Induction Of Tryptophan Oxygenase Research Articles

Maturation of fetal hepatocytes in vitro by extracellular matrices and oncostatin M: Induction of tryptophan oxygenase

Previously, we described that embryonic day 14.5 (E14.5) mouse fetal hepatocytes differentiate to express tyrosine amino transferase (TAT) and glucose-6-phosphatase, which are expressed in the perinatal liver, in response to oncostatin M (OSM) or in high-cell-density culture. However, under such conditions, fetal hepatic cells failed to express genes for adult liver-specific enzymes, such as tryptophan oxygenase (TO). Although phenobarbital (PB) and dimethylsulfoxide (DMSO) have been known to maintain the functions of adult hepatocytes in vitro, they failed to induce TO expression in fetal hepatic cells. Thus far, no system has been developed that reproduces terminal differentiation of fetal hepatocytes in vitro. Here, we describe that extracellular matrices derived from Engelbreth-Holm-Swarm sarcoma (EHS) in combination with OSM or high-cell-density culture induced expression of TO as well as cytochrome P450 genes that are involved in detoxification. However, EHS alone was insufficient to induce expression of TO, although it induced TAT expression in fetal hepatocytes. In addition, high-density culture further augmented differentiation. In conclusion, the combination of signals by cytokines, cell-cell contact, and cell-matrix interaction is required for induction of adult liver functions in fetal hepatocytes in vitro. This primary culture system will be useful for studying the mechanism of liver development. (H EPATOLOGY 2002;35:1351-1359.)

Sphingosine inhibition of tyrosine aminotransferase and tryptophan oxygenase induction by dexamethasone in primary culture of rat hepatocytes

The effect of sphingosine, a known selective inhibitor of protein kinase C, on the induction of tyrosine aminotransferase (TAT) and tryptophan oxygenase (TO) by dexamethasone was studied in the primary culture of rat hepatocytes to determine the possible involvement of protein kinase C in the expression of glucocorticoid action. Sphingosine inhibits the induction of TAT by dexamethasone in a concentration-and time-dependent manner in primary culture of rat hepatocytes. It does not inhibit the induction of TAT by Bt 2cAMP. Sphingosine inhibits also the induction of TO by dexamethasone in a manner similar to TAT inhibition. It has no effect on the activity of lactate dehydrogenase, a cytosolic marker enzyme and on the protein content of the cultured hepatocytes. These findings indicate that endogenous modulator of protein kinase C, such as sphingosine, may influence the expression of glucocorticoid action in rat hepatocytes.

Involvement of Protein Synthesis in the Action of Glucocorticoids on the Working Capacity of Adrenalectomized Rats

The administration of cortisol for 3 or 5 days increased the maximal duration of swimming under additional load, equal to 3% of body weight, in adrenalectomized but not in intact Wistar rats. The liver tryptophan oxygenase activity also increased. The blockade of RNA synthesis by actinomycin D excluded both results of cortisol administration. High doses of progesterone (25 or 75 mg/day) decreased the working capacity, rendering the action of cortisol on the working capacity of adrenalectomized rats insignificant. However, the antagonism between two hormones was not striking at the level of liver tryptophan oxygenase induction. The results suggest that the action of glucocorticoids on the working capacity is mediated by RNA and, further, by protein synthesis. The competition for glucocorticoid cytoplasmic receptors, to be found between glucocorticoids and a high level of progesterone, can disturb this mechanism. The synthesis of hepatic enzyme proteins is not responsible for the action of glucocorticoids on the working capacity.

Effects of oestradiol and vitamin B6 on tryptophan metabolism in the rat: implications for the interpretation of the tryptophan load test for vitamin B6 nutritional status.

