Chronic treatment with polychlorinated biphenyls (PCB) during pregnancy and lactation in the rat Part 2: Effects on reproductive parameters, on sex behavior, on memory retention and on hypothalamic expression of aromatase and 5alpha-reductases in the offspring.

The gender-specific expression pattern of aromatase and 5alpha-reductases (5alpha-R) during brain development provides neurons the right amount of estradiol and DHT to induce a dimorphic organization of the structure. Polychlorinated biphenyls (PCBs) are endocrine disruptive pollutants; exposure to PCBs through placental transfer and breast-feeding may adversely affect the organizational action of sex steroid, resulting in long-term alteration of reproductive neuroendocrinology. The study was aimed at: a) evaluating the hypothalamic expression of aromatase, 5alpha-R1 and 5alpha-R2 in fetuses (GD20), infant (PN12), weaning (PN21) and young adult (PN60) male and female rats exposed to PCBs during development; b) correlating these parameters with the time of testicular descent, puberty onset, estrous cyclicity and copulatory behavior; c) evaluating possible alterations of some non reproductive behaviors (locomotion, learning and memory, depression/anxiety behavior). A reconstituted mixture of four indicator congeners (PCB 126, 138, 153 and 180) was injected subcutaneously to dams at the dose of 10 mg/kg daily from GD15 to GD19 and then twice a week till weanling. The results indicated that developmental PCB exposure produced important changes in the dimorphic hypothalamic expression of both aromatase and the 5alpha-Rs, which were still evident in adult animals. We observed that female puberty onset occurs earlier than in control animals without cycle irregularity, while testicular descent in males was delayed. A slight but significant impairment of sexual behavior and an important alteration in memory retention were also noted specifically in males. We conclude that PCBs might affect the dimorphic neuroendocrine control of reproductive system and of other neurobiological processes.

Prospective study on prognostic significance of DNA ploidy and Ki-67 expression in colorectal cancer.

The aim of the study was to correlate tumoral DNA ploidy and Ki-67 expression with therapy response, Overall Survival (OS), Disease Specific Survival (DSS) and Disease Free Survival (DFS). Three samples of colorectal cancer were collected from each patient. One sample of normal tissue was our internal control. DNA ploidy was evaluated by FACSCalibur cytometer and Ki-67 by immunohistochemistry. We studied 67 patients and we found aneuploidy in 65,7 percent of carcinoma with a Ki-67 median expression of 55 percent. After surgery and chemotherapy in 35 percent of the patients with aneuploid carcinoma and high proliferative activity (Ki-67 greater than 55 percent) there were no evidence of disease versus 100 percent of patients with DNA diploidy and low proliferative activity (Ki-67 less than 55 percent). Tumoral aneuploidy significantly correlated with lower OS, DSS and DFS (18 percent vs 86 percent at 30 months). Univariated analysis demonstrated a significant correlation between aneuploidy and develop disease progression (p=0,033, odd ratio=5.7), while the cut-off of 55 percent for Ki-67 expression did not correlate with OS, DSS and DFS. Preliminary results (the study is still in progress) seemed to suggest that DNA ploidy has a prognostic and predictive significance in colorectal carcinoma.

Air quality biomonitoring: assessment of air pollution genotoxicity in the Province of Novara (North Italy) by using Trifolium repens L. and molecular markers.

Mixed air pollutants are considered a major cause of DNA damage in living species. In this study Trifolium repens L. cv Regal was used as a bioindicator to assess the genotoxicity of air stressors in the Italian province of Novara. Two on-site biomonitoring experiments were performed during the spring and autumn of 2004. Test plants were exposed at 19 monitoring sites distributed homogeneously throughout the province, and each experiment lasted for a period of 6 weeks. Genotoxicity was evaluated with Amplified Fragment Length Polymorphism (AFLP) molecular markers. The results show the predominantly rural central-west region of the Novara Province to have the worst air quality with regard to genotoxicity. Analyses of geomorphology, land use and climatic factors suggest that the compromised air quality in the region could be attributed to wind strength and direction, transporting pollution from vehicular traffic on the A4 highway and from the urban/industrialized centres of Novara and Vercelli. Plant growth, changes in plant photochemical efficiency and the presence of ozone related leaf injuries were also measured to better interpret the results of genotoxicity. Statistical analyses show that although climatic factors such as light intensity and temperature influence plant growth, they do not contribute to atmospheric stressor-induced DNA damage. Further analyses indicated that, as expected, a mixture of genotoxic and non-genotoxic pollutants coexist in the Novara Province troposphere, and that the elevated ozone concentrations experienced during the study may have contributed to the DNA damage in the tested plants by enhancing genotoxicity via interaction with other air stressors.

