Injury of rat thymocytes caused by exogenous peroxyl radicals in vitro.

The aim of this study was to investigate oxidative cell injury in rat thymocytes under conditions of radical generation exterior to the cell utilizing the thermolabile azocompound 2,2'-azobis(2-amidinopropane) dihydrochloride to generate peroxyl radicals at a constant and reproducible rate. This initiator, being water-soluble and endowed with a positive charge, is suitable for studies on oxidative damage of biomembranes induced in the external water environment. The relationship between cell viability, lipid and thiol oxidation and chain-breaking antioxidant depletion was studied. During the first hour of treatment cell viability decreased slightly, protein sulfhydryl groups were consumed slowly and no significant production of conjugated dienes occurred. After 90 min of incubation, when thymocyte permeability started to increase, the concentration of alpha-tocopherol decreased gradually, significant changes of polyunsaturated fatty acids occurred and a rapid phase of thio oxidation commenced. It can be concluded that, under conditions of an exogenous oxidant challenge, initially the cell membrane provides a physical barrier to the entrance of radicals to the thymocyte. When peroxyl radicals gain access to the membrane and the molecular barrier begins to disorganize, the oxidizable cellular components become susceptible to massive attack.

The two-electron quinone reductase DT-diaphorase generates and maintains the antioxidant (reduced) form of coenzyme Q in membranes.

The experiments reported here were undertaken to test the hypothesis that the antioxidative, reduced form of hydrophobic phase coenzyme Q (CoQ) may be generated and maintained by the two-electron quinone reductase, DT-diaphorase [NAD(P)H:(quinone-acceptor) oxidoreductase, EC 1.6.99.2] by catalyzing formation of the hydroquinone form of CoQ. This enzyme was isolated and purified from rat liver cytosol and its reduction of several CoQ homologs incorporated into large unilamellar vesicles (LUVETs) was demonstrated. The addition of NADH and DT-diaphorase to LUVETs and to multilamellar vesicles (MLVs) containing CoQ homologs, including CoQ9 and CoQ10, resulted in essentially complete reduction of the CoQ. Incorporation of either CoQ9H2 or CoQ10H2 and the lipophylic radical generator 2,2'-azobis(2,4-dimethylvaleronitrile) (AMVN) into MLVs in the presence of DT-diaphorase and NADH maintained the reduced state of CoQ and inhibited lipid peroxidation. The reaction between DT-diaphorase and CoQ was also demonstrated in isolated rat liver hepatocytes in which incorporation of CoQ10 provided protection from adriamycin (adr)-induced mitochondrial membrane damage. The role of DT-diaphorase in the antioxidant activity of CoQ was demonstrated by the co-incorporation of dicoumarol (dic), a potent inhibitor of DT-diaphorase, resulting in a loss of protection by incorporated CoQ10. These results support the antioxidant function of DT-diaphorase in both artificial and natural membrane systems by acting as a two-electron CoQ reductase which forms and maintains CoQ in the reduced state.

DT-Diaphorase maintains the reduced state of ubiquinones in lipid vesicles thereby promoting their antioxidant function.

The activity of purified DT-diaphorase in the reduction of ubiquinone homologues of different side-chain length incorporated in uni- and multilamellar vesicles was determined. The direct relationship between the reduced state of ubiquinones and the inhibition of lipid autoxidation induced by thermolabile azocompounds was also demonstrated. Results demonstrate that DT-diaphorase is able to generate and to maintain the reduced, antioxidant form of ubiquinones in both types of vesicles. Furthermore, the results reported herein show that, in the presence of nicotinamide adenine dinucleotide (NADH) and DT-diaphorase, ubiquinol-containing multilamellar vesicles exposed to a lipophilic azocompound did not undergo lipid peroxidation, whereas in vesicles lacking either NADH or DT-diaphorase, thiobarbituric acid reactive substances (TBARS) formation occurred. It is suggested that DT-diaphorase may be responsible for maintaining the reduced state of ubiquinones in various nonmitochondrial cellular membranes.

In vivo and in vitro binding of 1,2-dibromoethane and 1,2-dichloroethane to macromolecules in rat and mouse organs.

