Exogenous Angiotensin I Metabolism in Aorta Isolated from Streptozotocin Treated Diabetic Rats.

Purpose. Products of angiotensin (ANG) I metabolism may predispose to vascular complications of diabetes mellitus. Methods. Diabetes was induced with streptozotocin (75 mg/kg i.p.). Rat aorta fragments, isolated 4 weeks later, were pretreated with perindoprilat (3 µM), thiorphan (3 µM), or vehicle and incubated for 15 minutes with ANG I (1 µM). Products of ANG I metabolism through classical (ANG II, ANG III, and ANG IV) and alternative (ANG (1-9), ANG (1-7), and ANG (1-5)) pathways were measured in the buffer, using liquid chromatography-mass spectrometry. Results. Incubation with ANG I resulted in higher concentration of ANG II (P = 0.02, vehicle pretreatment) and lower of ANG (1-9) (P = 0.048, perindoprilat pretreatment) in diabetes. Preference for the classical pathway is suggested by higher ANG III/ANG (1-7) ratios in vehicle (P = 0.03), perindoprilat (P = 0.02), and thiorphan pretreated (P = 0.02) diabetic rat. Within the classical pathway, ratios of ANG IV/ANG II (P = 0.01) and of ANG IV/ANG III (P = 0.049), but not of ANG III/ANG II are lower in diabetes. Conclusions. Diabetes in rats led to preference toward deleterious (ANG II, ANG III) over protective (ANG IV, ANG (1-9), and ANG (1-7)) ANG I metabolites.

Perindoprilat changes ANG (1-9) production in renal arteries isolated from young spontaneously hypertensive rats after ANG I incubation.

We used mass spectrometry to quantitate production of angiotensinogen metabolites in renal artery of 3- and 7-month-old Wistar-Kyoto (WKY) and Spontaneously Hypertensive Rats (SHR). Tissue fragments were incubated for 15 min in oxygenated buffer, with added angiotensin I. Concentrations of angiotensins I (ANG I), II (ANG II), III (ANG III), IV (ANG IV), angiotensin (1-9) [ANG (1-9)], angiotensin (1-7) [ANG (1-7)], and angiotensin (1-5) [ANG (1-5)], excreted into the buffer during experiment, were measured using liquid chromatography-mass spectrometry (LC/MS) and expressed per mg of dry tissue. Effects of pretreatment with 10 microM perindoprilat on the production of ANG I metabolites were quantitated. Background production of any of ANG I metabolites differed neither between WKY and SHR rats nor between 3- and 7-month-old rats. Perindoprilat pretreatment of renal arteries resulted, as expected, in decrease of ANG II production. However, renal arteries of 7-month-old SHR rats were resistant to ACE inhibitor and did not change ANG II production in response to perindoprilat. In renal arteries, taken from 3-month-old rats, pretreated with perindoprilat, incubation with ANG I, resulted in the level of ANG (1-9) significantly higher in SHR than WKY rats. Our conclusion is that in SHR rats, sensitivity of renal artery ACE to perindoprilat inhibition changes with age.

Measurement of angiotensin metabolites in organ bath and cell culture experiments by liquid chromatography - electrospray ionization - mass spectrometry (LC-ESI-MS).

The metabolism of renin-angiotensin system (RAS) is more complicated than previously expected and understanding the biological phenomena regulated by variety of angiotensin metabolites requires their precise and possibly comprehensive quantitation. Physiological concentrations of angiotensins (Ang) in biological fluids are low, therefore their accurate measurements require very sensitive and specific analytical methods. In this study we developed an accurate and reproducible method of quantitation of angiotensin metabolites through coupling of liquid chromatography and electrospray ionization - mass spectrometry (LC-ESI-MS). With this method main angiotensin metabolites (Ang I, II, III, IV, 1-9, 1-7, 1-5) can be reliably measured in organ bath of rat tissues (aorta, renal artery, periaortal adipose tissue) and in medium of cultured endothelial cells (EA.hy926), exposed to Ang I for 15 minutes, in the absence or in the presence of angiotensin converting enzyme inhibitor, perindoprilat. Presented LC-ESI-MS method proved to be a quick and reliable solution to comprehensive analysis of angiotensin metabolism in biological samples.

Immunological findings in cigarette smokers.

