Mitochondrial genome depletion dysregulates bile acid- and paracetamol-induced expression of the transporters Mdr1, Mrp1 and Mrp4 in liver cells.

BACKGROUND AND PURPOSE: Mitochondria are involved in the toxicity of several compounds, retro-control of gene expression and apoptosis activation. The effect of mitochondrial genome (mtDNA) depletion on changes in ABC transporter protein expression in response to bile acids and paracetamol was investigated. EXPERIMENTAL APPROACH: Hepa 1-6 mouse hepatoma cells with 70% decrease in 16S/18S rRNA ratio (Rho cells) were obtained by long-term treatment with ethidium bromide. KEY RESULTS: Spontaneous apoptosis and reactive oxygen species (ROS) generation were decreased in Rho cells. Following glycochenodeoxycholic acid (GCDCA) or paracetamol, Rho cells generated less ROS and were more resistant to cell death. Apoptosis induced by GCDCA and Fas was also reduced. The basal expression of Mdr1 was significantly enhanced, but this was not further stimulated by GCDCA or paracetamol, as observed in wild-type (WT) cells. Basal expression of Mrp1 and Mrp4 was similar in WT and Rho cells, whereas they were up-regulated only in WT cells after GCDCA or paracetamol, along with the transcription factors Shp and Nrf2, but not Fxr or Pxr. Increased expression of Nrf2 was accompanied by its enhanced nuclear translocation. Glycoursodeoxycholic acid failed to cause any of the effects observed for GCDCA or paracetamol. CONCLUSIONS AND IMPLICATIONS: The Nrf2-mediated pathway is partly independent of ROS production. Nuclear translocation of Nrf2 is insufficient to up-regulate Mdr1, Mrp1 and Mrp4, which requires the participation of other regulatory element(s) whose activation in response to GCDCA and paracetamol is impaired in Rho cells and hence probably sensitive to ROS.

Biological variation of interleukin-1beta, interleukin-8 and tumor necrosis factor-alpha in serum of healthy individuals.

Components of biological variation can be used to assess the usefulness of reference values, to evaluate the significance of changes in serial results from an individual and to define objective analytical goals. The aim of the study was to assess, in 15 healthy subjects studied at regular monthly intervals over a period of 6 consecutive months, the biological variation of interleukin-1beta (IL-1beta), interleukin-8 (IL-8), and tumor necrosis factor-alpha (TNF-alpha). Biological variation data (within-subject and between-subject coefficient of variation (CV)) were determined using a simple nested analysis of variance. Derived parameters (index of individuality, reliability coefficient and critical diferences) were calculated from within-subject and between-subject CV. The mean and standard deviation (SD), within-subject CV, between-subject CV, index of individuality and reliability coefficient were as follows: for IL-1beta, 0.67 (0.32) pg/ml, 30%, 36%, 0.85, and 0.76; for IL-8, 3.68 (1.45) pg/ml, 24%, 31%, 0.85 and 0.75; and for TNF-alpha, 3.14 (1.87) pg/ml, 43%, 29%, 1.56 and 0.50, respectively. We conclude that between-subject variation and within-subject variation are quite similar for IL-1beta and IL-8 and are relatively high for the three cytokines studied. Index of individuality is less than 1.4 for IL-1beta and IL-8, and thus reference intervals based on population studies are of limited value. On the contrary, the index of individuality for TNF-alpha is greater than 1.4 and reference values can be used for diagnosis. Quality goals for imprecision are easily achieved for the three cytokines with current methodology.

Biological variation of interleukin 6 (IL-6) and soluble interleukin 2 receptor (sIL2R) in serum of healthy individuals.

The biological variation of interleukin 6 (IL-6) and soluble interleukin 2 receptor (sIL2R), measured by automated enzyme immunoassay, in fifteen subjects studied at regular monthly intervals over a period of 6 consecutive months was measured. The mean and standard deviation (SD), within-subject CV, between-subject CV, individuality index (II) and reliability coefficient (R) were as follow: for sIL2R 571 (231) U/ml, 5.84%, 38.81%, 0.21 and 0.93; and for IL-6 1.43 (0.9) pg/ml, 48.48%, 39.38%, 1.44, and 0.37. The data indicate a relatively high between-subject CV, quite similar in both cases, and a within-subject CV much higher for IL-6 than for sIL2R. Thus, reference values can be used for diagnosis for IL6 (high II), while not for sIL2R (low II). However, the low R for IL-6 implies that more than one measurement are needed. sIL2R has a very high R and a relatively small critical differences, a circumstance appropriate for follow-up.