The comparative interaction of equimolar amounts of 1,2-dichloroethane and 1,2-dibromoethane with rat and mouse nucleic acids was studied in both in vivo (liver, lung, kidney and stomach) and in vitro (liver microsomal and/or cytosolic fractions) systems. In vivo, liver and kidney DNA showed the highest labeling, whereas the binding to lung DNA was barely detectable. Dibromoethane was more highly reactive than dichloroethane in both species. With dichloroethane, mouse DNA labeling was higher than rat DNA labeling whatever the organ considered: the opposite was seen for the bioactivation of dibromoethane. RNA and protein labelings were higher than DNA labeling, with no particular pattern in terms of organ or species involvement. In vitro, in addition to a low chemical reactivity towards nucleic acids shown by haloethanes per se, both compounds were bioactivated by either liver microsomes and cytosolic fractions to reactive forms capable of binding to DNA and polynucleotides. UV irradiation did not photoactivate dibromoethane and dichloroethane. The in vitro interaction with DNA mediated by enzymatic fractions was PB-inducible (one order of magnitude, using rat microsomes). In vitro bioactivation of haloethanes was mainly performed by microsomes in the case of dichloroethane and by cytosolic fractions in the case of dibromoethane. When microsomes plus cytosol were used, rat enzymes were more efficient than mouse enzymes in inducing a dibromoethane-DNA interaction: the opposite situation occurred for dichloroethane-DNA interaction, and this is in agreement with the in vivo pattern. In the presence of both metabolic pathways, addition or synergism occurred. Dibromoethane was always more reactive than dichloroethane. An indication of the presence of a microsomal GSH transferase was achieved for the activation of dibromoethane. No preferential binding in vitro to a specific polynucleotide was found. Polynucleotide labeling was higher than (or equal to) DNA binding. The labeling of microsomal RNA and proteins and of cytosolic proteins was many times lower than that of DNA or polynucleotides. The in vivo and in vitro data reported above give an unequivocal indication of the relative reactivity of the haloethanes examined with liver macromolecules from the two species and agree, on the whole, with the relative genotoxicity (DNA repair induction ability, mutagenicity and carcinogenicity) of the chemicals.

Multiple steroid hormone receptors in normal and abnormal human endometrium.

The cytoplasmic concentrations of ER, AR, PR, and GR were determined in 124 specimens of normal and abnormal endometrium and other uterine human tissues by the DCC technique. In the endometrial carcinoma group, we observed that pretreatment with MAP leads to low cellularity, higher amount of AR, lower amounts of detectable ER, GR, and PR: the last receptor was almost always absent. A positive correlation between ER presence and tumor grade of differentiation was found in endometrial tumors from hormone-untreated patients. With the value of 142 fmol/mg DNA as the cut off point between high and low binding capacity, the frequency of the single receptors within the hormone-untreated cancer group ranged from 61% to 88%; ER and PR were simultaneously present in 55% of cases (they are tightly correlated in the different biopsies with respect to frequency and amount); ER-AR-PR were present in 45% and all the four receptors in 40% of cases. Slightly higher values were found in normal endometrium collected from hormone-untreated patients.

Correlation between clinical response to antiandrogenic therapy and occurrence of receptors in human prostatic cancer.

Occurrence of steroid hormone receptor has been evaluated in 30 prostatic cancer-bearing patients: 5 alpha-dihydrotestosterone receptor (DHTR) occurrence was correlated with both tumor grade of differentiation and clinical response to hormone therapy.

Antioxidant activity of reduced menadione in solvent solution and in model membranes.

The antioxidant activity of reduced menadione was investigated and compared with that of alpha-tocopherol both in solvent solution and in large unilamellar vesicles by using azocompounds as free radical generators. The results show that: i) reduced menadione behaves as a chain-breaking antioxidant; ii) its inhibition rate constant is similar to that of alpha-tocopherol in homogeneous solution, whereas it is 4 times larger in egg yolk lecithin vesicles; iii) the stoichiometric factor is found lower than 1 in both systems, since a substantial portion of menadiol is consumed by autoxidation and does not contribute to radical trapping; iv) when both alpha-tocopherol and menadiol are present in vesicles, reduced menadione can spare alpha-tocopherol. Data presented here suggest that the reduced form of vitamin K may protect, when present, cellular membranes from free radical damage.