The aim of our study was to assess the effects of cigarette smoking on selected indices of immunity. The study comprised 116 men divided into three groups: 37 subjects smoking for not more than 10 yr, 39 subjects smoking for more than 10 yr, and control group consisting of 40 age-matched men who never used to smoke. The following parameters were studied: total number of lymphocytes, B-cells, T-cells subpopulations: (CD3+)T-, (CD4+)T-helper, (CD8+)T-cytotoxic and (CD16+)natural killer (NK)-cells and serum concentration of immunoglobulins A, D, G and M, C3c and C4 complement components, acute phase proteins: alpha(1)-acid glycoprotein, haptoglobin, ceruloplasmin and lysozyme. The (CD4+)/(CD8+) ratio was also calculated. The suppressive effect of tobacco smoke on human immunity was seen as decreased serum concentration of immunoglobulins and lysozyme, especially in men smoking for more than 10 yr, decreased (CD16+)NK-cells absolute number and elevated population of (CD8+)T-cytotoxic lymphocytes entailing a decrease in CD4+/CD8+ ratio.

The activity of erythrocyte enzymes and basic indices of peripheral blood erythrocytes from workers chronically exposed to mercury vapours.

The influence of occupational exposure to mercury vapours on the activity of the red cell enzymes [glucose-6-phosphate dehydrogenase (G-6PD), acetylcholinesterase (AChE), glutathione reductase (GR) and superoxide dismutase (SOD)], as well as on peripheral blood indices [erythrocyte number (RBC), HCT, Hb, MCHC] and on serum concentrations of iron, ferritin, transferrin and total iron binding capacity (TIBC), was assessed. Studies were carried out on 46 men aged between 21 and 56 years (X = 39 +/- 10.4) exposed to mercury vapours during their work from 7 months to 32 years (= 14.7 +/- 10.8). The control group consisted of 35 healthy workers aged between 20 and 54 years (X = 33.6 +/- 9.8) not exposed to chemical nor physical agents. In both groups studied, there were 50% and 34.3% smokers, respectively. The activity of studied red cell enzymes--G-6PD, AChE, GR and SOD--was estimated according to the colorimetric methods described by Beutler and expressed as international units per gram of hemoglobin (IU g Hb(-1)). Peripheral blood cell parameters were determined using an automatic cell counter. The concentration of serum iron and TIBC was determined using colorimetric methods (Beckman), while that of ferritin and transferrin by nephelometric methods. The time-weighted average (TWA) of mercury concentration in the air determined before the study was 0.0028 mg m(-3). Statistical analysis of the data was performed using either the Cochran and Cox C-test or the Student's t-test. The medium mercury concentration in the urine was 77.44 +/- 48.15 microg l(-1). In the group exposed to mercury vapours, a significant decrease was found in G-6PD activity (23.9%, P<0.001), GR (18.8%, P<0.001), and SOD (5%, P<0.001) with a concomitant increase in AChE activity (35.9%, P<0.001) was found. Moreover, a statistically significant increase occurred in HCT and RBC, and a decrease in MCV and MCHC as well as increases of ferritin (130.9%, P<0.001), transferrin (118.4%, P<0.001) and TIBC (11.2%, P<0.05). Our results indicate that long-term exposure to mercury vapours induces changes in the activity of red cell enzymes--G-6PD, AChE, GR and SOD--and may also influence other important hematological parameters of the peripheral blood.

[The effects of occupational exposure to nitrogen dioxide (NO2) on the immunological parameters in workers]. / Wplyw zawodowego narazenia na dwutlenek azotu (NO2) oraz tlenek azotu (NO) na parametry ukladu odpornosciowego pracowników.

