A transgenic mouse model for the detection of cellular stress induced by toxic inorganic compounds.

Transgenic mice for genotoxicity testing have been developed, although no such models have been produced for the evaluation of toxic, nongenotoxic chemical compounds. We have developed a transgenic mouse model for the analysis of toxic inorganic compounds. We engineered a mouse lineage with the human growth hormone (hGH) gene under the control of the human hsp70 promoter, in which a plasma-detectable hGH response can be elicited by exposure to heat shock. In primary cell cultures from these mice, hGH release was observed following treatment with several toxic inorganics. Transgenic mice injected intraperitoneally with sodium arsenite, cadmium chloride, copper sulphate, or methylmercurium chloride showed significant hGH levels in plasma.

The activity of mammalian polynucleotide ligase on x-irradiated DNAs.

Selected samples of heterogeneous DNA from calf thymus with similar number-average molecular weight, Mn, and a low incidence of single-strand breaks were exposed in aqueous solutions to a mild X-ray dose of 1500 rads. The irradiation produced on the average about 0.2 bihelical and 2.2 monohelical scissions per DNA molecule of 1708 000 Mn. The percent distribution of the chemical termini released at the radiation nicks of DNA was as follows: 64.0 OH, 9.0 PO4 and 27.0 unknowns at the 3' ends: 3.8 OH, 68.2 PO4 and 28.0 unknowns at the 5' ends. A nuclease-free polynucleotide ligase I purified about 3000-fold over the crude homogenate from calf thymus succeeded in rejoining 50% of the breaks in the X-irradiated DNA. The ability of the enzyme to close radiation nicks in DNA directly was confirmed also by experiments on synthetic poly(dA).poly([3H]dT),poly(dT)-cellulose substrates with an irradiated dT chain at either the 3' or the 5' side of the functional break. The poor discrimination of mammalian ligase versus nicked DNA containing radiation damage is of practical relevance. While rejoining altered nucleotide chains in the helices of DNA, the enzyme might contribute to the fixation of premutational lesions in the genetic material.

Studies on terminal deoxynucleotidyl transferase and adenosine deaminase in myasthenic thymus.

Thymic function in myasthenic patients was examined using two biochemical markers which specifically define a population of cortisone-sensitive cortical thymocytes. The enzymatic activities of terminal deoxynucleotidyl transferase (TdT) and adenosine deaminase (ADA) were determined in 13 samples. High contents of both enzymes were found in young patients. The enzymatic activities were easily detectable also in the oldest patients, despite the morphological involution and the decrease in TdT which are known to occur with age in the normal thymus. TdT and ADA-containing cells were almost completely depleted in all the 3 treated patients by the corticosteroid treatment which provides a non-surgical alternative to the elimination of this lymphoid population by thymectomy. The persistence of TdT and ADA activity in old age, and their inhibition by the corticosteroid treatment.

Nickel-induced proteins in human HaCaT keratinocytes: annexin II and phosphoglycerate kinase.

It has been established in previous in vitro experiments with human HaCaT keratinocytes that nickel becomes cytotoxic at concentrations higher than 100 microM and that it is accumulated mainly in the cytosolic fraction (Ermolli et al., 2000). The aim of this work was to search possible biomarkers of metal insult, i.e. nickel-binding proteins or proteins differentially expressed in the cytosolic fraction of nickel-exposed cells (up to 1 mM nickel) as compared to controls. Cytosolic proteins were studied by isoelectric focusing (IEF) and two-dimensional gel electrophoresis (2-DE). Separation by IEF revealed nickel-induced changes in the abundance of cytosolic proteins as visualised with nickel-nitrilo-triacetic-alkaline phosphatase (Ni-NTA-AP) in blots. The cytosolic fraction of cells incubated with nickel, at concentrations over 100 microM, showed nickel binding components which were absent or present in significantly lower amounts in control cells. These proteins had isoelectric points (pIs) 6.9, 7.7 and 8.5. After 2-DE silver- and protein staining significantly increased abundance of four proteins was observed. Their pI values corresponded to those of the nickel binding ones seen after IEF. A protein with pI 6.9 had a molecular weight estimated to 38 kDa, two proteins with pI around 7.7 showed molecular weights of 57 and 22 kDa, respectively and another protein with pI of 8.5 had a molecular weight of 33 kDa. The increased abundance of these components, both in IEF experiments and in 2-DE, correlated with the nickel concentration in the culture media. N-terminal amino acid sequencing and database search allowed identification of one a protein as phosphoglycerate kinase and another one as annexin II. The involvement of these proteins in cellular functions and their possible implications in the mechanism of nickel toxicity in keratinocytes are discussed. Some of these proteins may be biomarker candidates for effects of nickel exposure in human keratinocytes.

