Josef Hurych

Effects of various chelating agents, quinones, diazoheterocyclic compounds and other substances on proline hydroxylation and synthesis of collagenous and non-collagenous proteins.

Abstract The effect of metal chelating agents on [ 14 C]proline hydroxylation and [ 14 C]-proline incorporation into collagenous and non-collagenous proteins in chick embryo skin slices varies according to the substance studied. α,α′-Dipyridyl (0.1–1 mM) and sodium diethyldithiocarbamate (1 mM) block proline hydroxylation and lead to the formation of a typical hydroxyproline-deficient collagen containing approximately twice as much proline as normal collagen. 1,10-Phenanthroline in 0.1–0.05 mM concentrations inhibits the hydroxylation of proline and the proline incorporation into collagen by 50%; it has no effect on the specific activity of proline in non-collagenous proteins. EDTA, d -penicillamine and metopiron in 1 mM concentrations do not affect the three above-mentioned reactions. Glycine, cysteine, methionine and 8-hydroxyquinoline as well as quinones (1,4-benzo-, 1,2-naphtho-) and other oxidation-reduction substances (glutathione, cytochrome c ) inhibit all three reactions to the same extent in relation to the dose. An attempt is made to correlate some physicochemical parameters of diazoheterocyclic compounds ( e.g. dipyridyl) with their biological reactivity. A hypothesis is proposed for specific inhibition of proline hydroxylation by some agent assuming the intracellular complexing of Fe 2+ enzymes. The finding of two differing sensitives, namely of collagenous and non-collagenous proline, to the action of 1,10-phenanthroline favours the concept of differing metabolic pathways in the synthesis of both groups of proteins. This finding could be the basis for selective inhibition of collagen synthesis.

Quantum chemical study of the mechanism of collagen proline hydroxylation.

Abstract Quantum chemical Extended Huckel Theory (EHT) model calculations agree with the finding that, in collagen proline hydroxylation, one atom of molecular oxygen is incorporated into peptidyl proline and the other one into α-ketoglutarate. Intermediate steps consist of the activation of molecular oxygen by Fe 2+ bound to the enzyme, the formation of an OOH particle in anionic or radical form and the nucleophilic addition of this particle to the carbonyl group of α-ketoglutarate.

Quantum chemical study of properties and reactivity of quartz dust: I. Electronic structure of α-quartz

Abstract An attempt was made to obtain data for testing a hypothesis [Robock, K. (1967) Beitr. Silikose-Forsch, 92, 1–96] concerning the cytotoxic and fibrogenic actions of silica. According to this hypothesis electron transfer reactions (between cell biopolymers and the surface silica layer) are responsible for the development of the given actions. Electronic structure of α-quartz and of analogous systems derived either by the substitution of Si atoms by Al atoms or by introducing an additional ion (Li 4 , Be 2+ , B 3+ , Na + , Mg 2+ , Al 3+ , Fe 2+ , Fe 3+ , K + , Mn 2+ , F − , Cl − , S 2− ) into the lattice was studied by means of semiempirical quantum chemical methods (EHT, CNDO/2). The passing from α-quartz to its analogs is accompanied by significant changes of reactivity indices.

Laboratory testing of biological activity of ore mine dust: Fibrogenicity, cytotoxicity, and hemolytic activity

Abstract The biological activity of respirable dust (particle size below 5 μm) from three ore mines was compared with its quartz content (14 to 36%) and with the occurrence of lung silicosis in miners. Three months after intratracheal instillation of 50 mg of dust suspensions, silicotic changes of the first and second degree (Kings classification) developed in the lungs of rats. The content of hydroxyproline in the lungs increased by 60 to 110% in comparison to control values. No correlation was found between the quartz content of the dust and the histological changes or hydroxyproline levels. Cytotoxicity of the dust for guinea pig peritoneal macrophages and hemolytic activity in vitro for human erythrocytes did not correlate with its fibrogenicity. No new cases of lung silicosis have developed in the last 20 years in miners exposed in these ore mines to total dust concentrations below 10 mg/m3, although the incidence of silicosis was frequent in the past, when the dust concentrations were much higher.

Changes in pulmonary connective tissue proteins after a single intratracheal instillation of cadmium chloride in the rat

Changes of soluble and insoluble fractions of pulmonary connective tissue proteins were studied in rats for 2-84 days following a single intratracheal instillation of cadmium chloride (10 micrograms Cd2+/lung). A transient decrease in body weight and an immediate increase in lung wet weight (200% of control value, P less than 0.01) were observed. Incorporation of [14C]proline and its conversion to [14C]hydroxyproline in vivo into different soluble and insoluble fractions of connective tissue revealed an increased metabolic turnover elicited by cadmium intoxication. A lag in the maturation of collagen into higher functional forms in the early phase of the process was demonstrated. A striking decrease in elastin was found in first 7 days (40-50%). However, this acute damage of pulmonary connective tissue was followed by a permanent increase of collagen and elastin concentration in the later phase of recovery. Histopathologic examination 14-84 days after cadmium instillation confirmed the presence of lesions in pulmonary tissue with an initial inflammation followed by reparatory changes.

Enzyme changes during experimental silicotic fibrosis: I. PZ peptidase and collagen deposition in the lungs

Abstract In various time intervals from 2 to 22 weeks after intratracheal quartz dust instillation (25 mg DQ12) the content of collagen proteins, deoxyribonucleic acid, soluble proteins, and collagen peptidase activity (PZ substrate) in rat lungs were estimated. In the period from 2 to 8 weeks the total amount of collagen as well as enzyme activity increased in parallel by about 200% in comparison to controls. In later periods (16 and 22 weeks after dust instillation) disproportions occurred. Collagen content in silicotic lungs continued to increase; the total activity of collagen peptidase in lungs started to decrease as did the specific enzyme activity. There was also observed a decrease in the content of lung proteins soluble in isotonic KCl. From 6 weeks onward, disc electrophoresis showed changes in the proportions of these proteins in dusted rats. The changes were particularly manifest in postalbumins, α2- and γ-globulins, indicating more severe disorders in lung metabolism. There were found no differences in the amount and in the electrophoretic characteristics of serum proteins as well as in specific activity of PZ peptidase in serum of control and quartz-dusted rats. Neither control nor silicotic rat sera disclosed inhibitory effects on the activity of lung PZ peptidase. It appears that the increase of collagen proteins in lungs of animals dusted with quartz is also accelerated by the impairment of collagen breakdown.

Molecular-orbital study of hydroxylation of collagenous proline and lysine

EHT and CNDO/2 types of calculations permit the interpretation of the course of hydroxylation of collagenous proline and lysine. Calculations were performed for the models of proline (I), zwitterion of proline (II), proline-containing peptide (III), and lysine (IV). The theoretical results are consistent with an electrophilic mechanism.

Sodium bis(hydroxyethyl)dithiocarbamate reduces acute lung tissue damage induced by cadmium in rats

The protective effect of three dithiocarbamates against lung tissue damage induced by a single intratracheal instillation of cadmium chloride was examined in rats. The relative efficacy of these compounds was tested by comparing characteristic features of lung tissue damage: the increase of lung weight, and the changes in the synthesis and content of structural proteins. Of three compounds administered intraperitoneally at a dose of 2.46 mmol/kg body weight, the most effective in suppressing lung damage was sodium bis(hydroxyethyl)dithiocarbamate (DEDTC). Its efficacy was dependent on the time interval between administration of cadmium chloride and the DEDTC. The parameters of lung tissue damage which were examined approached control values when DEDTC and cadmium chloride were administered simultaneously.