Monoclonal antibody panels for acute leukemia and myelodysplastic syndrome diagnosis. Results of a co-operative quality control group.

The need for standardization criteria and result reproducibility in immunophenotyping hematological diseases has increased along with their clinical importance. Our group "Policentric Study Group on Immunological Markers", is composed of 40 laboratories. Its aim, over recent years, has been to find a standardized way of immunophenotypic analysis applicable to various hematological diseases. The objective of this study is to contribute to the debate concerning standardization of monoclonal antibody panels and immunophenotypic analysis procedures in acute leukemia (AL) and myelodysplastic syndrome (MDS), with the following targets: to improve interlaboratory reproducibility of the immunophenotyping data, and interpretative results; to study, with improved feasibility, correlation between immunophenotype and clinical or biological findings on a large number of AL and MDS cases; to verify the utility of the proposed monoclonal antibody panels for proper AL and MDS classification, and to detect minimal residual disease. In the field of AL and MDS our experience is based on about 1800 and 700 cases respectively analyzed over the last five years. Starting from these experiences and data of the literature we have elaborated the proposed panels of monoclonal antibodies and the methods of analysis. We have suggested a standardized immunophenotypic approach to study AL and MDS. In particular our work has focused on the gating strategy. This aims at drawing a gate of analysis having high purity and recovery, and on the choice of monoclonal antibody combinations for multiparametric analysis, particularly the normal antigen expression on each step of lineage differentiation or their clinically relevant aberrant expressions. A standardized criteria has become a necessary starting point in any kind of analytical process. In the field of acute leukemias and myelodysplastic syndromes the work of this polycentric group has focused on the pre-analytical and analytical steps to be taken in cytometric evaluation of hematological malignancies. The results obtained may contribute to reaching intra and inter-laboratory reproducibility.

DCEP (dexamethasone, cyclophosphamide, etoposide, and cisplatin) is an effective regimen for peripheral blood stem cell collection in multiple myeloma.

DCEP (dexamethasone, cyclophosphamide, etoposide, and cisplatin) has proved to be an effective salvage therapy for refractory-relapsed MM patients. Little is known, however, about its potential as mobilizing therapy. The aim of this study was to evaluate the efficacy of DCEP in mobilizing PBSC and to define its toxicity. Fifty-five MM patients received DCEP followed by G-CSF as part of high-dose programs including autologous transplantation. At the time of mobilization, 40 patients had previously received VAD only, and 15 alkylating agents. Mobilization was successful (minimum number of CD34(+) cells 2 x 10(6)/kg) in 48/55 patients (87%), and 41/55 patients (75%) collected >4 x 10(6)/kg CD34(+) cells. Of the seven patients who did not mobilize stem cells, five (71%) had been previously exposed to alkylating agents. The median number of CD34(+) cells harvested was 5.8 x 10(6)/kg (range 2.1-22.4). There was no treatment-related mortality. The side-effects of DCEP were always tolerable. No neutropenia <1000/microl nor thrombocytopenia <50,000/microl were observed. No patient required transfusion as a consequence of therapy, or hospitalization for septic complications. In conclusion, DCEP, in addition to its demonstrated anti-tumor activity, is an effective regimen for mobilizing peripheral blood progenitor cells in myeloma patients, with little or no side-effects. These properties render DCEP a useful regimen for the debulking and mobilization phase of high-dose programs for multiple myeloma.

Metal biomonitoring with mosses in the surroundings of an oil-fired power plant in Italy.

