Hepatic Tyrosine Aminotransferase Research Articles

Dual function of glucocorticoid hormones in the body and using this duality for preventing complications of glucocorticoid therapy

Glucocorticoid preparations are used in all areas of medicine for more than 70 years as the most effective anti-inflammatory drugs possessing also anti-allergic and immunosuppressive actions. However, the use of these preparations, unique in the combination and expression of therapeutic properties, is associated with nearly inevitable serious adverse effects. The complications of glucocorticoid therapy are caused by the duality of glucocorticoid hormone functions. Glucocorticoid hormones in the body act: · As the most universal regulators of virtually all metabolic and physiological processes and · As hormones mainly responsible for the organism behavior under stress conditions. In clinical practice, glucocorticoid preparations are used mainly as stress-responsible agents, and their regulatory activities are neglected, but they are manifested inevitably as complications. To prevent these complications, it is necessary to follow the regulatory properties of glucocorticoid preparations which are realized through regulation of activities of many enzymes. The hepatic tyrosine amino transferase seems to be, possibly, the best example of the regulatory activity of glucocorticoids. Based on the literature data and results of the author’s studies, it is proposed to use blood level of tyrosine as a promising laboratory parameter for monitoring glucocorticoid therapy.

Antioxidants reverse the changes in energy metabolism of rat brain after chronic administration of L.-tyrosine

Tyrosinemia type II is a rare autosomal recessive disease caused by deficiency of hepatic tyrosine aminotransferase and is associated with neurologic and development difficulties in numerous patients. Considering that the mechanisms underlying the neurological dysfunction in hypertyrosinemic patients are poorly known and that high concentrations of tyrosine provoke mitochondrial dysfunction and oxidative stress, in the present study we investigated the in vivo influence of antioxidants (N-acetylcysteine, NAC; and deferoxamine, DFX) administration on the inhibitory effects on parameters of energy metabolism in cerebral cortex, hippocampus and striatum of rats, provoked by chronic administration of L.-tyrosine. Our results showed that chronic administration of L.-tyrosine results in a marked decrease in the activity of citrate synthase in all the analyzed structures and succinate dehydrogenase activities in hippocampus and striatum, and that antioxidants administration can prevent this inhibition in hippocampus and striatum. Moreover, chronic administration of L.-tyrosine inhibited the activity of complex I, II-III and IV in the striatum, which can be prevented by antioxidant treatment. However, the co-administration of NAC plus DFX could not prevent the inhibition of creatine kinase activity in the striatum. In conclusion, the present study demonstrates that the administration of antioxidants NAC and DFX attenuates the L.-tyrosine effects on enzymes of the Krebs cycle and the mitochondrial respiratory chain, suggesting that impairment of energy metabolism can be involved with oxidative stress. These results also indicate a possible neuroprotective role for NAC and DFX as a potential adjuvant therapy to the patients with Tyrosinemia type II.

PT/Y Mice Are Sensitive to the Inhibitory Effect of o-Aminoazotoluene on Glucocorticoid Induction of Tyrosine Aminotransferase and Its Hepatocarcinogenic Effect.

PT/Y mice used for studies of the effects of mutagens are characterized by the absence of spontaneous tumors of the liver, but often develop these tumors in response to chronic oaminoazotoluene treatment. The level of glucocorticoid induction of adaptive hepatic enzyme tyrosine aminotransferase decreases by more than 70% 24 h after acute injection of o-aminoazotoluene to these animals. These mice can serve as a model for studies of the relationship between the effect of carcinogens on the regulation of activity of adaptive hepatic enzymes and their capacity to induce the development of liver tumors.