Nickel, cobalt and chromium-induced cytotoxicity and intracellular accumulation in human hacat keratinocytes.

Nickel, cobalt and chromium can induce allergic contact dermatitis (ACD) and may provoke irritant reactions in the skin. This study aimed at investigating cytotoxicity and cell viability along with intracellular metal accumulation in HaCaT human keratinocytes exposed to soluble forms of nickel, cobalt or chromium. The EC50 (24 h) values as detected by MTT test were 30 microM for sodium chromate (Na2CrO4), 475 microM for cobalt chloride (CoCl2) and 600 microM for nickel chloride (NiCl2). Chromium chloride (CrCl3) was not toxic up to 1 mM. No clear effects were observed after 4 h, but 24-h treatments with 1 mM CoCl2 or 10 microM Na2CrO(4) were found to almost completely abolish the ability of the cells to form colonies, whilst 1 mM NiCl2 reduced cellular survival to only 70% of control cultures. Intracellular accumulation of metals was evaluated by the use of radioisotopes at the EC50 value and at 1/10-1/5 of this concentration. Accumulation of Na2(51)CrO4 was linear with increasing dose. This was not the case for 63NiCl2 and 58CoCl2. All the metals were accumulated preferentially in the cytosols; 96% or more for 63NiCl2, approximately 90% for 58CoCl2 and 60-70% for Na2(51)CrO4. Finally, it was observed that HaCaT human keratinocytes can concentrate the metals present in the media up to 3.9 and 12.5 times for NiCl2 and CoCl2, respectively, and up to 167 for Na2CrO4. These striking metal intracellular accumulation patterns, which have not been earlier described in keratinocytes, highlight the relevance of searching for specific biomarkers of early cellular toxic effects, such as cytosolic proteins that bind the metals.

Acetaldehyde activation of poly(ADP-ribose)polymerase in hepatocytes of mice treated in vivo.

The activities of poly(ADP-ribose) polymerase and of DNA polymerases alpha and beta and the level of cytochrome P450 were determined in mouse parenchymal liver cells 5 h after treatment with 0.1, 0.3, 1.0, and 3.0 mumole of acetaldehyde. Injection with 1.0 and 3.0 mumole of acetaldehyde induced an increase in poly(ADP-ribose) polymerase activity and in the P450 level, but had no effect on DNA polymerases. The stimulation of poly(ADP-ribose) polymerase activity can be used as an index of induced DNA damage. The possibility of using this experimental approach with other cells derived from mice treated in vivo with different xenobiotics is discussed.

The use of pre-implantation mouse embryos cultured in vitro in toxicological studies.

