Arginase as a useful factor for the diagnosis of colorectal cancer liver metastases.

The present work is a continuation of studies on arginase as a marker in the diagnosis of colorectal cancer liver metastases (CRCLM). The purpose of the study was the evaluation of the arginase test in comparison with other colorectal cancer tests such as CEA, CA 19-9 and biochemical markers of liver function such as aspartate aminotransferase (AST) and alanine aminotransferase (ALT). The studies were conducted on blood serum from 85 patients with CRCLM obtained one to two days before tumor resection. The control group comprised 140 healthy blood donors and 81 patients with various non-malignant gastrointestinal diseases. Raised arginase activity was observed in serum of 85% of CRCLM patients, whereas elevated levels of CEA and CA 19-9 were found in 63% and 42% of patients, respectively. The combination of CEA or CA 19-9 with the arginase assay improved their sensitivity, but the sensitivity of the combined parameters was not higher than that of the arginase test itself. AST and ALT activities were increased in about 30% of CRCLM patients. The specificity of the arginase test calculated for 221 control subjects was 76%. It can thus be concluded that the determination of serum arginase activity can be helpful in the diagnosis of patients with colorectal cancer liver metastases.

The effect of serotonin, its precursors and metabolites on brain glutathione-S-transferase.

The isoelectric point and substrate specificity of the main isoform of glutathione-S-transferase (GST, EC 2.5.1.18) isolated from brain stem, hippocampus and parietal cortex of pig brain were determined. The effect of serotonin, its precursors (Try, 5-HTry), physiologically active derivative (melatonin) and final metabolite (5-HIAA) on the activity of this form was examined. Investigation indicated that serotonin did not affect the activity of GST in all studied regions of brain. The inhibitory effect of Try was stronger than that of 5-HTry, but weaker than the one expressed by melatonin and especially by 5-HIAA. Studies on the type of inhibition showed that Try, melatonin and 5-HIAA can compete for the active site with the electrophilic substrate but not with glutathione. Therefore precursors and endogenous derivatives of serotonin but not serotonin itself may affect the detoxification function of brain glutathione-S-transferase and increase the exposure of brain to toxic electrophiles.

Arginase isoforms in human colorectal cancer.

Arginase (EC 3.5.3.1) activity was determined in 54 colorectal tissues obtained from patients with primary colorectal adenocarcinoma as well as in serum of 45 patients and 65 healthy individuals. In patients, the preoperative values of the mean serum arginase activity and the activity in colorectal tumors were much higher than in serum of healthy subjects and control tissues. Two isoforms of arginase, anionic and cationic, were identified in colorectal tissues (normal and cancerogenous), and only one, the cationic form, in serum. These arginases were different from the main human liver cationic arginase (pI 9.3). The anionic colorectal arginase was identical with the human liver anionic isoform (pI 7.7), and the cationic arginase from colorectal tissues and blood serum with the human kidney cationic enzyme (pI 8.9). The total activity and the level of protein of the cationic arginase in colorectal cancer was higher than in control tissue, and it was also higher in serum of patients with colorectal cancer than in healthy subjects. Thus, it can be concluded that the increased arginase activity in blood serum and colorectal cancer in studied patients was due to the raised level of the cationic arginase and this isoform seems to be a discriminating parameter for assessing the presence of colorectal cancer.

[Tricyclic antidepressants as inhibitors of brain glutathione-S-transferase]. / Trójpierscieniowe leki przeciwdepresyjne jako inhibitory mózgowej transferazy glutationowej.

Tricyclic antidepressants, amitriptyline, doxepin--derivatives of cycloheptadiene as well as imipramine and clomipramine, derivatives of dibenzazepine inhibit the activity of glutathione-S-transferase pi isolated from different regions of human brain (parietal cortex, frontal cortex, brain stem). The inhibitory effect of studied drugs depends more on their chemical structure than on brain localization of the enzyme. All tricyclics bind nonspecifically to the effector site of glutathione-S-transferase (GST). The obtained results indicate that the inhibitory effect of tricyclic antidepressants on brain GST may decrease the efficiency of the enzymatic barrier, which protects brain against toxic electrophiles, and contribute in their adverse effects. On the other hand, brain GST may decrease the therapeutic effects of tricyclic antidepressants by binding them as ligands.