Hepatic tyrosine aminotransferase and glucocorticoid abuse in meat cattle

Besides being extensively applied as therapeutical remedies, glucocorticoids (GCs) - most notably dexamethasone or prednisolone - are also illegally used in livestock for growth-promoting purposes. This study was designed to assess the suitability of liver tyrosine aminotransferase (TAT), a gluconeogenic enzyme known to be induced by GCs, to act as a reliable candidate biomarker to screen for GC abuse in cattle. Enzyme activity was measured spectrophotometrically in liver cytosols or in cell extracts, and TAT gene expression was determined by real-time PCR. Compared with untreated veal calves, a notable scatter (20-fold) and much higher median values (3-fold) characterized TAT specific activity in liver samples from commercially farmed veal calves. A time-related increase in both enzyme activity and gene expression was detected in rat hepatoma cell lines treated with dexamethasone concentrations (10(-8) or 10(-9) m) in the range of those recorded in noncompliant samples from EU official controls. In experimental studies in which finishing bulls were administered GCs at growth-promoting dosages, however, no such changes were recorded in dexamethasone-treated animals; a statistically significant rise in liver TAT activity (+95%) only occurred in prednisolone-treated bulls. Although further research is needed to characterize the GC-mediated response in cattle liver, TAT does not appear to be a specific and sensitive biomarker of GC abuse in the bovine species.

Effects of sulcotrione [2-(2-chloro-4-mesylbenzoyl)-cyclohexane-1,3-dione] on enzymes involved in tyrosine catabolism and the extent of the resulting tyrosinemia and its relationship with corneal lesions in rats

We aimed to investigate the effects of subchronic exposure to sulcotrione on the enzymes involved in tyrosine catabolism and the extent of the resulting tyrosinemia and corneal lesions in rats. Daily oral administration of sulcotrione at 0.1, 0.5, 5, 50 and 100 mg/kg/day markedly inhibited hepatic 4-hydroxyphenylpyruvate dioxygenase (HPPD). In response to the tyrosinemia, the activity of hepatic tyrosine aminotransferase (TAT) at each dose level was insignificantly induced and hepatic homogentisic acid oxidase (HGO) at 5, 50 and 100 mg/kg/day was markedly reduced. Repeated oral administration of sulcotrione to rats at doses of 5, 50, and 100 mg/kg/day for 90 days produced corneal lesions with an incidence of 18.8%, 75.0% and 56.3%, respectively. The significant increase in tyrosine levels indicates that the occurrence of corneal lesions in rats exposed to sulcotrione was not only associated with the concentration of tyrosine in the ocular fluid, but also with other, unidentified factors.

Dimethyl-diphenyl-propanamide Derivatives As Nonsteroidal Dissociated Glucocorticoid Receptor Agonists

A series of 2,2-dimethyl-3,3-diphenyl-propanamides as novel glucocorticoid receptor modulators is reported. SAR exploration led to the identification of 4-hydroxyphenyl propanamide derivatives displaying good agonist activity in GR-mediated transrepression assays and reduced agonist activity in GR-mediated transactivation assays. Compounds 17 and 30 showed anti-inflammatory activity comparable to prednisolone in the rat carrageenan-induced paw edema model, with markedly decreased side effects with regard to increases in blood glucose and expression of hepatic tyrosine aminotransferase. A hypothetical binding mode accounting for the induction of the functional activity by a 4-hydroxyl group is proposed.

Dietary protein differentially regulates the kinetic behaviour of serine dehydratase and tyrosine aminotransferase of liver and white muscle of rainbow trout (Oncorhynchus mykiss)

We have determined the kinetic behaviour of serine dehydratase (SerDH) and tyrosine aminotransferase (TyrAT) in the liver and white muscle of juvenile rainbow trout (Oncorhynchus mykiss) fed on a low-protein/high-fat (LP/HF) and control diets. The relationship between the kinetic parameters and tissue-DNA concentration has also been determined. SerDH and TyrAT showed hyperbolic kinetics in all cases. The partial replacement of protein with fat significantly increased hepatic SerDH-specific activity, maximum velocity (V(max)) and Michaelis constant (K(m)) while no significant changes were detected in the values of these parameters in white muscle. Nevertheless, hepatic and white-muscle TyrAT specific activity and V(max) decreased in trout fed on LP/HF diet with respect to control. The K(m) of hepatic TyrAT was also lower in trout fed on LP/HF diet than in control. The TyrAT kinetic parameters expressed per cell unit also changed in the same sense as previously described. These results show that, in trout, SerDH and TyrAT are two enzymes regulated by the partial replacement of dietary protein by fat.