Characterization of large unilamellar vesicles as models for studies of lipid peroxidation initiated by azocompounds.

The aim of this work was to characterize large unilamellar vesicles (LUVETs) prepared by a hand-driven extrusion device in order to use them for studies of lipid peroxidation and antioxidant activity. Vesicle structure and size were examined by electron microscopy. Lipid and antioxidant content was determined before and after the extrusion procedure. Then LUVETs were subjected to autoxidation initiated by both the lipid-soluble 2,2'-azobis(2,4-dimethylvaleronitrile) and the water-soluble 2,2'-azobis(2-amidinopropane hydrochloride) azocompounds. The results demonstrated that: i) LUVETs prepared with lipid concentrations ranging between 25 and 150 mM were essentially unilamellar and reasonably homogeneous, with an average diameter of 90 nm; ii) the phospholipid, cholesterol and antioxidant amounts retained by filters were about 10-15%; iii) LUVETs were suitable for autoxidation studies initiated by the water-soluble azocompound both in the absence and presence of antioxidants. The lipid-soluble azocompound could be used only at low concentrations and its vesicle content had to be determined since part of the initiator was not incorporated into the lipid bilayer. These data suggest that LUVETs seem to be recommended for studies of lipid peroxidation and antioxidant activity.

Autoxidation and antioxidant activity of ubiquinol homologues in large unilamellar vesicles.

The antioxidant activity of ubiquinol homologues with different side-chain length such as ubiquinol-3 and ubiquinol-7 was compared with that of alpha-tocopherol when peroxidation was induced by the water-soluble initiator 2,2'-azobis-(2-amidinopropane hydrochloride). In large unilamellar vesicles containing equal amounts of alpha-tocopherol, ubiquinol-3 and ubiquinol-7 the rates of inhibition were very similar but the stoichiometric factor of quinols was approximately 1. To explain this low value, which is one-half of that found when the autoxidation was performed in apolar solvents (Chem. Phys. Lipids (1992) 61, 121-130), the oxidation of alpha-tocopherol and ubiquinol-3 initiated by the azocompound was studied both in methanol and in dimiristoyl-lecithin vesicles. The results obtained show that the ubiquinol homologues undergo a radical chain reaction taking place at the polar interface and suggest that the average preferred location of both quinol headgroups is near to the outer surface of the bilayer.

17 beta-estradiol, 5 alpha-dihydrotestosterone, progesterone and cortisol receptors in normal and neoplastic human endometrium.

Using the value of 0.24 fmoles/microgram DNA as breaking point between high and low binding capacities, we quantified receptors for 17 beta-estradiol (ER), 5 alpha-dihydrotestosterone (DHTR), progesterone (PR) and cortisol (CR) in normal and neoplastic human uterine tissues. Concerning receptors occurrence, significant relationships were observed between ER and PR, ER and DHTR, and DHTR and PR. A direct correlation between the presence of ER and tumor grading was found: PR was less frequent in grade II and absent in grade III endometrial carcinoma, however this was not a significant correlation. In endometrial carcinoma at least 1 of the receptors was detected in 67-91% of the cases, 3 receptors (ER, DHTR, PR) in 56%, and all 4 receptors in 45%. The simultaneous detection of multiple receptors could play an important role in determining hormone response.

[Specific cytoplasmic receptors for steroid homones in benign and malignant breast diseases, in normal glandular tissue and pectoral muscle]. / Recettori citoplasmatici specifici per ormoni steroidi in affezioni mammarie benigne e maligne, in tessuto ghiandolare normale e muscolo pettorale.

An improvement to the DCC (destran-coated charcoal) method for determining receptors for 17-beta-oestradiol, 5-alpha-dihydrotestosterone and progesterone in benign and malignant breast conditions, normal glandular tissue and muscular tissue is reported. Preliminary results on 16 malignant cancers and 4 fibrocystic mastopathies are discussed together with a critical review of the technique employed and the results obtained in hormone competition.