Glutathione conjugation in male reproductive system: studies on glutathione-S-transferase of bull and boar epididymis.

Male reproductive organs are extremely sensitive to the negative influence of toxic environmental factors as well as drugs, and until now not many attempts have been made at studying the detoxication enzymes and the relationship between the activity of those enzymes and spermatozoa fertility. In the present work we studied cytosolic glutathione-S-transferases (GST, EC 2.5.1.18) from different parts (head, corpus and tail) of bull and boar epididymis. We isolated two molecular forms of GST from each part of epididymis, characterized their biochemical properties and examined the mechanism of the catalyzed reaction. On the basis of their substrate specificity and isoelectric point, the isoforms were found to belong to the near neutral GST class mi. All examined GST forms exhibited higher affinity towards GSH than towards 1-chloro-2,4-dinitrobenzene (CDNB) and bull epididymis GST forms showed biphasic Lineweaver-Burk double reciprocal curves in the presence of GSH as a variable substrate. Boar epididymis anionic GST had the -SH groups both in the GSH and the CDNB binding place, whereas the cationic GST form--arginine residues in the CDNB binding place. Bull epididymis GST forms contained neither thiol nor arginine residues essential for catalytic activity.

Biotransformation in monkey brain: coupling of sulfation to glutathione conjugation.

Phenol sulfotransferase (PST, EC 2.8.2.1) and glutathione-S-transferase (GST, EC 2.5.1.18), the phase II biotransformation enzymes inactivate many exo- and endogenous compounds. The effect of PST substrates (catecholamines, simple phenols, selected phenolic drugs) and PST products (phenolic sulfates) on GST activity was investigated to identify possible interactions between sulfation and glutathione conjugation in the brain. Two soluble forms of PST and two forms of GST were isolated from monkey (Rhesus macacus) brain cortex. Catecholamines, hypertensive and hypotensive drugs which are sulfated by monkey brain PSTs slightly inhibit the activity of brain GSTs. The greatest inhibitory effect was observed with neurotoxic compounds such as 6-OHDA and manganese. The commonly used analgesic drugs inhibit both GST forms. These enzymes are also inhibited by phenacetin, the precursor of paracetamol, and prototype salicylates such as sodium salicylate and acetylsalicylic acid. The effect of simple phenols and their sulfated metabolites on GST activity varies. The obtained results point to a possible interaction between sulfation and glutathione conjugation in vivo since many physiologically, therapeutically and toxicologically active compounds which are sulfated by brain phenol sulfotransferases may be bound by brain glutathione-S-transferases. These compounds may lose their activity (on being bound to GST) and expose the brain to the toxic electrophiles (by decreasing GST activity).

Sulfation of hypertensive and hypotensive drugs by monkey brain phenol sulfotransferase.

The substrate specificity and affinity of two forms of phenol sulfotransferase (PST) from Rhesus macaque brain cortex were studied. Catecholamines, their methylated metabolites (normetanephrine, metanephrine) and methylated precursor, alpha-methylDOPA, were examined as substrates for both the cationic (PST I) and the anionic (PST II) forms of the enzyme. Sulfation of hypertensive drugs (phenylephrine, octopamine, metaraminol), hypotensive drugs (alpha-methylDOPA, minoxidil), and related agents without a free hydroxy group on the benzene ring were also studied. Results indicated that both PST forms sulfated alpha-methylDOPA and minoxidil, but only PST II transferred the sulfate group to catecholamines and most of the adrenergic agents examined.

Effects of selected vasoactive substances on adenylate cyclase activity in brain, isolated brain microvessels, and primary cultures of brain microvessel endothelial cells.

The specific activity of adenylate cyclase was assayed in homogenates of gray matter, freshly isolated and primary cultured microvessel endothelial cells from bovine cerebral cortex. Specific activities for the tissues were 14.6 +/- 2.1, 15.6 +/- 2.7, and 8.4 +/- 1.5 pmol cAMP/mg protein/min +/- SD for gray matter, cultured microvessels, and freshly isolated microvessels, respectively. Adenylate cyclase associated with gray matter and cultured microvessels was sensitive to histamine and selected catecholamines. Perhaps due to metabolic deficiencies, adenylate cyclase of freshly isolated microvessels exhibited little or no response to either the catecholamines or histamine. Angiotensin II stimulated adenylate cyclase of both freshly isolated and cultured microvessels but had no effect on gray matter. Bradykinin did not stimulate cAMP generation in any of the tissues. Overall results support the role of cAMP in regulating brain microvessel functions and suggest that primary cultures of brain microvessels may be useful in examining cAMP-mediated biochemical pathways at the blood-brain barrier.