1. The effects of the administration of oestradiol and vitamin B6on tryptophan metabolism in the rat have been assessed by measurement of the release of14CO2from [14C]tryptophan, in vivo, in order to determine whether, and to what extent, the abnormalities of tryptophan metabolism that are associated with oestrogen administration can be attributed to drug-induced vitamin B6deficiency or depletion. Two positional isomers of [14C]tryptophan have been used; [ring-2-14C]tryptophan as an index of the activity of tryptophan oxygenase (L-tryptophan: oxygen oxidoreductase (decyclizing),EC1.13.11.11) and [methylene-14C]trytophan as an index of the activity of kynureninase (L-kynurenine hydrolase,EC3.7.1.3).2. The administration of 500 μg oestradiol/kg body-weight led to a reduction in the release of14CO2from both positional isomers of tryptophan, suggesting that the activities of both tryptophan oxygenase and kynureninase are reduced following oestrogen treatment. The kinetics of the release of14CO2from [methylene-14C]tryptophan after the administration of oestradiol were compatible with competitive inhibition of kynureninase by oestradiol or a metabolite.3. The administration of 10 mg pyridoxine hydrochloride/kg body-weight also reduced the production of14CO2from both positional isomers of14C]tryptophan, suggesting some toxicity of such a high dose of the vitamin.4. In animals which had received the supplementary dose of vitamin B6, the administration of oestradiol led to further reduction in the production of14CO2from [ring-2-14C]tryptophan, suggesting a further reduction in the activity of tryptophan oxygenase, and an increase in the production of14CO2from [methylen-14C]tryptophan, but with a delay in the peak of production.5. These results confirm that there is no induction of tryptophan oxygenase by oestradiol, but rather reduced activity of the enzyme after the administration of a relatively high dose of the hormone. They also confirm that the inhibition of kynureninase by oestrogen metabolites that has been reported previously in partially-purified enzyme preparations also occurs in vivo.6. It is suggested that the abnormal results of the tryptophan load test that have been reported in women receiving oestrogens, and which have been interpreted as indicating some extent of drug-induced vitamin B6deficiency, can be accounted for by the inhibition of tryptophan metabolism by oestrogens or their metabolites. Therefore it seems likely that the practice of administering supplements of vitamin B6to women receiving oestrogens may not be appropriate, and indeed may exacerbate the changes in tryptophan metabolism that result from the administration of oestrogens. The tryptophan load test would appear to be unreliable as an index of vitamin B6nutritional status in women receiving oestrogens.

The effects of oestrogen administration on tryptophan metabolism in rats and in menopausal women receiving hormone replacement therapy

The effects of the administration of oestrogens on the activity of hepatic tryptophan oxygenase have been assessed both directly (by measurement of enzyme activity in vitro) and indirectly (by measurement of urinary excretion of tryptophan metabolites) in rats, and indirectly in menopausal women receiving hormone replacement therapy. Intraperitoneal administration of 500 μg of oestradiol or ethinyl oestradiol/kg body wt had no effect on the activity of tryptophan oxygenase in homogenates of liver from mature (13-week-old) female rats. Both adrenalectomy and ovariectomy led to a reduction in the activity of tryptophan oxygenase in homogenates of liver from mature rats; again there was no effect of giving 500 μg of oestradiol/kg body wt by intraperitoneal injection. Intraperitoneal administration of 210 μg of oestrone sulphate/kg body wt for 1 or 2 days before killing, or its incorporation in the diet for up to 8 weeks at an equivalent dose rate, had no effect on the activity of tryptophan oxygenase in homogenates of liver from ovariectomized 6–14-week-old female rats. Intraperitoneal administration of 500 μg oestradiol/kg body wt to intact mature female rats together with 500 mg tryptophan/kg body wt caused a reduction in the urinary excretion of xanthurenic and kynurenic acids, kynurenine and N 1-methyl nicotinamide. When peri- and post-menopausal women were treated with ethinyl oestradiol (20 μg/day) or piperazine oestrone sulphate (3 mg/day) for 3 months, there was an increase in the concn of tryptophan in plasma, with no change in the urinary excretion of xanthurenic and kynurenic acids and kynurenine. This study provides no evidence for the induction of tryptophan oxygenase by oestrogens in rats or human beings.