The study of T-cell subpopulation: T CD3+, T CD4+, T CD8+; NK CD16+ and B CD19+ cells as well as of serum concentrations of immunoglobulins G, A, M, E, complement components C3c and C4, total circulating immune complexes (CIC), CRP, haptoglobin, ceruloplasmin and transferin, was carried out in 16 males, smokers and non-smokers. The control group of smokers and non-smokers comprised 36 males not exposed to any chemical compound. The study was performed also in two groups of non-smokers: 5 workers occupationally exposed and 7 non-exposed controls. For the determination of T-cells, NK- and B CD19+ cells populations monoclonal antibodies were used in indirect immunofluorescence tests. Passive sampling spectrophotometric method was employed to determine NO2 and NO concentrations in the work environment air. Their mean values were 0.0867 +/- 0,0585 mg x m-3 (from 0.0165 to 0.1960) and 0.0614 +/- 0.0263 mg x m-3 (from 0.0220 to 0.1090), respectively. The number of total leucocytes and lymphocytes in the group of 16 smokers and non-smokers exposed to NO2 and NO was increased. Stimulation T-cells line in the men exposed to NO2 and NO was evidenced by an increased number of T CD3+ cells, about two-fold increase in absolute number of T CD4+ cells (p < 0.001), an increased number of T CD8+ cells (p < 0.001) and by an enhanced value (by 24.7%) of the T CD4+/T CD8+ ratio. In the group of 16 persons exposed to NO2 and NO serum IgG concentration was elevated (p < 0.01), C3c C4 decreased (p < 0.001), and serum CIC enhanced by about two times (p < 0.001). Stimulation T-cells line in the groups of 5 non-smoking workers exposed to NO2 and NO was evidenced by an increased number of T CD3+ (p < 0.05), T CD4+ (p < 0.05) and T CD8+ cells but without any change in the value of the T CD4+/T CD8+ ratio. In addition, decreased serum C3c and C4 levels together with serum CIC elevated by 76.7% (p < 0.05) were evidenced in this group on non-smokers. Moreover, significant positive correlations between NO2 concentrations in the air and the numbers of total lymphocytes, T CD3+, TCD4+, T CD8+ cells or IgG: (magnitude of r in the range between 0.31 and 0.71), as well as significant negative correlations between NO2 concentrations in the air and C3c (r = 0.44) in the group of 16 smoking and non-smoking workers were calculated. In this group significant positive correlation between NO concentrations in the air of the workplace and counts of T CD3+, T CD8+, B CD19+ (magnitude of r in the range between 0.51 and 0.63), haptoglobin and ceruloplasmin (magnitude of r from 0.37 to 0.58), as well as significant negative correlation between NO concentrations in the air and serum levels of IgG, IgA and IgM (magnitude of r from -0.67 to -0.47) and CIC (magnitude of r = -0.39) were also observed. In the group of 5 exposed non-smoking workers, the values of correlation coefficients magnitude of r between NO2 and NO concentrations in the work environment air and immunological parameters were similar to those in the whole group. The results obtained suggest that during occupational exposure NO2 may play a more important role mainly in the process of inflammation but exogenous NO seems to act as modulating factor of this proinflammatory NO2 effect through a greater and exposure-dependent influence mainly on B CD19+ cells and other parameters of humoral immunity.

[Usefulness of determining urinary markers of early renal damage for monitoring nephrotoxicity during occupational exposure to mercury vapors]. / Przydatnosc oznaczania w moczu markerów wczesnego uszkodzenia nerek do monitorowania nefrotoksycznosci par rteci w trakcie zawodowego narazenia.

Using nephelometry, concentration of albumin, IgG, transferrin, retinol binding protein (RBP) alpha 1-microglobulin were determined in urine of 83 males with history of occupational exposure to metallic mercury vapours from 0.6 to 37 years, and in 30 non-exposed males. The weighted average of mercury air concentrations was 0.028 mg/m3. Duration of occupational exposure to mercury vapours did not elevate urine excretion of proteins. The urine concentration of proteins in question were higher (especially beta 2-m) in workers with urine mercury concentration between 51 and 150 microliter-1 and highest in workers with urine mercury concentrations above 150 micrograms l-1 and the differences were significant. In addition, a positive correlations between urine mercury concentrations and alpha 1-m (r = 0.33) as well as between urine mercury concentration and albumin (r = 0.31) were observed. In conclusion, the determination of proteins in urine as markers of early renal damage may be useful for monitoring occupational exposure to mercury vapours, especially in the group of workers with elevated values of urine mercury concentrations.

Immunological effects of occupational exposure to metallic mercury in the population of T-cells and NK-cells.

This paper presents a study of the counts of lymphocytes, (CD3+)T-cells, (CD4+)T-helper and (CD8+)T-suppressor and (CD16+)NK-cells in the peripheral blood of 101 males with a history of occupational exposure to metallic mercury vapours (Hg0) and in 36 males without this exposure. These workers were divided depending on the duration of exposure: 37 males with a short-term history of exposure to Hg0 (up to 10 years) and 64 males with a history of long-term exposure (10 to 37 years). For the determination of T-cell populations monoclonal antibodies were used in indirect immunofluorescence tests. The time weighted average of mercury concentrations in air was 0.028 mg m-3. Mercury concentration in the urine of the exposed subjects ranged from 20-260 micrograms dm-3, and in blood it was from 4 to 72 micrograms dm-3. Stimulation of the T-cell line was noted as evidenced by increased numbers of (CD3+)T-cells, (CD4+)T-helper and (CD8+)T-suppressor cells in the workers with < 10 or > 10 years' exposure to Hg0. Lower increase count of (CD3+)T-cells and (CD4+)T-helper cells than (CD8+)T-suppressor cells was the cause of decreased values in the (CD3+)T/(CD8+)T-suppressor ratio and (CD4+)T-helper/(CD8+)T-suppressor ratio in the workers with < 10 or > 10 years' of exposure. Moreover, no changes were observed in the T-cell populations between workers with < 10 and those with > 10 years' exposure. In addition, statistical analysis of the effects of age and duration of exposure to Hg0 on the studied immunological parameters indicates that exposure duration may affect some of the values. These quantitative changes of T-cell population as well as changes of the (CD3+)T/(CD8+)T-suppressor and (CD4+)T-helper/(CD8+)T-suppressor ratio have been proposed as immunological indicators of exposure to Hg0, which can be used for monitoring and to explain the origin of autoimmunity disorders induced by metallic mercury.