Levels of 12 trace elements were measured in samples of the bryophyte Hypnum cupressiforme Hedw. and in soil collected in the surroundings of an oil-fired power plant in Northern Italy. Metal bioaccumulation in moss was estimated after soil correction in order to obtain deposition patterns and individuate potentially toxic metals emitted from the plant. V and Ni, occurring together in fuel oil, showed highest bioaccumulation values near the stacks. Mean contamination of the study area for these elements is 5.5 (V) and 3.3 (Ni) times the background levels of the reference site. Other elements showed only limited alterations of bioaccumulation values, in relation to agricultural and industrial activity in the study area.

An Italian national multicenter study for the definition of reference ranges for normal values of peripheral blood lymphocyte subsets in healthy adults.

BACKGROUND AND OBJECTIVE: Reference ranges are necessary in clinical chemistry and hematology to compare an observed value and to provide meaningful information. The aim of this multicenter study was the definition of reference ranges of the relative and absolute numbers of lymphocyte subsets by evaluating a large cohort of healthy adults and by using a standard protocol to reduce the variability in both sample preparation methodology and flow cytometer operation. Other aims of this study were the evaluation of the influence of sex, age, obesity, smoking, sport and some methodological variables on lymphocyte subsets and the comparison of differential white blood cell values obtained by flow cytometry and those obtained by hematology counters. DESIGN AND METHODS: Blood samples from 1311 healthy adults (blood donors and volunteers chosen according to the Italian law for donor selection) were analyzed to study, by flow cytometry, the immunophenotype of lymphocyte subsets and their distribution in terms of percentages and absolute values. Pre-analytical and analytical phases were performed according to the guidelines of the International Federation of Clinical Chemistry (IFCC) and the Italian Group of Cytometry (GIC). T cells were defined by the expression of CD3; T subpopulations by the coexpression of CD4 or CD8 or HLA-DR; B-lymphocytes were identified by the expression of CD19 while natural killer lymphocytes were identified by positivity of CD16 and/or CD56 without CD3. We calculated, for each laboratory and for all data collected, the frequency distribution percent values and absolute values of each lymphocyte subset. The influence of age, sex, smoking, obesity and sport was calculated by the t-test. The influence of some methodological variables was calculated by the t-test and multiple regression test. RESULTS: Fifty-three flow cytometry laboratories at different institutions in Italy participated in this study. Data was obtained from 1311 healthy adults aged from 18 to 70; 968 phenotype analyses (74%) were considered eligible for statistical analysis. Significant results were found as regards sex, smoking and some methodological variables (quantity of sample, washing procedures, brand of monoclonal antibodies and kind of instruments used). The comparison between hematology counters and cytometers showed no difference for any of the parameters considered. INTERPRETATION AND CONCLUSIONS: The large number of cases, the different kinds of laboratories and their distribution throughout the country make our sample representative of the Italian adult population. The standardization criteria of pre-analytical and analytical phases (the most important issues in evaluating reference values for an indicator) assured good reproducibility among laboratories so that the obtained reference ranges may be useful for interlaboratory comparison of results. Instruments and the brand of monoclonal antibodies may represent an inevitable cause of variability.

Biochemical characterization of the effects of the benzodiazepine, midazolam, on mitochondrial electron transfer.

Midazolam, a water soluble benzodiazepine used as a preanaesthetic and hypnotic drug, showed a concentration-related (0.1-0.75 mM) depressant effect on both Adenosine 5'-diphosphate (ADP)-induced oxygen consumption and oxidative phosphorylation of rat liver mitochondria if the substrate was oxidized at different steps in the oxidation chain, but not when the substrate was ascorbate plus tetramethyl-p-phenylenediamine (complex IV). Furthermore, midazolam did not affect citrate synthase activity, but inhibited the 2,4 dinitrophenol (DNP)-uncoupled mitochondrial respiration. This result shows that midazolam primarily acts as a mitochondrial electron transport inhibitor. This inhibition is mainly due to the fact that midazolam decreases NADH ubiquinone reductase (complex I) and ubiquinol cytochrome c reductase (complex III) activities, but it also inhibits complex II activity. Spectrophotometric measurements of redox states of rat skeletal muscle mitochondria cytochromes show a decrease in the reduction of aa3 and c+c1 cytochromes in the presence of the benzodiazepine. Midazolam significantly decreased the reduced ubiquinone/total ubiquinone ratio (evaluated by means of HPLC and electrochemical detection) in rat liver mitochondria in both beta-hydroxybutyrate and succinate. Ubisemiquinone may be the redox component affected by midazolam, whether or not bound to the iron-sulfur proteins present in all three mitochondrial complexes. These effects of midazolam, not necessarily related to the preanaesthetic and hypnotic action are probably mediated via mitochondrial benzodiazepine receptors.