Characterization of ZK 245186, a novel, selective glucocorticoid receptor agonist for the topical treatment of inflammatory skin diseases

Glucocorticoids are highly effective in the therapy of inflammatory diseases. Their value, however, is limited by side effects. The discovery of the molecular mechanisms of the glucocorticoid receptor and the recognition that activation and repression of gene expression could be addressed separately opened the possibility of achieving improved safety profiles by the identification of ligands that predominantly induce repression. Here we report on ZK 245186, a novel, non-steroidal, low-molecular-weight, glucocorticoid receptor-selective agonist for the topical treatment of inflammatory dermatoses. Pharmacological properties of ZK 245186 and reference compounds were studied in terms of their potential anti-inflammatory and side effects in functional bioassays in vitro and in rodent models in vivo. Anti-inflammatory activity of ZK 245186 was demonstrated in in vitro assays for inhibition of cytokine secretion and T cell proliferation. In vivo, using irritant contact dermatitis and T cell-mediated contact allergy models in mice and rats, ZK 245186 showed anti-inflammatory efficacy after topical application similar to the classical glucocorticoids, mometasone furoate and methylprednisolone aceponate. ZK 245186, however, exhibits a better safety profile with regard to growth inhibition and induction of skin atrophy after long-term topical application, thymocyte apoptosis, hyperglycaemia and hepatic tyrosine aminotransferase activity. ZK 245186 is a potent anti-inflammatory compound with a lower potential for side effects, compared with classical glucocorticoids. It represents a promising drug candidate and is currently in clinical trials.

New forms of hereditary tyrosinemia type II in mink: hepatic tyrosine aminotransferase defect.

Three different forms of hereditary tyrosinemia type II, with some of the features described for the Richner-Hanhart's syndrome, occur in mink (Mustela vison Schreb.). These disorders are inherited as simple autosomal recessive characters. The main symptoms of the diseases are watery dull eyes, frequent urination, alteration of the hair/skin on the toes and a highly elevated level of tyrosine in blood and urine. An enzyme defect in hepatic tyrosine aminotransferase (EC 2.6.1.5) is considered as the reason of these three forms of the mink disease. Differences between these forms of hereditary tyrosinemia in mink now described lie among other things in the time of onset, duration and severity of the disese.

Modeling receptor/gene-mediated effects of corticosteroids on hepatic tyrosine aminotransferase dynamics in rats: dual regulation by endogenous and exogenous corticosteroids

Receptor/gene-mediated effects of corticosteroids on hepatic tyrosine aminotransferase (TAT) were evaluated in normal rats. A group of normal male Wistar rats were injected with 50 mg/kg methylprednisolone (MPL) intramuscularly at the nadir of their plasma corticosterone (CST) rhythm (early light cycle) and sacrificed at various time points up to 96 h post-treatment. Blood and livers were collected to measure plasma MPL, CST, hepatic glucocorticoid receptor (GR) mRNA, cytosolic GR density, TAT mRNA, and TAT activity. The pharmacokinetics of MPL showed bi-exponential disposition with two first-order absorption components from the injection site and bioavailability was 21%. Plasma CST was reduced after MPL dosing, but resumed its daily circadian pattern within 36 h. Cytosolic receptor density was significantly suppressed (90%) and returned to baseline by 72 h resuming its biphasic pattern. Hepatic GR mRNA follows a circadian pattern which was disrupted by MPL and did not return during the study. MPL caused significant down-regulation (50%) in GR mRNA which was followed by a delayed rebound phase (60-70 h). Hepatic TAT mRNA and activity showed up-regulation as a consequence of MPL, and returned to their circadian baseline within 72 and 24 h of treatment. A mechanistic receptor/gene-mediated pharmacokinetic/pharmacodynamic model was able to satisfactorily describe the complex interplay of exogenous and endogenous corticosteroid effects on hepatic GR mRNA, cytosolic free GR, TAT mRNA, and TAT activity in normal rats.

