving at the same time E, ME and tryptophan (5 mg/d p. 100 g) or E, Me and ATP (1.65 mg/d), and in the series 3, groups were injected with E + adenosine (31.2 mg/d p. 100 g) and (or) uridine (284 mg/d p. 100 g). All animals were immunized i.p. 1 week before killing with sheep red blood cells (SRBC): either 2.4 X 10(9) SRBC p. 100 g without adjuvant (series 1 and 2) or 1 X 10(9) SRBC with Freund's adjuvant added (series 3). Rosette-and plaque-forming cells (RFC and PFC) were counted in the spleen, and titers of serum hemagglutinins and hemolysins were determined with separation of 2-mercaptoethanol-resistant IgG from non-resistant immunoglobulins. E provoked within 2 weeks a drastic inhibition of the immune responses: drop in the RFC and PFC levels p. 10(6) (approximately 2900 leads to approximately 320 and approximately 415 leads to 40 respectively, in the study 3), and significant decrease in the serum antibody titer, especially IgG. Addition of Me still amplified the immunosuppression. Supplementation with tryptophan, ATP, adenosine and (or) uridine was also ineffective. Though not displaying any immunoprotective potency, Me partly neutralized the non-immunological effects of E: arrest of the body growth and involution of lymphoid organs and male genital organs, particularly apparent after 6 weeks of treatment. In conclusion, the ethionine-induced immunosuppression does not result from a metabolic exclusion of labile methyl groups or from an acute ATP deficiency due to an excess trapping of adenine as S-adenosyl-ethionine. Lack of pyrimidines or tryptophan must also be discarded. On the other hand, the possibility of maintaining the immunosuppressive activity of E, while reducing the toxicity of this compound, by addition of Me deserves to be emphasized.

Effects of ethanol and ethionine on DNA synthesis during experimental liver regeneration.

Ethanol reversed the ethionine-induced inhibition of 3H-thymidine incoroporation into hepatic DNA in the male and female rat after partial hepatectomy.