Glucocorticoid induction of tryptophan oxygenase: Attenuation by intragastrically administered carbohydrates and metabolites

In vivo tryptophan 2,3-dioxygenase (TPO) activity in male rats was estimated from the rate of production of 14CO 2 after intragastric administration of [ 14C-2]tryptophan. The synthetic glucocorticoids hydrocortisone-21-sodium succinate or Triamcinolone acetonide were injected to elevate hepatic TPO activity on an acute (1–6 hr) or chronic (24 hr) basis. Glucose, fructose, or glycerol was intragastrically intubated in doses ranging from 4 to 16 mmoles to assess their abilities to attenuate acute or chronic increases of TPO activity by these glucocorticoids. Hydrocortisone-21-sodium succinate at doses of 0, 25,and 50 mg/kg produced dose-dependent elevations of TPO. A 50 mg/kg dose produced a 3-fold elevation of enzyme activity when measured in vitro as product produced by liver homogenates and a 2-fold elevation when assessed from expired radioactie carbon dioxide from radiolabeled tryptophan in vivo. Enzyme activity measured by 14CO 2 production reached peak values in 2–3 hr and returned to baseline in 5 hr. Glucose, fructose or glycerol completely prevented the rise in conversion of [ 14C-2]tryptophan produced by hydrocortisone hemisuccinate when administered at doses of 12 or 16 mmoles 0.5 hr before the steroid. Lower doses had less effect. The potencies of the compounds in inhibiting acute increases in TPO activity produced by hydrocortisone hemisuccinate were in the order glycerol > fructose > glucose. Chronic Triamcinolone treatment elevated in vivo TPO activity by 2.5-fold and in vitro TPO activity by 5-fold. The chronic elevation of in vivo TPO by Triamcinolone could be arrested within 1 hr by an intragastric fructose load. The present finding, that acute or chronic glucocorticoid-induced increases in in vivo TPO activity were rapidly blocked by intragastric carbohydrate loads, is consistent with the view that dietary carbohydrates modulate hepatic TPO activity via feedback repression and not by a cessation of TPO enzyme synthesis.

Alteration in the inducibility by dexamethasone of the activity of tyrosine aminotransferase and tryptophan oxygenase in regenerating rat liver after partial hepatectomy.

In adrenalectomized rats, the activity of tryptophan oxygenase in liver cytosol was increased 4 h after dexamethasone injection, with a peak on day 2. It then gradually decreased to the value obtained in normal animals treated with dexamethasone. In adrenalectomized, 70% hepatectomized rats, tryptophan oxygenase activity decreased significantly during the period of DNA synthesis after partial hepatectomy; it then increased to above the level existing before hepatectomy, reaching a maximum on day 5, and subsequently returned to the level before hepatectomy. These changes were well correlated with those of dexamethasone receptors at all times after adrenalectomy and partial hepatectomy. In adrenalectomized rats, tyrosine aminotransferase activity was increased 4 h after dexamethasone injection, reached a maximum on day 2, and then remained elevated. The level was increased by subsequent 70% hepatectomy, reached a maximum on day 5, and then began to decrease, but activity was still high on day 10 after hepatectomy when the receptor level had returned to the value existing before hepatectomy. When actinomycin D or cycloheximide was injected with dexamethasone, the induction of the two enzymes was completely inhibited. The activities of the two enzymes in regenerating liver of rats without adrenals and without glucocorticoid treatment were as low as those in adrenalectomized rats without glucocorticoid treatment. Thus, there was a good correlation between the dexamethasone receptor level and the induction of tryptophan oxygenase, but there was a dissociation between the receptor level and the induction of tyrosine aminotransferase after adrenalectomy and partial hepatectomy. The mechanism of this dissociation is unknown.