Pharmacokinetics of methylprednisolone after intravenous and intramuscular administration in rats

Methylprednisolone (MPL) pharmacokinetics was examined in adrenalectomized (ADX) and normal rats to assess the feasibility of intramuscular (i.m.) dosing for use in pharmacodynamic studies. Several study phases were pursued. Parallel group studies were performed in normal and ADX rats given 50 mg/kg MPL (i.v. or i.m.) and blood samples were collected up to 6 h. Data from studies where normal rats were dosed with 50 mg/kg MPL i.m. and killed over either 6 or 96 h were combined to determine muscle site and plasma MPL concentrations. Lastly, ADX rats were dosed with 50 mg/kg MPL i.m. and killed over 18 h to assess hepatic tyrosine aminotransferase (TAT) dynamics. MPL exhibited bi-exponential kinetics after i.v. dosing with a terminal slope of 2.1 h(-1). The i.m. drug was absorbed slowly with two first-order absorption rate constants, 1.26 and 0.219 h(-1) indicating flip-flop kinetics with overall 50% bioavailability. The kinetics of MPL at the injection site exhibited slow, dual absorption rates. Although i.m. MPL showed lower bioavailability compared with other corticosteroids in rats, TAT dynamics revealed similar i.m. and i.v. response profiles. The more convenient intramuscular dosing can replace the i.v. route without causing marked differences in pharmacodynamics.

Hepatotoxin-induced hypertyrosinemia and its toxicological significance

A (1)H Nuclear Magnetic Resonance (NMR) spectroscopic investigation of the effects of single doses of four model hepatotoxins on male Sprague-Dawley rats showed that hypertyrosinemia was induced by three of the treatments (ethionine 300 mg/kg, galactosamine hydrochloride 800 mg/kg and isoniazid 400 mg/kg) but not by the fourth (thioacetamide 200 mg/kg). Concomitant histopathological and clinical chemistry analyses showed that hypertyrosinemia could occur with or without substantial hepatic damage and that substantial hepatic damage could occur without hypertyrosinemia. However, in the rats dosed with galactosamine hydrochloride, which showed highly variable amounts of liver damage at ca. 24 h after dosing, a clear relationship was found between the degree of hypertyrosinemia and the extent of the hepatic necrosis induced. In line with the cause of clinically observed Type II Tyrosinemia, we consider that the critical event in the onset of hepatotoxin-induced hypertyrosinemia is likely to be a reduction in hepatic tyrosine aminotransferase (TAT) activity. We discuss mechanisms by which TAT activity could be lost with special consideration given to pyridoxal 5'-phosphate (P5P) depletion and to the inhibition of protein synthesis. This analysis may have implications for the interpretation of clinical measures of liver status such as Fischer's ratio and the branched-chain tyrosine ratio (BTR).

Hepatic tyrosine aminotransferase activity is affected by chronic heat stress and dietary tyrosine in broiler chickens

1. Two experiments were conducted to investigate the impact of high temperature and dietary tyrosine (Tyr) content on performance and activity of hepatic tyrosine aminotransferase (EC 2.6.1.5.), an enzyme that catalyses the first step in the metabolic degradation of Tyr in broiler chickens. 2. Two-week-old birds were allocated to one of three temperature treatments: 24 ° C (control), 36 ° C (heat stress, HS) and 24 ° C pair-fed (24PF) for 2 weeks and fed on diets containing 100% (Experiment 1) and 50, 100 and 200% (Experiment 2) of the NRC requirement for Tyr. 3. In Experiment 1, exposure of chickens to 36 ° C for 2 weeks caused significant increase in hepatic tyrosine aminotransferase activity but no significant change in activity of hepatic phenylalanine 4-hydroxylase (EC 1.14.16.1) (an enzyme that catalyses conversion of phenylalanine to Tyr) compared with the 24PF birds. No significant changes attributable to heat stress were detected in hepatic glutamic-oxaloacetic transaminase (EC 2.6.1.1) activity. 4. In Experiment 2, heat stress caused reductions in weight gain and feed intake in chickens on all diets, compared with their control counterparts. Hepatic tyrosine aminotransferase activity was increased by heat stress compared with their 24PF counterparts in chickens fed on the 100 and 200% Tyr diets, while in chickens fed the 50% Tyr diet, it was reduced by heat stress. 5. From these results, it is suggested that hepatic tyrosine aminotransferase activity is affected by heat stress and dietary Tyr content and the increased tyrosine aminotransferase activity with, in part, relatively low phenylalanine hydroxylase activity in hepatic tissues may be involved in the Tyr metabolism characteristic of heat-stressed chickens.