Relative susceptibility of the thymus and thymocytes to lipid peroxidation.

Thymus and thymocytes peroxidizability was studied in well defined conditions where lipid hydroperoxide dependent peroxidation occurs. The results show that: (i) lipid peroxidation is very low in thymocytes notwithstanding the higher content of arachidonic acid; (ii) the amounts of lipophilic chain-breaking antioxidants is higher in thymocytes; (iii) the thymus contains more total lipids and phospholipids. Thus the higher sensitivity to peroxidation of the thymus can be due to the replacement of thymus parenchyma by fatty tissue not correlated to an increase of lipophilic antioxidants.

Peroxidation potential of rat thymus during development and involution.

The thymus of rats of ages between 1 and 7 months was homogenised and subjected to oxidative stress induced by iron salts. Lipid peroxidation, protein thiols and glutathione status were evaluated. The thymus of rats of 1 month of age exhibited lower susceptibility to the radical attack with respect to the thymus of rats between 3 and 7 months of age. This susceptibility was correlated with the content of polyunsaturated fatty acids and of lipophylic chain-breaking antioxidants.

The biology of stem cell factor, a new hematopoietic growth factor involved in stem cell regulation.

Recently, a new hematopoietic growth factor, stem cell factor, the ligand for the c-kit-proto-oncogene, has been cloned. The gene for this factor or for its receptor are deleted in two well known series of mice mutants which display pleiotropic stem cell defects. Therefore, this factor supposedly plays an important role in stem cell biology. This paper reviews some of the elegant genetic work which led to the discovery of the factor and of its receptor, the biological effects that this factor exerts in the hematopoietic system in normal individuals and in patients with Diamond-Blackfan anemia and speculates on some of its potential clinical applications.

Sequential metabolic events and morphological changes during in vivo large granular lymphocyte activation and proliferation.

Interferon (IFN) and IFN inducers are known to boost natural killer (NK) activity in vivo and in vitro. In vivo enhancement of NK activity results from activation of preexisting NK cells as well as from an increased number of large granular lymphocytes (LGL), with a portion of them undergoing cell division. Our study was addressed to analyze the sequence of metabolic events occurring within the LGL population of Fischer rats treated with poly(I:C), as an IFN inducer. The increase in cytotoxic activity and LGL number in the peripheral blood already reached maximal levels by 12 hr after poly(I:C) injection, remained on a plateau 24 to 48 hr later, then slightly decreased on Day 4, and returned to control levels by Day 6. A similar kinetics was observed for RNA synthesis. In contrast DNA synthesis first increased at 24 hr, peaked at 48 hr, then decreased on Day 4, and was not detectable on Day 6. Percoll fractionation resulted in 92-97% of LGL in fraction 1, and cells in this fraction accounted for the increase of cytotoxicity as well as for newly synthesized RNA and DNA. However, LGL recovered on Day 1 or 2 after poly(I:C) stimulation displayed quite heterogeneous morphology, and a number of mitotic configurations were seen on Day 2 within the LGL population. Our results indicate that the boosting of NK activity by poly(I:C) is always associated with an increase in LGL numbers, the enhanced lytic capacity is associated in vivo with new RNA synthesis by the NK cells, and only in a later phase NK cell proliferation may account for the increase in LGL numbers.

[Determination of cytoplasmic receptors for specific steroid hormones. II) Androgen receptor]. / Dosaggio di recettori citoplasmatici specifici per ormoni steroidei. II) Androgeno-recettore.

Cytoplasmic receptors for 5 alpha-dihydrotestosterone (3H-DHT) were determined in normal and hypertrophic human prostate using the slightly modified DCC method we previously standardized for 17beta-estradiol-receptor. Incubations were always performed at 0 degree C for 1 hr. Discrimination between 3H-DHT binding to cytoplasmic receptor and to Sex Hormone Binding Globulin (SHBG) was achieved on the basis of binding affinity, thermolability and pattern of specificity by various steroid hormones. In particular, 5 beta-DHT did not bind to cytoplasmic receptor, while it did to SHBG.