The preimplantation mouse embryo has been found to be a good model for various toxicological investigations. This communication deals with two examples of research activities carried out in our laboratory to detect the embryotoxic properties of tritium and of 1,2:3,4-diepoxybutene (DEB). Exposures of blastocysts for 24 hr to concentrations as high as 0.296 kBq/ml tritiated amino acids or nucleoside induced a statistically significant reduction in the percentage of embryos that reached the stage of two-layer inner cell mass (ICM). The same quantity of tritiated arginine, but not of thymidine or tryptophan, also induced a lower percentage of differentiating ICM than the control when added to culture medium during the second cleavage division. These findings support the idea that tritium released by nuclear power plants as HT or as tritiated water (HTO) could become a radiotoxicological problem since it can be converted easily into organic compounds by living organisms. DEB was found to be highly embryotoxic in preimplantation mouse embryos in vitro at micromolar concentrations. This compound is formed in mammalian cells by the oxidative metabolism of 1,3-butadiene, a chemical used in rubber industries and present in tobacco smoke. Again, the most sensitive stages of preimplantation development were found to be the two- and the four-cell embryos. These results were confirmed by in vivo measurements.

Studies of terminal deoxynucleotidyl transferase in normal and neoplastic human cells.

Optimized biochemical assays and cytoimmunofluorescence tests were used to detect terminal deoxynucleotidyl transferase, TdT, in malignant cells of 36 leukemias and 75 lymphomas from patients not receiving chemotherapy. TdT was virtually absent from normal lymph nodes and from leukocytes of chronic lymphocytic leukemia, CLL, taken as controls. Its quantitative distribution in the neoplasms matched the current knowledge. Appreciable amounts of TdT were found in all the 10 lymphomas of lymphoblastic type, LL, and in the white blood cells of: 16 out of 19 acute lymphoblastic leukemia, AAL, perhaps with modulation in the various phenotypes; 2 out of 3 acute undifferentiated leukemias, AUL; and 3 out of 7 blastic crises in chronic myelogenous leukemia, b.c. CML. Biochemical and cytoimmunological analyses yielded concordant responses and even roughly comparable estimates in the same patients. TdT immunofluorescence was clearly nuclear in most cells and was cytoplasmic occasionally. Definite correlations between concentrations of enzymatic activity and percentage of immunofluorescent cells could not e established. Further detailed work will be required to identify putative subgroups in TdT-positive blast populations.

Poly(A) polymerase distribution in normal and malignant lymphoid cells.

The incorporation of ATP on poly(A) primers catalyzed by poly(A) polymerase was investigated in normal and neoplastic lymphoid cells from animal and human sources. High levels of the enzyme were found in mouse thymus, in chicken bursa and thymus, as well as in neoplastic cells from patients affected by lymphoblastic and Burkitt's lymphomas. Low or very low quantities were found in peripheral blood lymphocytes, chronic lymphocytic leukemia cells, normal lymph nodes and solid lymphoid tissues of Hodgkin's disease. In general, the enzymatic content of neoplastic lymphoid cells reflected those of their normal counterpart. No effect of fasting or cortisone treatment on poly(A) polymerase in mouse spleen, thymus or liver was found. No particular relationships with B, T or non-T, non-B lineages were observed, but some relationship with DNA polymerase alpha was found. Therefore, it may be that poly(A) polymerase levels are related to the proliferative activity of the cellular populations.

Alkaline phosphatase and gamma glutamyltranspeptidase in human lymphomas.

Alkaline phosphatase (AP) and gamma glutamyltranspeptidase (GGT) were studied in normal lymphoid cells and in 28 cases of human lymphomas (23 of non-Hodgkin's and 5 of Hodgkin's disease). The expression of AP was enhanced in several samples with a high proportion of mature B cells, particularly in centroblastic-centrocytic lymphoma, whereas tissues mainly composed of T cells always showed low levels of this enzyme. GGT levels were high in thymus, as well as in centroblastic-centrocytic lymphoma and other NHL, thus demonstrating no restriction to a particular cell lineage. Some B-cell neoplasms with cellular origin different from that of centroblastic-centrocytic lymphoma, such as chronic lymphocytic leukemia and centrocytic lymphoma, had low levels of both enzymes. The role of investigation with specific antibodies against these two enzymatic activities in the physiology of lymphoma cell membrane is discussed.