Endotoxin inhibition of dexamethasone induction of tryptophan oxygenase in suspension culture of isolated rat parenchymal cells: Involvement of the hepatic nonparenchymal cell fraction

The effect of endotoxin on the induction of tryptophan oxygenase activity by dexamethasone has been studied in suspension cultures of isolated rat hepatic parenchymal cells incubated alone or mixed with hepatic nonparenchymal cells. Hepatic cellular fractions were isolated from untreated rats and from animals injected 2 hr prior to killing with 2 mg/kg of endotoxin. Endotoxin (400 μg/ml) added to suspensions of parenchymal cells from untreated animals did not decrease the induction of tryptophan oxygenase by dexamethasone. Endotoxin addition to cell suspension containing parenchymal and nonparenchymal cells from untreated rats caused a significant diminution in the induction of enzyme activity elicited by 0.1 μM, but not by 20 μM, dexamethasone. The nonparenchymal cell fraction from untreated animals had no effect on enzyme induction in the absence of added endotoxin. However, nonparenchymal cells isolated from rats pretreated in vivo with endotoxin diminished the induction of tryptophan oxygenase activity elicited by 0.1 and 20 μM dexamethasone even in the absence of added endotoxin. The inhibitory effect of nonparenchymal cells from endotoxin-pretreated rats was related to cell concentration and was accentuated by the in vitro addition of endotoxin. Parenchymal cells from the endotoxin-pretreated animals had an altered sensitivity to dexamethasone. The ec 50 concentration for dexamethasone induction of tryptophan oxygenase was 40 and 500 μm for parenchymal cells from untreated and endotoxin-pretreated animals respectively. Parenchymal cells from the pretreated animals were more sensitive to the inhibitory effect of the nonparenchymal cells from the pretreated animals. These results are interpreted as indicating that endotoxin does not directly antagonize the induction of tryptophan oxygenase. The results suggest that this inhibition of tryptophan oxygenase induction is a mediated event involving an interaction of endotoxin with cellular constituents of the hepatic nonparenchymal cell fraction.

Induction of tryptophan oxygenase and tyrosine aminotransferase by metabolites of hydrocortisone

Metabolites of hydrocortisone were isolated from rat liver on a preparative scale, fractionated by column chromatography on Sephadex LH-20 and silica gel and tested for biological activity. Apart from the well known neutral metabolites, steroid glucuronides and sulfates, we obtained metabolite fractions containing non-conjugated steroidal carboxy acids and acid metabolites of unknown structure. One of these fractions induced tyrosine aminotransferase (EC 2.6.1.5) in adrenalectomized female rats but not trptophan oxygenase (EC 1.13.11.11), whereas another one mainly increased activity of tryptophan oxygenase. The doses necessary to significantly induce both enzymes were much lower in case of these metabolites than in the case of hydrocortisone itself. The active fractions eluting from silica gel column were analyzed by thin-layer chromatography in two different solvent systems. Absence of hydrocortisone in these fractions could be clearly demonstrated. Furthermore, the active fractions eluting from the silica gel column were characterized by treatment with an extract from Helix pomatia and/or diazomethane and subsequent analysis by thin-layer chromatography. We conclude, considering the biological activity of some synthesized derivatives of hydrocortisone, that the biologically active components are acid metabolites of hydrocortison which are not identical to any of the known metabolites.

Effects of an acute ethanol load on cortisol- or tryptophan-mediated induction of tryptophan oxygenase and tyrosine aminotransferase in rat liver

Effects of an acute ethanol load on cortisol- or tryptophan-mediated induction of tryptophan oxygenase and tyrosine aminotransferase in rat liver

The kynurenine load test, an adjunct to the tryptophan load test.