Fifth-generation model for corticosteroid pharmacodynamics: application to steady-state receptor down-regulation and enzyme induction patterns during seven-day continuous infusion of methylprednisolone in rats.

A fifth-generation model for receptor/gene-mediated corticosteroid effects was proposed based on results from a 50 mg/kg i.v. bolus dose of methylprednisolone (MPL) in male adrenalectomized rats, and confirmed using data from other acute dosage regimens. Steady-state equations for receptor down-regulation and tyrosine aminotransferase (TAT) enzyme induction patterns were derived. Five groups of male Wistar rats (n = 5/group) were subcutaneously implanted with Alzet mini-pumps primed to release saline or 0.05, 0.1, 0.2, and 0.3 mg/kg/hr of MPL for 7 days. Rats were sacrificed at the end of the infusion. Plasma MPL concentrations, blood lymphocyte counts, and hepatic cytosolic free receptor density, receptor mRNA, TAT mRNA, and TAT enzyme levels were quantitated. The pronounced steroid effects were evidenced by marked losses in body weights and changes in organ weights. All four treatments caused a dose-dependent reduction in hepatic receptor levels, which correlated with the induction of TAT mRNA and TAT enzyme levels. The 7 day receptor mRNA and free receptor density correlated well with the model predicted steady-state levels. However, the extent of enzyme induction was markedly higher than that predicted by the model suggesting that the usual receptor/gene-mediated effects observed upon single/intermittent dosing of MPL may be countered by alterations in other aspects of the system. A mean IC50 of 6.1 ng/mL was estimated for the immunosuppressive effects of methylprednisolone on blood lymphocytes. The extent and duration of steroid exposure play a critical role in mediating steroid effects and advanced PK/PD models provide unique insights into controlling factors.

Tissue distribution of 2-(2-nitro-4-trifluoromethylbenzoyl)-cyclohexane-1,3-dione (NTBC) and its effect on enzymes involved in tyrosine catabolism in the mouse

Administration of a single oral dose of 2-(2-nitro-4-trifluoromethyl-benzoyl)-cyclohexane-1,3-dione (NTBC) to mice increases the concentration of tyrosine in the plasma and aqueous humour. The tyrosinaemia is both time and dose-dependent with a single dose of 30 μmol NTBC/kg (10 mg/kg) producing maximal concentrations of tyrosine in plasma of about 1200 nmol/ml and in aqueous humour of about 2200 nmol/ml at 16 h after dosing. Analysis of the key hepatic enzymes involved in tyrosine catabolism, following a single dose of 30 μmol NTBC/kg, showed that 4-hydroxyphenylpyruvate dioxygenase (HPPD) was markedly inhibited soon after dosing and that the activity recovered very slowly. In response to the tyrosinaemia, the activity of hepatic tyrosine aminotransferase (TAT) was induced about two-fold, while the activity of hepatic homogentisic acid oxidase (HGO) was reduced at 4 and 5 days after dosing. Daily oral administration of NTBC at doses up to 480μmol NTBC/kg (160mg/kg/day) to mice produced a maximal tyrosinaemia of about 600-700nmol/ml plasma, showing some adaptation relative to a single dose. Unlike the rat, no treatment-related corneal lesions of the eye were seen at any dose levels up to 6 weeks. Administration of a single oral dose of [ 14C]-NTBC at 30μmol/kg led to selective retention of radiolabel in the liver and to a lesser extent the kidneys. Our studies show that NTBC is a potent inhibitor of mouse liver HPPD, which following repeat exposure produces a marked and persistent tyrosinaemia, which does not result in ocular toxicity.