Hydrolytic enzyme of the alveolar macrophage in diffuse pulmonary interstitial disease.

Hydrolytic enzymes [acid phosphatase, beta-glucuronidase, beta-D-N-acetyl glucosaminidase (beta-D-NAGA), lysozyme and angiotensin-converting enzyme (ACE)] are the major constituents of alveolar macrophages (AM). These enzymes play a crucial role in the pathogenesis of interstitial lung diseases. Cell-associated activity of several enzymes in alveolar macrophages obtained from control subjects (n = 5) and patients suffering five representative types of interstitial pulmonary diseases [sarcoidosis (n = 10), extrinsic allergic alveolitis (n = 5), idiopathic pulmonary fibrosis (n = 5), neoplastic infiltration of the lung (n = 5) and Pneumocystis carinii pneumonia (n = 5)] were evaluated. Cells were obtained by bronchoalveolar lavage and isolated by Ficoll-Hypaque gradient. Enzymatic activity was assessed by standardized tests. Bronchoalveolar lavage (BAL) lymphocyte counts were significantly elevated in the patients with active sarcoidosis (median: 57%), allergic extrinsic alveolitis (median: 51%) and neoplastic infiltration (median: 31%) as compared with the other groups, whereas BAL neutrophil and eosinophil counts were significantly elevated in the patients with idiopathic pulmonary fibrosis (neutrophil median: 29%; eosinophil median: 3%). The highest alveolar macrophage enzymatic activities were obtained in the active sarcoidosis group (median ACE: 23.38 microKat 10(-6) AM; median lysozyme: 8.64 nKat 10(-6) AM; median beta-glucuronidase: 324.22 U 10(-6) AM; median acid phosphatase: 0.78 nKat 10(-6) AM; median beta-D-NAGA: 1.85 nKat 10(-6) AM) which was significantly greater than in the control group (median ACE: 6.69 microKat 10(-6) AM; median lysozyme: 1.95 nKat 10(-6) AM; median beta-glucuronidase: 39.88 U 10(-6) AM; median acid phosphatase: 0.38 nKat 10(-6) AM; median beta-D-NAGA: 0.44 nKat 10(-6) AM). However, intracellular lysosomal enzymatic activities of alveolar macrophages from patients with allergic extrinsic alveolitis, a disease in which the degree of alveolar macrophage activation is maximal, were similar to those of the control group. These findings demonstrated a different pattern of expression of alveolar macrophage's hydrolytic enzymes in lymphocytic diffuse pulmonary interstitial disease. In sarcoidotic patients, hydrolytic enzymes were increased whereas in allergic extrinsic alveolitis, hydrolytic enzyme activities were similar to control groups. Indirect data suggest that the release of lysosomal enzymes by alveolar macrophages during allergic extrinsic alveolitis may be a factor involved in the pulmonary lesions appearing in this disease.

Reversal of cyclosporine A-induced alterations in biliary secretion by S-adenosyl-L-methionine in rats.

This study examines the ability of S-adenosyl-L-methionine to prevent or antagonize the cyclosporine A-induced adverse effects on biliary secretion in the rat. S-adenosyl-L-methionine was administered as a single bolus 3, 5 and 8 hr before administering a single dose of cyclosporine A, and also concurrently administered with cyclosporine A for 1 or 2 wk. Acute S-adenosyl-L-methionine preadministration attenuated the cyclosporine-induced cholestasis and inhibition of bile acids, cholesterol and phospholipid biliary secretion. S-adenosyl-L-methionine pretreatment for 1 wk and simultaneous cotreatment with cyclosporine for 1 or 2 wk not only maintained the beneficial effects reported above but further improved them because the adverse effects of the immunosuppressor drug were prevented or antagonized by S-adenosyl-L-methionine. These results provide the first direct evidence of the ability of exogenously administered S-adenosyl-L-methionine to antagonize cyclosporine-induced abnormalities in biliary bile acids, lipids and protein secretion. The beneficial effects of S-adenosyl-L-methionine could be related, at least in part, to the improvement in the hepatobiliary transport of bile acids.

Carbohydrate antigen 15-3 and mucinous carbohydrate antigen immunoreactivities in human seminal plasma.