[Determination of cytoplasmic receptors for specific steroid hormones. III) Progestin receptor]. / Dosaggio di recettori citoplasmatici specifici per ormoni steroidei.--III) Progestinico-recettore.

A method for determining cytoplasmic progesterone receptor was standardized in normal human endometrium comparing two different tracers, 3H-progesterone (3H-P) and 3H-medroxyprogesterone acetate (3H-MAP), a synthetic progestin which does not bind to Corticosteroid Binding Globulin (CBG). Receptor assays were performed as previously reported for 17beta-estradiol receptor, with slight modifications: incubation lasted 1 hr at 0 degree C, followed by 5 min DCC exposure under the same conditions. When 3H-P was employed as tracer, blanks performed with cold MAP gave similar results as using cortisol in incubation tubes and progesterone and cortisol in blanks. 3H-MAP was a good tracer for progesterone receptor because it neither bound to CBG nor to androgen or cortisol receptors; it had very high affinity and specificity for P-R; it was not metabolized by cytosol at 0 degree C and, finally, it detected receptor amounts quite comparable to those obtained using 3H-P.

[Measurement of cytoplasmic receptors specific for steroid hormones. IV) Glucocorticoid receptor]. / Dosaggio di recettori citoplasmatici specifici per ormoni steroidei.- IV) Glucocorticoide-recettore.

Binding of 3H-cortisol (3H-C) and 3H-dexamethasone (3H-DX), a fluorinated glucocorticoid which does not bind to Corticosteroid Binding Globulin (CBG), was investigated in two different target tissues, human breast cancer and human leukemic lymphocytes, using a modified DCC assay method. Maximal binding to cytosol occurred, when increasing concentrations of 3H-C or 3H-DX were incubated in the presence or in the absence of an excess of cold cortisol or dexamethasone, at 0 degrees C for 1 hr or 4 hr, respectively. A 50 sec exposure to DCC was sufficient to separate bound from free labelled steroid. Data presented suggest that two different classes of glucocorticoid exist, which are differently distributed in target tissues, and may be identified on the basis of affinity for the tracer, binding specificity and thermolability.

HPLC and light scattering detection allow the determination of phospholipids in biological samples and the assay of phospholipase A2.

Some applications to biological samples of a method for the separation and the quantitative analysis of phospholipids by high performance liquid chromatography (HPLC) and light scattering mass detection are described. Results obtained in the determination of phospholipid classes from rat tissues such as liver, heart and kidney have been compared with data from the literature. The method has been applied to the evaluation of phospholipids in human low-density lipoproteins (LDL), about which little is known. The procedure is also suitable for a rapid and reliable assay of the water-soluble phospholipase A2 activity; the relationship between the aggregation state of substrate phospholipids (mixed micelles, multilamellar and unilamellar vesicles) and the enzyme activity has been studied.

Differential regulation of interleukin-1 gene expression in human CD3- large granular lymphocytes.

Our laboratory analyzed the expression of lymphokine and cytokine mRNA in CD3- peripheral blood large granular lymphocytes (LGL). Herein we present evidence that this subset of lymphocytes can synthesize IL-1 beta mRNA constitutively and that the cytoplasmic mRNA levels of IL-1 beta can be increased rapidly by interleukin (IL)-2. IL-1 alpha mRNA is expressed constitutively very infrequently and increases in IL-1 alpha mRNA are seen only after prolonged incubation with IL-2. Furthermore, IL-1 activity could not be detected in LGL culture supernatants, indicating that other processes may be involved in releasing biologically active IL-1 from LGL. In addition, MAb to the p75 IL-2 receptor on LGL abrogated IL-2 induction of IL-1 beta mRNA, suggesting that IL-2 signaling via the p75 IL-2 receptor induced IL-1 beta gene expression in LGL. Since, in contrast to T cells, LGL are capable of mediating effector functions without prior stimulation, they are said to be already "primed" for response. Overall, these data suggest that constitutive lymphokine gene expression may be involved in the in vivo priming of LGL.