Vitamin B-6 Deficiency and Level of Dietary Protein Affect Hepatic Tyrosine Aminotransferase Activity in Cats

Total activity [pyridoxal 5′-phosphate (PLP) added in the assay] of hepatic tyrosine aminotransferase (TAT) measured in cats at 0300, 0900, 1500 and 2100h was 10.3 ±1.1, 14.0 ± 0.7, 9.8 ± 1.3 and 11.0 ± 0.7 nkat/g liver, indicating little diurnal variation. Activity after 18 h of food deprivation was 10.0 ± 0.3 nkat/g liver, also not different from cats that were eating ad libitum. These findings support the idea that cats have only limited changes in the activity of hepatic TAT compared with rats. Total TAT activity was measured in cats fed high protein (550 g/kg) and low protein (180 g/kg) diets for 4 wk. Cats fed a high protein diet had activities significantly higher (about twice) than cats fed the low protein diet. Hepatic TAT activity of vitamin B-6-deficient cats (diet without pyridoxine for 9 wk) was compared with cats given the same diet with 8 mg pyridoxine/kg. Total hepatic TAT activity in deficient cats was significantly (P < 0.05) lower per gram soluble or total protein (but not per gram liver) than control cats; holoenzyme activity and percentage of active enzyme in deficient cats were also significantly lower by 75 and 64%, respectively. The apparent Km of TAT from cats for tyrosine (2.1 mmol/L) was similar to that for rats (1.9 mmol/L), but higher for PLP in cats (0.16 μmol/L) than rats (0.034 μmol/L). Part of the reason for the higher plasma tyrosine in vitamin B-6–deficient cats than rats is the higher Km of TAT for PLP in cats than rats.

The Effect of a Low-Protein Diet and Dietary Supplementation of Threonine on Tyrosine and 2-(2-Nitro-4-trifluoromethylbenzoyl) cyclohexane-1,3-dione-Induced Corneal Lesions, the Extent of Tyrosinemia, and the Activity of Enzymes Involved in Tyrosine Catabolism in the Rat

Rats fed a low-protein diet and administered 2-(2-nitro-4-trifluoromethylbenzoyl)cyclohexane-1,3-dione (NTBC) orally at 30 μmol/kg/day (10 mg/kg/day) or fed a low-protein diet containing 5 ppm NTBC develop lesions to the cornea of the eye within 3–8 days of exposure with an incidence of about 80%. This treatment also produces a marked inhibition of both hepatic and renal 4-hydroxyphenylpyruvate dioxygenase (HPPD) activity, an induction of hepatic but not renal tyrosine aminotransferase activity, and a marked tyrosinemia in the plasma and aqueous humor. The extent of tyrosinemia and changes in the activity of tyrosine catabolic enzymes are similar to those reported for rats fed a normal protein diet and administered NTBC orally at 30 μmol/kg/day. However, the onset of corneal lesions occurs much earlier in rats fed a low-protein diet. The adverse ocular effects of NTBC can be alleviated by supplementing the low-protein diet with 1% w/w threonine. The protection afforded by threonine inclusion in the diet was not due to any amelioration in the extent of inhibition of hepatic HPPD activity or reduction in the extent of the tyrosinemia as measured 8 days after treatment. Rats fedl-tyrosine at 5% w/w in a low-protein diet rapidly develop lesions to the cornea of the eye, which are associated with a marked tyrosinemia, increased hepatic tyrosine aminotransferase activity, and about a 50% reduction in the activity of hepatic HPPD. The onset of corneal lesions produced by feeding a high tyrosine diet could be delayed, but not prevented, by inclusion of 1% w/w threonine in the low-protein diet. The basis for the beneficial effect of dietary supplementation of threonine in alleviating the corneal lesions produced by NTBC is unclear. However, our findings do illustrate that protein deficiency limits the ability of the rat to respond to a tyrosine load produced by inhibition of HPPD.

Novel and recurrent tyrosine aminotransferase gene mutations in tyrosinemia type II.