The effect of cigarettes smoking on the blood counts of T and NK cells in subjects with occupational exposure to organic solvents.

The study was carried out in a population of 139 men, divided into two control groups: 40 non-smokers and 39 smokers not exposed to chemical compounds, and two groups exposed to them: 19 non-smokers and 41 cigarette smokers with occupational contact with organic solvents. The results of toxicological analyses of air and chromatographic analyses of solvents demonstrated the presence of benzene, toluene, xylene and their partly hydrogenated derivatives, paraffin hydrocarbons, oleins, naphthenes (components of painter's naphtha), monohydric and polyhydric alcohols (butanol, cyclohexanol, butylene glycol) esters (ethyleneglycol acetate, butyl acetate) and ketones (methylisobutyl ketone, cyclohexanone). In the time of the studies the TWA values for benzene were 0 to 38 mg x m-3 (0 to 12 ppm), with arithmetic mean averages of about 19 mg x m-3 (6 ppm) and for the level of benzene 0-351 mg x m-3 (0-110 ppm) with arithmetic mean annual averages of about 48 mg x m-3 (15 ppm). Mean phenol concentration in the urine of the workers in groups I, II, III and IV respectively was: 7.9 +/- 3.5; 10.0 +/- 5.8; 16.8 +/- 6.2 and 18.4 +/- 9.7 mg x l-1. Hippuric acid concentration in the urine of the workers in groups I to IV was: 496 +/- 326, 538 +/- 341, 982 +/- 420 and 1107 +/- 507 mg x l-1 respectively. The absolute counts were determined of T-cells (CD 3+), T-helper (CD 4+), T-suppressor (CD 8+) cells and natural killer (NK) cells (CD 16+) in the peripheral blood by indirect immunofluorescence. In the subjects with occupational exposure to organic solvents the counts of T-cells and NK-cells were reduced, and the number of T-suppressor cells was raised which resulted in a decrease of the T-helper/T-suppressor ratio. These changes were more pronounced in cigarette smokers. The assessment of the immunotoxic effect of organic solvents during occupational exposure should take into consideration the possibility of a synergistic action with tobacco and may be of practical use for monitoring the toxic effect of organic solvents on the lymphocyte system.

Effects of occupational exposure to mercury vapors on T-cell and NK-cell populations.

The counts of lymphocytes, (CD3+) T-cells, (CD4+) T-helper and (CD 8+) T-suppressor and (CD 16+) NK-cells were determined in the peripheral blood of 81 males with a history of occupational exposure to metallic mercury vapors and in 36 males without this exposure. For the determination of T-cell populations monoclonal antibodies were used in indirect immunofluorescence tests. The weighted mean of mercury concentrations in air was 0.028 mg x m-3. Mercury concentration in the urine of the exposed subjects ranged from 10-240 micrograms x l-1, and in blood it was from 4-30 micrograms x l-1. Stimulation of the T-cell line was noted as evidenced by increased number of T-cells by 35% in the workers with exposure to mercury vapors below or by 38% in the workers over 10-years, by increased number of T-helper cells by 42% (p < 0.001) in the workers with exposure below or by 60% (p < 0.001) in the workers over 10 years and by increased number of T-suppressor cells by 85% (p < 0.001) in the workers below or by 96% (p < 0.001) in the workers over 10 years exposure. Lower increase of T-helper cells population than T-suppressor cells population was the cause of decreased value of the T-helper/T-suppressor ratio by about 21% (p < 0.01) in the workers with exposure below and over 10 years. No changes were observed in the T-cell populations between workers with up to 10 and those with over 10 years exposure. The quantitative changes of T-helper cells and T-helper/T-suppressor ratio may represent an immunological indicator of exposure to mercury vapors. Presented changes in human T-lymphocytes population associated with occupational exposure to mercury vapors have been proposed to explain the origin of more frequent autoimmunity induced by mercury.