Aerodynamic characteristics of tracheostomy speaking valves: an updated report.

Two analyses were conducted to define some aerodynamic properties of one-way speaking valves designed for use with the tracheotomized patient. In the first analysis, the resistance to airflow of six different valves was determined during steady-state flow testing at rates of .450, .500, and .550 I/s. Significant differences among the valves were established only at the lowest flow rate. All valves exhibited relatively low resistance in the range of nasal resistance reported for normal adults. In the second analysis, the aerodynamic integrity of the valves was assessed during repetition of the syllable /pa/ under a condition used to simulate tracheostomy speech production. Significant differences were found among the valves in terms of air loss occurring during the rise in pressure associated with the production of the consonant /p/. Valves with diaphragms open at atmospheric pressure consistently exhibited air loss. Average slope of the rise in pressure for one of the valves tested was significantly greater, suggesting increased work during speech production. The results of these analyses suggest that although the inspiratory resistance to airflow was similar among various one-way speaking valves, some valves exhibit air loss during speech production.

Different effects of vanadium ions on some DNA-metabolizing enzymes.

The effects of vanadium on some enzymes involved in DNA metabolism were investigated in vitro. Vanadate (V) ions competitively inhibit calf thymus terminal deoxynucleotidyl transferase with Ki = 2.5 microM. A binding of vanadium to the enzyme with no change of the amount of the Zn constituent of the protein was found at concentrations of vanadate causing inhibition. The catalytic activity of mammalian DNA polymerase alpha was also inhibited by vanadate ions at an I50 of 60 microM, while the bacterial (E. coli) DNA polymerase I was affected to the same extent only when the concentration of vanadate was raised to about 0.5 mM. In contrast to the inhibitory effects caused by vanadium on the nucleotidyl transferases, concentrations of pentavalent vanadium ions of the order of 10 microM increase 2.4-fold the hydrolytic activity of deoxyribonuclease I from bovine pancreas. These findings suggest that vanadium can interact with enzymes involved in nucleic acid metabolism.

Terminal deoxynucleotidyl transferase in human lymphomas: possible existence of forms with high and low molecular weights.

Optimized methods for extraction and enzyme assay in crude tissue preparations were used to determine the amounts of terminal deoxnucleotidyl transferase (TdT) in malignant lymphomas. The TdT concentration was increased only in lymphoblastic lymphomas (LL) and was as high in these tumours as in the white blood cells from untreated patients with acute lymphoblastic leukaemia (ALL). The enzymes extracted from such lymphomas and from the leukaemic lymphoblasts had the same properties. Moreover, forms of TdT with low and high mol. wt were found in the LL tumours, similar to other reports of TdT-positive leukaemias. The overall study points at some basic biochemical identity of certain lymphoblastic malignancies, irrespective of whether the transformed cells are in solid tumours or are disseminated in the blood.

Aerodynamic characteristics of tracheostomy speaking valves.

Pressure-flow characteristics were determined for four different one-way valves used for speech production in the patient who has had a tracheotomy. Each valve was tested at steady-state flow rates of 150, 250, 350, and 450 ml/sec in isolation and attached to a tracheostomy tube. Results indicated significant differences in resistance among the valves. The resistance of one valve was substantially greater than that of the normal upper airways. It is suggested that future research determine the clinical significance, if any, of these differences relative to speech and respiratory behaviors in tracheostomized patients.

[Abortion and the man. Psychological and psychopathological manifestations in the face of lost fatherhood]. / L'aborto al maschile. Manifestazioni psicologiche e psico-patologiche in occasione della mancata paternità.