Carbohydrate antigen 15-3 (CA 15-3) and mucinous carbohydrate antigen (MCA) immunoreactivities were detected in human seminal plasma. Mean values for CA 15-3 (8.2 +/- 3.7 U/ml, range 2.6 - 18.4 U/ml) and MCA (13.8 +/- 8.2 U/ml, range 2.1-31.9 U/ml) in the seminal plasma were of the same magnitude as that found in serum. No correlation was obtained between seminal plasma, either CA 15-3 or MCA immunoreactivities, and volume of seminal plasma, sperm count or percent of motile spermatozoa. Seminal plasma CA 15-3 and MCA levels were significantly (p less than 0.001) correlated (r = 0.55). The nature and origin of CA 15-3 and MCA immunoreactivities in human plasma are unknown.

Hormonal regulation of potassium shifts during graded exhausting exercise.

Serum potassium, aldosterone and insulin, and plasma adrenaline, noradrenaline and cyclic adenosine 3':5'-monophosphate (cAMP) concentrations were measured during graded exhausting exercise and during the following 30 min recovery period in six untrained young men. During exercise there was an increase in concentration of serum potassium (4.74 mmol.l-1, SEM 0.12 at the end of exercise vs 3.80 mmol.l-1, SEM 0.05 basal, P less than 0.001), plasma adrenaline (2.14 nmol.l-1, SEM 0.05 at the end of exercise vs 0.30 nmol.l-1, SEM 0.02 basal, P less than 0.001), plasma noradrenaline (1.10 nmol.l-1, SEM 0.64 at the end of exercise vs 1.50 nmol.l-1, SEM 0.05 basal, P less than 0.001), serum aldosterone (0.92 nmol.l-1, SEM 0.14 at the end of exercise vs 0.36 nmol.l-1, SEM 0.05 basal, P less than 0.01), and plasma cAMP (35.4 nmol.l-1, SEM 2.3 at the end of exercise vs 21.4 nmol.l-1, SEM 4.5 basal, P less than 0.05). While concentrations of serum potassium, plasma adrenaline and cAMP returned to their basal levels immediately after exercise, those of plasma noradrenaline and serum aldosterone remained elevated 30 min later (1.90 nmol.l-1, SEM 0.01, P less than 0.01; and 0.85 nmol.l-1, SEM 0.12, P less than 0.01, respectively). Serum insulin concentration did not change during exercise (6.47 mlU.l-1, SEM 0.58 at the end of exercise vs 5.47 mlU.l-1, SEM 0.41 basal, NS) but increased significantly (P less than 0.02) at the end of the recovery period (7.12 mlU.l-1, SEM 0.65).(ABSTRACT TRUNCATED AT 250 WORDS)

Inter-relationship between serum potassium and plasma catecholamines and 3':5' cyclic monophosphate in alcohol withdrawal.

Serial analyses of serum potassium and plasma epinephrine, norepinephrine and adenosine 3':5'-cyclic monophosphate (cyclic AMP) concentrations were measured in 13 patients with alcohol withdrawal, six of whom presented delirium tremens. Patients with delirium showed at admission levels of potassium (3.45 +/- 0.45 mmol/l) lower (P less than 0.02) than patients without delirium (3.81 +/- 0.14 mmol/l). Three patients were hypokalemic, all of them with delirium. Serum potassium increased significantly in all the patients during evolution. A close negative correlation (r = -0.751) between the intensity of withdrawal and serum potassium was observed. Plasma epinephrine concentrations were increased at admission (623 +/- 192 pmol/l), patients with delirium showing greater values (705 +/- 137 pmol/l). As the alcohol withdrawal improved, plasma epinephrine concentration decreased. Plasma norepinephrine concentrations were also increased at admission (3422 +/- 1451 pmol/l), but did not change significantly during evolution, being similar in patients with and without delirium. Plasma cyclic AMP levels were high at admission (40.4 +/- 24.3 nmol/l) and increased significantly (P less than 0.05) during evolution. The data obtained suggest that in patients with alcohol withdrawal, as symptomatology improves, plasma epinephrine decreases, while plasma norepinephrine remains increased. The combined actions of the two facts--less beta-stimulus, maintaining of alpha-stimulus--would comprise a significant increase of kalemia, that in cases of initial hypokalemia would lead to normal values of serum potassium.

Role of vasoactive substances in the segmentary vasomotor response following spinal cord stimulation. An experimental study.