Vulnerability of mitochondrial complex I in PC12 cells exposed to manganese.

The present findings provide experimental evidence for the hypothesis that an impairment of mitochondrial function may be involved in manganese neurotoxicity. Specifically, the treatment of dopaminergic neuronal-derived cell line (PC12) with MnCl2 produced a significant inhibition of some mitochondrial complexes of the respiratory chain, while in the glial-derived cell line (C6) this effect was not observed. In PC12 the decrease in complex I activity was more pronounce than in other mitochondrial complexes. However treatment of cells with ZnSO4 exerted no significant variations in enzymatic activities. A direct exposure of mitochondrial fraction to MnCl2 reduced enzymatic activities of mitochondria in both cell lines adding further support to the proposed theory that the different sensitivity of the cells to manganese may be explained by a difference in uptake or intracellular storage. These data indicate that manganese neurotoxicity could be the result of a direct effect just on complex I activity or due to a secondary effect of oxidative stress induced by an excess of this transition metal.

Benomyl affects the microtubule cytoskeleton and the glutathione level of mammalian primary cultured hepatocytes.

Rat primary hepatocyte cultures have been used to study the effect of Benomyl alone or in combination with Pirimiphos-methyl. The results presented demonstrate that Benomyl alone is responsible for the microtubular disorganization in both a time- and dose-dependent manner, that the effect is reversible after the agent is removed, and that Benomyl is a potent glutathione-depleting agent. Pirimiphos-methyl, alone or combined with Benomyl had no effect on microtubule organization, but reinforced the decrease in glutathione.

Acute myeloid leukemia and diabetes insipidus: results in 5 patients.

The clinical and hematological characteristics of 5 patients affected with both acute myeloid leukemia (AML) and diabetes insipidus (DI) are described. Banded chromosomal analysis demonstrated monosomy 7 in 2 patients and a complex cytogenetic rearrangement in another. No patient entered complete remission with standard induction chemotherapy. These data confirm that in patients with AML, the association of DI (with or without monosomy 7) is an unfavorable prognostic factor.

Mitochondrial toxicity of iron and the protective role of ferritin on dopaminergic PC12 cell line.

A specific defect of imtochondrial Complex I activity has recently been identified in the substantia nigra of parkinsonian brain. Using an in vitro dopaminergic PC12 cell line model, a possible pathogenic link has been investigated between this finding and increased levels of iron actually observed in the substantia nigra of parkinsonian patients. Exposure of PC12 cells to FeSO(4) for 168 hr induced a significant inhibition of Complex I, whereas succinate dehydrogenase activity was not reduced by this treatment. Furthermore, the non-dopaminergic cell line (SK-N-BE) was not sensitive to the mitochondria! inhibitory effect of iron. Direct treatment of the mitochondrial fraction with FeSO(4) decreased Complex I activity in both cell lines, suggesting that the difference in sensitivity of cells to iron could be due to the difference in uptake or intracellular storage of this element.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) affects the actin cytoskeleton and calcium level of Swiss 3T3 mouse fibroblasts.

Organization of the actin cytoskeleton, the cytosolic free-calcium concentrations and ATP levels were analyzed in 3T3 mouse fibroblasts treated with 0.75 or 1.5 mM MPTP. In the presence of the drug actin filaments were time- and dose-dependently disorganized, ATP level was unaffected and intracellular calcium increased within 5 s. The correlation between MPTP cytotoxicity and [Ca2+]i level emerging from these results, suggests that the primary effect of the molecule itself is on the plasma membrane's integrity for calcium ion regulation.