[Usefulness of immunologic and protein determinations for assessment of exposure to mercury]. / Przydatnosc badan immunologicznych i proteinologicznych do oceny skutków narazenia na rtec.

The count of lymphocytes, lymphocytes T (CD 3+), lymphocytes T-helper (CD 4+), T-suppressor (CD 8+) and NK-cells (CD 16+) as well as immunoglobulins A, D, G, M, complements C3c and C4, alpha 1-acid glycoprotein, haptoglobin, ceruloplasmin and transferrin were detected in males exposed to mercury vapours and in males of the control groups. The shift time weight average (TWA) of mercury in ambient air was 0,028 mg x m-3. Mean mercury concentration was from 0 to 240 micrograms x 1(-1) in urine of the study group and from 0-30 micrograms x 1(-1) in blood. Stimulation of lymphocytes system, manifested by an increase number of lymphocytes T, T-helper and T-suppressor in the peripheral blood was observed. Higher increase of T-suppressor cells than T-helper populations was the case of decreased value of the T-helper/T-suppressor ratio by 20.5% (p < 0.05) in workers exposed below 10 or by 21% (p < 0.05) in workers exposed over 10 years. Decrease of immunoglobulin A, G and M concentration in serum of workers exposed to mercury vapours over 20 years was also observed. The concentrations of IgG, C3c, C4 and determined acute phase proteins in serum of exposed subjects did not differ from that in the non-exposed subjects.

[Effect of cigarette smoking on T and NK cell count in blood of persons occupationally exposed to organic solvents]. / Wplyw palenia papierosów na liczbe komórek t i nk w krwi osób zawodowo narazonych na rozpuszczalniki organiczne.

The studies covered 139 men including two control groups: 40 nonsmokers and 41 smokers occupationally exposed to organic solvents. The results of toxicological analysiss of the air revealed the presence of benzene, toluene, xylene and their hydrogenated derivatives, paraffin hydrocarbons, oleins, naphthenes, alcohols (butanol, cyclohexanol), esters (ethylglycol acetate, butyl acetate) and ketones methyl isobutyl ketone, cyclohexanon). Absolute number of lymphocytes, T lymphocytes (CD 3+), T-helper (CD 4+), T-suppressor (CD 8+) and NK-cells (CD +16) were determined in the peripheral blood by indirect immunofluorescence test. A decreased number of lymphocytes and NK-cells as well as an increased number of T-suppressor lymphocytes were noted. That contributed to the decrease of T-helper/T-suppressor ratio in the subjects occupationally exposed to organic solvents. Cigarette smoking enhanced this disorder.

[Occupational exposure to mercury vapors stimulates T lymphocytes]. / Zawodowa ekspozycja na pary rteci stymuluje limfocyty T.

The number of lymphocytes, lymphocytes T, lymphocytes T helper and suppressor were determined in 55 males exposed to mercury vapours and in 36 males of the control group, using monoclonal antibody and indirect immunofluorescence assay. The shift time weighted average was 0.0028 mg x m-3. Mean mercury concentration was 54 +/- 44 micrograms x l-1 in urine of the study group and 5 +/- 7 micrograms x l-1 in blood. Stimulation of lymphocytes, manifested by an increased number of lymphocytes T helper in the peripheral blood, was observed in men exposed to mercury. The results suggest that autoimmunological reactions may occur in the exposed men.

Effect of prostaglandin E2 and 3-morpholinosydnonimine (SIN-1) on arachidonic acid metabolism in fMLP-stimulated rat neutrophils and on thrombin-induced human platelet aggregation.

The effects of prostaglandin (PG) E2 and the nitric oxide (NO) donor SIN-1 on leukotriene (LT) release from formyl-methionyl-leucyl-phenylalanine (fMLP) (100 nM)-stimulated rat peritoneal neutrophils (RPN) and on thrombin-induced aggregation of washed human platelets were investigated. Both PGE2 (1-100 nM) and SIN-1 (30-300 microM) inhibited release of LTB4 and cysteinyl-LT from RPN in a concentration-dependent manner. The combined effects of PGE2 and SIN-1 were not greater than expected by summation. On the other hand, the inhibitory effect of SIN-1 (0.5 or 1.0 microM) on platelet aggregation was potentiated by PGE2 (0.3-5 microM) in a concentration-dependent manner, while PGE2 alone in the concentrations used had only marginal effects. The results suggest differential regulation of platelet and leukocyte functions by the mediators PGE2 and NO, which could be relevant for various physiological and pathophysiological conditions.