The kynurenine load test was introduced to distinguish, in vivo, between abnormal tryptophan metabolism caused by induction of tryptophan oxygenase or by impaired kynureninase function. This test was carried out by giving a loading dose of 700 mumol L-kynurenine sulphate followed by determination of urinary metabolites of the tryptophan-nicotinic acid ribonucleotide pathway. The results of the kynurenine load test in a group of healthy post-menopausal female subjects show a consistant increase in urinary excretion of some metabolites of the tryptophan-nicotinic acid ribonucleotide pathway (kynurenine, 3-hydroxy-kynurenine, and 3-hydroxyanthranilic acid) of a magnitude comparable to the increase seen after a loading dose of 9800 mumol L-tryptophan (the tryptophan load test).

Suppression by Δ9-tetrahydrocannabinol of induction of hepatic tyrosine aminotransferase and tryptophan oxygenase

The present study characterizes the action of Δ 9-THC on enzyme induction by studying its effects on the induction of hepatic tyrosine aminotransferase (TAT) by steroids. In none of our studies did Δ 9-THC inhibit TAT activity in the absence of steroid. Although treatment with hydrocortisone (HC, 150 mg/kg, 2 hr prior to sacrifice) caused a 2.1-fold induction of enzyme activity, pretreatment with Δ 9-THC (200 mg/kg, 2 hr prior to sacrifice) decreased this induction to 1.3-fold. When mice were treated with Δ 9-THC 1 hr prior to HC induction, TAT activity was induced only 1.1-fold over control while HC alone induced TAT activity 2.5-fold. Even when steroid treatment preceded Δ 9-THC administration by 3 hr, there was significant inhibitory activity. Enzyme activity at 0, 3, and 6 hr after steroid was 18.7, 41.4, and 55.5 μmol of PPA/g of liver/hr, respectively. When Δ 9-THC was administered at 3 hr after steroid and mice killed 3 hr later, enzyme activity was reduced to 36.2 μmol PPA/g liver/hr. Inhibition of steroid induction was dose-related over a range of 50–400 mg/kg of Δ 9-THC. Δ 9-THC had little effect on induction of TAT or tryptophan oxygenase in mouse liver by tryptophan and had no effect on tryptophan induction of tryptophan oxygenase in rat liver.

Schistosoma mansoni and Schistosoma japonicum: Effects of infection on induction of tryptophan oxygenase in mouse livers

Neither l-tryptophan oxygenase nor d-tryptophan oxygenase activities could be detected in adult Schistosoma japonicum. 1. 2. The mean l-tryptophan oxygenase activity in livers from female mice after a 6 week S. japonicum or an 8 week S. mansoni infection were only 45% and 61% respectively of normal liver l-tryptophan oxygenase activity. 2. 3. Induction of l-tryptophan oxygenase enzyme protein synthesis by hydrocortisone 21-phosphate is nearly twice as great in female mouse livers after an 8 week S. mansoni infection as in a 6 week S. japonicum infection.

Effects of pyrazole on rat liver tryptophan oxygenase

The effects of pyrazole administration on rat liver tryptophan oxygenase have been studied both under basal conditions and after induction by cortisol or activation by tryptophan. Pyrazole administration is followed by a decrease of the basal holoenzyme and total enzyme activities. It induces furthermore a considerable inhibition of the cortisol mediated tryptophan oxygenase induction. These effects are not mediated by a modification of a tryptophan oxygenase effector, as shown by mixed homogenate experiments. The tryptophan enhancement of total tryptophan oxygenase activity is not affected by pyrazole administration contrary to the holoenzyme activity. Pyrazole added in vitro inhibits liver tryptophan oxygenase activity only when used at concentrations which are considerably higher that those occuring in vivo after pyrazole administration.