This study examines the psychological and psychopathological reactions that may appear in a man when his partner aborts. The small amount of literature that exists on the subjects was examined, and several men were, interviewed at the time their partners aborted voluntarely. It turns out that in spite of the man's lack of recognition of any reactions (reinforced by current attitudes and by the scant psychological and medical interest in the subject), there is considerable emotional involvement in the lost parenthood, both for the man and the woman. As revealed by the interviews, this involvement may manifest itself in persecutory or depressive anxiety and psychosomatic symptoms. Moreover, but very rarely, real and typical psychopathological symptoms may appear, such as depression and behavioural disorders. The appearance of these reactions is linked to the problems and conflicts aroused by prospective fatherhood, leading to a comparison between the experiences of fuliwed fatherhood and those of fatherhood lost.

PIP: Only recently has any study evaluated the psychological and psychopathological reactions of men whose partners have chosen to have an abortion. Recent studies have shown that fatherhood causes deep personal and social changes and conflicts. It is therefore natural that an interruption of this process, such as through abortion, can inspire feelings of being partially "killed", thus bringing back childhood anxieties and feelings of castration. One study on women who had abortions showed that only 1/6 were accompanied by their partners, and that for those who face abortion as a couple, the future did not seem to hold negative consequences. A study of a number of young men emphasized the frustration of 2 desires: that of becoming a father and, consequently, of becoming a man. Another study of 50 childless young men indicated that the "lack of fatherhood" after the 1st abortion was the origin of frustration and pain that, in some cases, drove unmarried couples to discontinue their relationship. In a 4th study, of 52 women who went to an abortion clinic, 29 were accompanied by their partners. 20 partners agreed to be interviewed. Although none were asked the reason for the abortion, each explained the motivation for it. The observations show "persecution anxieties", especially with regard to the partner, depression anxieties and psychosomatic symptoms. Depression and behavioral disorders, although rarer, may also manifest themselves. The study suggests a comparison between reactions and feelings of potential fatherhood and fatherhood lost.

The toxicity of tritium: the effects of tritiated amino-acids on preimplanted mouse embryos.

The effects of tritiated amino-acids, arginine, lysine, histidine and aspartic acid on the growth and development of two-cell mouse embryos, cultured in vitro, were investigated. The LD50 for the dibasic amino acids, measured on the third day of growth, ranged from 30 to 130 nCi/ml. This was compared with the DNA precursor, thymidine, for which the LD50 was 80 nCi/ml.

Preparation of polydeoxynucleotides linked to a solid support by coupling CNBr-activated cellulose with 5'-NH2-terminated oligo and poly(pdT)'s.

Synthetically-prepared 5'-NH2-dT(pdT)n oligomers (66,n=4 or 7) were immobilized on cyanogen bromide activated cellulose. The influence of temperature, pH, and ionic strength on the rate of the coupling process was studied. The oligomer 5'-NH2-DT(pdT)8 could be elongated enzymatically to the polymers 5'-NH2-dT(pdT)n (n=20, 51 and 84), which could be immobilized on cellulose. The cellulose-NH-dT(pdT)84 polymer thus obtained could be assembled to a new solid-state polymer e.g. poly(dA)290 . poly(/3H/dT)200, poly(dT)85-cellulose which, in turn, was a very convenient substrate for assaying DNA-ligase.

Multienzymatic analyses of human malignant lymphomas. Correlation of enzymatic data with pathologic and ultrastructural findings in Burkitt's and lymphoblastic lymphomas.