Analysis of long-term results and prognostic factors among 138 patients with advanced Hodgkin's disease treated with the alternating MOPP/ABVD chemotherapy.

BACKGROUND: A prospective study was conducted to assess (a) the long-term results and toxicity of the alternating MOPP/ABVD regimen in advanced Hodgkin's disease; (b) the prognostic value of pretreatment variables and of drug dose intensity. PATIENTS AND METHODS: A total 138 consecutive patients with advanced Hodgkin's disease entered this study; patient selection included stages IIB (33% of total), IIIB (26%), IV (25%), and stages IIA-IIIA (16%) with bulky disease and pulmonary hilum involvement. The MOPP/ABVD program was delivered in an 8-month program; adjuvant radiotherapy on sites of bulky disease was delivered in 24 patients. RESULTS: Complete remission was obtained in 106 (77%) patients; significant factors for CR in univariate analysis were stage, symptoms, histology, and bone marrow involvement. The five-year relapse-free survival (RFS) was 83%; in a multivariate analysis, histology only correlated with RFS (p = 0.04). The five-year freedom from tumor mortality and overall survival (OS) were 79% and 67%, respectively. An adverse prognostic significance for OS was observed for B symptoms and bone marrow involvement. The median percentage of relative dose intensity (RDI) was as follows: Adriamycin 86, mechlorethamine 85, vincristine 73, vinblastine 84, bleomycin 79, procarbazine 74, dacarbazine 81. No significant association was found between RDI and clinical outcome. No severe pancytopenia or life-threatening complications occurred during therapy. CONCLUSIONS: Alternating MOPP and ABVD cured more than 65% of patients with advanced HD; acute and late toxicity were acceptable. Prognostic analysis defined subgroups with a lower chance of cure which may deserve a more intensive initial therapy.

The role of systemic high-dose cytarabine in the treatment of central nervous system leukemia. Clinical results in 46 patients.

BACKGROUND: Given the good penetration of systemic high-dose cytarabine (HDara-C) into the cerebrospinal fluid (CSF), this approach was used to treat patients with central nervous system (CNS) leukemia, either isolated or with concurrent extraneurologic disease (END). METHODS: From 1983 to 1991, 46 adults with CNS involvement were treated with systemic HDara-C: 25 had acute lymphoblastic leukemia (ALL), 15 had high-grade non-Hodgkin lymphoma (NHL), 5 had acute myelogenous leukemia (AML), and 1 had lymphoid blast crisis of chronic myelogenous leukemia. Induction consisted of HDara-C 3 g/m2 every 12 hours, by 3-hour infusion, for 8 doses (30 patients), or 6 doses (16 patients), followed by 4 doses at day 21. RESULTS: Of 46 patients, 29 (63%) achieved complete remission (CR): 15/15 with isolated CNS leukemia, and 14/31 (45%) with CNS and concurrent marrow or lymph node disease. Of 17 patients not meeting CR criteria because of persistent END, 11 showed complete CNS response. The first 10 remitters were consolidated with monthly 4-dose courses of HDara-C. The remaining 19 received postinduction multidrug chemotherapy (including vincristine, doxorubicin, cyclophosphamide, L-asparaginase, etoposide plus intermediate-dose ara-C, mitoxantrone plus HDara-C) and intrathecal methotrexate (MTX) +/- cranial radiation therapy. One patient underwent autologous and one allogeneic bone marrow transplant. Median CR duration was 7 months (range, 2-56+): 8 months for patients with isolated CNS leukemia, and 4 months for those with concurrent END: In only two patients was CNS the primary site of relapse. Three patients with isolated CNS leukemia are disease-free at 23, 40, and 56 months. The main toxicity was myelosuppression. No patient showed dose-limiting neurologic toxicity. CONCLUSIONS: Systemic HDara-C appears effective therapy for CNS leukemia, maximally in cases with isolated CNS involvement. HDara-C may be combined safely with cranial radiation therapy and intrathecal MTX. This approach for CNS leukemia, however, needs to be combined with additional treatments to eradicate residual disease in extraneurologic compartments.