Substrate specificity and some properties of phenol sulfotransferase from human intestinal Caco-2 cells.

The phase II metabolic reactions, sulfation and glucuronidation, were studied in a human colon carcinoma cell line (Caco-2), which has been developed as a model of intestinal enterocytes. Phenol sulfotransferase (PST, EC 2.4.2.1) was isolated from Caco-2 cells cultured for 7, 14 and 21 days. The enzyme catalyzed the sulfation of both p-nitrophenol and catecholamines (e.g., dopamine) as well as most catecholamine metabolites. The affinity (Km) of PST for dopamine was much higher than for p-nitrophenol, and the specific activity of PST with both substrates increased with the age of the cells. The thermal stability of Caco-2 PST increased with cell age and was not dependent on the acceptor substrate used. The thermolabile PST from 7-day old cells was more sensitive to NEM than was the thermostable enzyme from 21-day old cells. No UDP-glucuronyltransferase (EC 2.4.1.17) activity was detected in 7-, 14- and 21-day old Caco-2 cells with any of the methods used.

Sulfation in male reproductive organs. Bull and boar testis phenol sulfotransferases.

Phenol sulfotransferases (PST) from bull and boar testis were partially purified and characterized. A single form of PST adsorbed on DEAE-cellulose was found in the bull testis, whereas from boar testis two different peaks of PST activity were separated. The bull testis PST and both boar testis enzymes were active with p-nitrophenol and adrenalin. They all showed higher affinity to pNP than to adrenalin and were inhibited by these substrates at higher concentrations. Their optimal pH was at 8.5. Bull testis PST and boar PST II which were adsorbed on DEAE-cellulose were thermostable, whereas boar PST I was thermolabile. Those three PST forms differed in sensitivity to 2,6-dichloro-4-nitrophenol (DCNP), N-ethyl maleimide (NEM), iodoacetamide (IAA) and phenylglyoxal (PG). Bull and boar PST II were more rapidly inactivated in the presence of DCNP than boar PST I. In the presence of NEM, the--SH groups reagent, the bull phenol sulfotransferase and boar PST I lost their activity, whereas the activity of boar PST increased. Also iodoacetamide, another--SH group modificator, raised boar PST II activity and decreased boar PST I activity. DTT, which protects thiol groups, had an opposite effect on the enzymes studied than NEM. Phenylglyoxal, a reagent specific for arginine residues inhibited bull testis PST and both boar phenol sulfotransferases. Substrate protection experiments were also performed to determine the localization of reactive groups in bull and boar testis phenol sulfotransferases.

Substrate specificity of phenol sulfotransferase from primary cultures of bovine brain microvessel endothelium.

The substrate specificity of the thermostable phenol sulfotransferase (PST) from primary cultures of brain microvessel endothelial cell monolayers was characterized. Selected catecholamines, catecholamine metabolites, and p-nitrophenol at 5, 50, and 500 microM were used as substrates in PST assays of cytosol extracts. Endogenous catecholamines, epinephrine, norepinephrine, and dopamine, exhibited no detectable activity as substrates (500 microM) compared to 500 microM p-nitrophenol as substrate (1.8 pmol/mg/min specific activity) for the PST. In contrast, 500 microM of either deaminated or 3-O-methylated metabolites of catecholamines exhibited intermediate (approximately 1.0 pmol/mg/min specific activity) to low (approximately 0.2 pmol/mg/min specific activity) activity, respectively, as substrates compared to p-nitrophenol as substrate for the PST. Additionally, 500 microM of metabolites of catecholamines that were both deaminated and 3-O-methylated exhibited high activity (greater than 3.0 pmol/mg/min specific activity) as substrates compared to p-nitrophenol as substrate for the PST. Qualitatively similar results were observed at lower substrate concentrations. Therefore, results from this study suggest a potential role for PST as part of the "enzymatic" blood-brain barrier in regulating transendothelial passage of endogeneous catecholamines between the blood and the brain.