Enzyme activity measurements are of great relevance to the classification and biochemical characterization of the various types of leukemias, but they have been much less studied in solid lymphoid tumors. The authors report investigations in human lymphomas. The levels of the following enzymes were determined: terminal deoxynucleotidyl transferase (TdT), deoxyribonucleic acid polymerase alpha (DP alpha), adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP), thymidine and uridine kinases (TK and UK, respectively), and thymidine phosphorylase (ThPh). Moreover, cytochemical investigations were done in the group of Burkitt's lymphoma (BL) and lymphoblastic lymphoma (LL), and ultrastructural studies were performed in seven of the nine LL of this series. These results were obtained: (1) TdT (90 cases) was highly specific for LL; eight of nine LL were positive, and all other histologic types were negative; the only TdT-, acid esterase (AcE) positive, nonconvoluted LL was probably related to TdT- normal medullary thymocytes, and had an unfavorable clinical course with resistance to a vincristine-and-prednisone-including treatment; (2) ADA (61 cases) could distinguish clearly between the high levels of LL and the low levels found in any other group of lymphomas; among LL, the highest values were found in T-cell-derived neoplasias, and the lowest value in a periodic acid-Schiff (PAS) positive, acid phosphatase negative case that showed the presence of large nucleoli at the ultrastructural analysis, a finding that is unusual for LL and possibly related to a more immature differentiation stage; (3) PNP (39 cases) values alone were not clinically relevant, but together with ADA levels, a subset of T-LL with high ADA:PNP ratio could be selected among LL; (4) DP alpha (61 cases), and TK and UK (37 cases) were found in concentrations reflecting the malignancy of the non-Hodgkin's lymphoma, and were more elevated in the high-grade malignant lymphomas; (5) ThPh (34 cases) was always elevated in Hodgkin's disease, but low in Burkitt's lymphoma and LL; thus, they had a high TK:ThPh ratio that could be useful in predicting clinical response to thymidine treatment. The authors think that taken together, multiple enzyme determinations could be useful in the characterization of human lymphomas.

Adenosine deaminase and terminal deoxynucleotidyl transferase in human lymphomas: an aid to the diagnosis and subclassification of lymphoblastic lymphomas.

Adenosine deaminase (ADA) and terminal deoxynucleotidyl transferase (TdT) activities were determined on 97 biopsy specimens obtained from patients with non-neoplastic diseases (12 cases), Hodgkin (30 cases), and non-Hodgkin lymphomas (55 cases). Thirty additional cases were tested only for TdT. TdT was positive in 10 out of 13 lymphoblastic lymphomas (LL) examined and negative in all the other specimens, including the ten cases of the immunoblastic type. Levels of ADA above 350 U/mg of protein were found in 10 out of 12 LL tested, but not in any other specimen. The 3 TdT- LL had high contents of ADA. Therefore, all LL can be detected using both ADA and TdT markers. The 3 TdT- LL had a heterogeneous phenotype and their possible origin is discussed in view of the possibility that they constitute a rare entity distinct from the more common TdT+ LL. Very low levels of ADA (below 100 U/mg of protein) were found in chronic lymphocytic leukemia and immunocytoma, and in Burkitt's lymphoma. In other B-cell non-Hodgkin lymphomas, intermediate values between 100 and 350 U were often found, and this finding could be relevant to the different cellular origin of the various B-cell neoplasias. We conclude that ADA distribution is solid lymphoid tumors reflects the cellular origin of these neoplasias. Adenosine deaminase alone and in combination with TdT can be useful in the diagnosis and classification of childhood lymphomas in which the immature hystotypes predominate.

Local regression of breast tumors following intramammary ganciclovir administration in double transgenic mice expressing neu oncogene and herpes simplex virus thymidine kinase.

Females from a mouse lineage transgenic for the activated rat neu oncogene under the control of the mouse mammary tumor virus (MMTV) long terminal repeat (LTR) all develop breast tumors with high reproducibility within the first 2-3 months of life. These animals were crossed with mice from a lineage transgenic for the herpes simplex virus thymidine kinase gene (HSVtk) under the control of its own promoter and polyoma enhancer. Double transgenic mice (for both neu and tk) developed breast neoplasias with the same kinetics as the neu-only mice. Tumor-bearing double transgenic mice, treated intratumorally with the antiviral agent ganciclovir (GCV), showed an inhibiting effect on tumor growth. However, this effect was not seen either on GCV-treated neu-only transgenic mice or on saline-injected controls. This suggests that tk-engineered breast tumors are susceptible to GCV administered locally, and implies that neu-mice could be a useful model for testing the effectiveness of HSVtk-bearing vectors followed by systemic GCV on breast cancer cells.