Induction of trytophan oxygenase by dexamethasome in isolated hepatocytes dependence on composition of medium and pH

Hepatocytes were isolated from perfused rat livers. 4 x 10-6 cells/ml were incubated at at 37 degrees C in different media in the absence and presence of a steroid hormone, dexamethasone phosphate (2 x 10-5 M). 1. Hormonal enzyme induction occurred in cells suspended in a simple salt medium, devoid of amino acids and macromolecules. This induction was completely blocked by addition of either actinomycin D (2 mu-g/ml) or cycloheximide (50 mu-g/ml). 2. Incubation of cells in media containing defatted albumin did not enhance hormonal enzyme induction, although disintegration of cells during incubation was reduced. Addition of a crude albumin fraction reduced tryptophan oxygenase induction and dextran completely blocked enzyme induction by dexamethasone. 3. An increase of dexamethasone concentration in the presence of albumin to 9 x 10-5 M was unable to raise enzyme induction further, and a still higher concentration of hormone, 3 x 10-4 M, resulted in reduced enzyme induction. 4. The hormonal induction of tryptophan oxygenase was most pronounced when the pH of the medium was between 7.0 and 7.6, with an optium at 7.3. No induction was found when the pH of the medium was either 6.6 or 7.8. The basal tryptophan oxygenase activity was much less influenced by similar pH variations. It is concluded that hepatocytes in suspension are able to carry out hormone-stimulated enzyme synthesis and that factors influencing this process may be studied under controlled conditions in such systems.

Depression of hepatic gluconeogenesis and the hypoglycemia of endotoxin shock

Depression of hepatic gluconeogenesis and the hypoglycemia of endotoxin shock

The effect of glucose ingestion on basal and induced enzyme levels in rat tissues

The ingestion of glucose ad libitum (for 19 h to 3 days) decreased the levels of enzymes concerned with amino acid metabolism in liver and intestine (but not in kidney and brain) and raised those of hepatic glucokinase, pyruvate kinase and NADP-malate dehydrogenase. Glucose feeding inhibited the substrate and cortisol induction of tryptophan oxygenase; it did not diminish the induction of tyrosine aminotransferase by glucagon (in adrenalectomized rats) or of renal ornithine aminotransferase by estrogen and it enhanced the response of NADP-malate dehydrogenase to thyroxine. In hepatomas, as opposed to normal liver, 24 h of glucose feeding increased the basal and the cortisol-induced levels of tyrosine aminotransferase. The results obtained with endocrinectomized and hormone-treated rats led to the following conclusions: (1) the effects of glucose ingestion on the quantitative pattern of hepatic enzymes is not mediated through altered secretion of pituitary, adrenal or pancreatic hormones but presumably by metabolites of glucose; (2) glucose, ingested in large amounts over 1–3 days, is not an inhibitor of enzyme inductions in general: its effect varies with the enzyme, the inducer and the tissue in which the enzyme is located.

Effects of Schistosoma mansoni infection on induction of tryptophan oxygenase in mouse livers

1. 1. l-Tryptophan oxygenase activity could not be detected in adult Schistosoma mansoni. 2. 2. Mouse livers infected with S. mansoni eggs had increased l-tryptophan oxygenase activities before and after treatment with hydrocortisone-21 phosphate.

Induction of tryptophan oxygenase in primary rat liver cell suspensions by glucocorticoid hormone

The effect of dexamethasone phosphate on tryptophan oxygenase activity has been studied in suspensions of enzymatically prepared rat liver cells. The hormone was found to induce a 2–3 fold increase in enzyme activity after incubating the cells for 7 h in Krebs-Ringer phosphate solution at 37 °C. Purified parenchymal cells and a mixture of parenchymal cells and reticuloendothelial cells gave comparable results, indicating that the hormonal effect was on the parenchymal cells.

Complexity of in vivo induction of tryptophan oxygenase and tyrosine aminotransferase: Effects of tryptophan metabolites

1. 1. In the intact rat, tryptophan oxygenase and tyrosine aminotransferase are both induced by various tryptophan metabolites, in particular by quinolinic, picolinic, quinaldic and xanthurenic acids. 2. 2. Inductions by tryptophan metabolites and by hydrocortisone are additive in the case of tyrosine aminotransferase, but not additive in the case of tryptophan oxygenase. 3. 3. Theophylline is able to induce tyrosine aminotransferase as well; however, no potentiation of the effects of tryptophan metabolites can be obtained by simultaneous administration of theophylline.