Tyrosinemia type II (Richner-Hanhart syndrome, RHS) is a disorder of autosomal recessive inheritance characterized by keratitis, palmoplantar hyperkeratosis, mental retardation, and elevated blood tyrosine levels. The disease results from deficiency in hepatic tyrosine aminotransferase (TAT). We have previously described one deletion and six different point mutations in four RHS patients. We have now analyzed the TAT genes in a further seven unrelated RHS families from Italy, France, the United Kingdom, and the United States. We have established PCR conditions for the amplification of all twelve TAT exons and have screened the products for mutations by direct sequence analysis or by first performing single-strand conformation polymorphism analysis. We have thus identified the presumably pathological mutations in eight RHS alleles, including two nonsense mutations (R57X, E411X) and four amino acid substitutions (R119W, L201R, R433Q, R433W). Only the R57X mutation, which was found in one Scottish and two Italian families, has been previously reported in another Italian family. Haplotype analysis indicates that this mutation, which involves a CpG dinucleotide hot spot, has a common origin in the three Italian families but arose independently in the Scottish family. Two polymorphisms have also been detected, viz., a protein polymorphism, P15S, and a silent substitution S103S (TCG-->TCA). Expression of R433Q and R433W demonstrate reduced activity of the mutant proteins. In all, twelve different TAT gene mutations have now been identified in tyrosinemia type II.

Differences in binding of hepatic nuclear proteins from lean and obese rats to the 5'-upstream region of tyrosine aminotransferase.

The glucocorticoid effects on liver tyrosine aminotransferase mRNA levels have been studied in young, lean, and obese Zucker (fa/fa) rats and 5'-upstream regions of the tyrosine aminotransferase (TAT) gene have been used in gel retardation studies to investigate nuclear protein binding. Hepatic TAT mRNA levels were increased in obese fa/fa rats but were normalized seven days after adrenalectomy. Corticosterone replacement to adrenalectomized rats restored the increased levels of TAT mRNA in the obese animals. A 60-bp fragment of upstream TAT DNA (-2463 to -2403) was identified which showed higher levels of band shifting after incubation with hepatic nuclear proteins of obese rats compared with the proteins from lean animals. This differential level of gel retardation was substantially reduced by alkaline phosphatase treatment of nuclear proteins. Gel retardation was reduced when nuclear proteins were prepared from adrenalectomized obese rats, and increased with nuclear proteins from adrenalectomized rats replaced with corticosterone. DNA affinity chromatography and gel electrophoresis identified three proteins of approximately 58, 62, and 65 kDa in the DNA-protein complex. Increased amounts of these three proteins were purified from nuclei of obese rats. HNF3 alpha antibodies induced hypershift of the gel retardation pattern implicating HNF3 alpha as one of the proteins that binds to the 60 bp DNA fragment. The data support the hypothesis that decreased phosphorylation of nuclear proteins in obese rats is glucocorticoid-dependent and may contribute to the altered transcriptional activity of glucocorticoid-responsive genes.

Pharmacokinetics and receptor-mediated pharmacodynamics of corticosteroids

The corticosteroids, such as prednisolone and methylprednisolone, provide diverse antiinflammatory and immunosuppressive effects which typically show responses with slow onset and prolonged duration. This report summarizes modeling efforts which are successful in describing such steroid effects. Clinical effects with such a pattern, including adrenal suppression and altered trafficking of basophils and helper T-cells, can be related to plasma drug concentrations by models containing an inhibition function and differential equations for controlling input and disposition of the response variable. Some responses have circadian-controlled inputs which add time-dependent complexities to the models. Kinetic/dynamic data for severat corticosteroid effects yield IC 50 values which agree well with receptor K D values. A relationship of linear AUC of effect versus log AUC of steroid in plasma is found with these models over a large range of doses. Gene-mediated effects of corticosteroids are initiated by receptor-binding which causes a cascade effect altering DNA transcription, RNA, mRNA and proteins or enzymes accounting for drug effects. Models for such behavior have been developed in animals for hepatic tyrosine aminotransferase (TAT) enzyme activity. Studies with methylprednisolone formulated in liposomes show tissue sequestration of steroid, prolonged receptor-binding and extended inhibition of splenocyte proliferation. The data and models usually show good correspondence of the AUC of receptor occupancy with the AUC of pharmacologic response.