It is presumed today that spinal cord stimulation induces local delivery of vasoactive substances, such as prostacyclins, histamine, substance P, and vasoactive neuropeptides, in the perivascular environment and the vascular wall to mediate the segmental vasodilator response. To investigate this mechanism, 9 dogs were subjected to low thoracic spinal cord stimulation. Venous and arterial blood samples from the paraesthesic area in the lower limbs were obtained before and 120 min after stimulation to measure changes in the plasma concentration of vasoactive intestinal peptide, substance P, and histamine. The results were compared with those obtained from vessels of the upper limbs. Blood flow changes following stimulation were recorded by electromagnetic flowmeters. Local arterial vasoactive intestinal peptide showed a mean increase of 33% after 60 min of stimulation. Changes concerning substance P were inconclusive. Local arterial and venous histamine concentrations increased 26 and 29%, respectively, after 60 min of stimulation.

Carpal tunnel syndrome and vitamin B6.

Twelve patients with carpal tunnel syndrome were studied. Clinical and electrophysiological data were obtained and an estimation of vitamin B6 (pyridoxine) status by an assay of erythrocyte aspartate aminotransferase and coenzyme stimulation assay were done. None of the patients was found to have vitamin B6 deficiency. Patients were treated with 150 mg of pyridoxine daily for 3 months. Erythrocyte aspartate aminotransferase increased significantly (p less than 0.001) in all the patients. In 6 patients there were clinical and electrophysiological improvement and erythrocyte aspartate aminotransferase increased more than in the other 6 patients. The data obtained appear to indicate that although vitamin B6 deficiency is not common in carpal tunnel syndrome patients, pyridoxine supplementation can be recommended as adjuvant treatment in those patients undergoing surgery.

Cytoplasmic enzyme activities in human hypernephroma compared with normal renal cortical tissue.

Cytoplasmic lactate dehydrogenase (LDH), alpha-hydroxybutyrate dehydrogenase (HBDH), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) enzymatic activities were measured in normal renal cortical tissue and in hypernephroma. Significantly lower activities were always found in tumoral tissue than in normal renal tissue. Their respective values (mean +/- SD) were: LDH, 4,333 +/- 747 (normal tissue) vs. 997 +/- 748 U/l (tumor); HBDH, 2,554 +/- 466 vs. 387 +/- 290 U/l; AST, 529 +/- 109 vs. 65 +/- 37 U/l, and ALT, 205 +/- 45 vs. 9.9 +/- 5.4 U/l. The LDH/HBDH ratio was significantly greater in tumoral (2.69 +/- 0.69) than in normal tissue (1.70 +/- 0.11). These results indicate that hypernephroma exhibits a low metabolic rate when compared to normal tissue. Their enzymatic activities suggest a decreased energy metabolism, predominantly of the anaerobic type, and a reduced synthesis of nonessential amino acids in the tumor. These findings could explain in part the slow growth rate of hypernephroma.

Cytoplasmic creatine kinase in normal and pathological human kidney tissue.

Cytoplasmic creatine kinase (CK) activity was measured in 39 renal cortex samples from various kidney diseases. The highest CK values were observed in normal tissues (507 +/- 71). The mean values of CK enzymatic activity in the other groups decreased in the following order: chronic pyelonephritis (273 +/- 98), hydronephrosis (233 +/- 102), renal tuberculosis (133 +/- 34), pyonephrosis (96 +/- 46), and hypernephroma (45 +/- 33). The decrease in CK activity in kidney tissue paralleled tissue damage, and affects cellular functionality in relation with the processes that use adenosine 5'-triphosphate, such as the ionic pumps.

Hormone levels in serum and seminal plasma of men with different types of azoospermia.

Hormone concentrations in the serum and seminal plasma of 15 normozoospermic, 17 excretory azoospermic and 14 secretory azoospermic men were measured. The results indicate that: (a) serum FSH and LH levels are markedly elevated in secretory azoospermia, as compared with excretory azoospermia and normozoospermia; (b) serum 17 alpha-hydroxyprogesterone levels are somewhat raised in secretory azoospermia as compared with excretory azoospermia and normozoospermia; (c) serum testosterone levels are lower in both types of azoospermia with respect to normozoospermia; (d) in secretory azoospermia the oestradiol serum levels are relatively high and dihydrotestosterone serum levels relatively low, whereas the serum levels of these hormones in excretory azoospermia are similar to those in normozoospermic men; (e) in the seminal plasma of azoospermic patients the levels of prolactin, progesterone, testosterone, dihydrotestosterone and oestradiol were depressed, but only dihydrotestosterone levels could be of value in differentiating types of azoospermia because they are lower in secretory azoospermia. We suggest that the measurement of FSH, LH, 17 alpha-hydroxyprogesterone, dihydrotestosterone and oestradiol in serum and dihydrotestosterone in seminal plasma may be used in the differential diagnosis between secretory and excretory azoospermia when invasive tests are unavailable.