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.

Dopamine and the depressant action of morphine on stimulated guinea-pig ileum.

1 Morphine reduces the amplitude of the contractions induced by electrical stimulation, in the myenteric plexus-longitudinal muscle preparation of guinea-pig ileum. Dopamine and apomorphine have the same effect but at much higher concentrations. 2 Dopamine, at concentrations lower than those which would normally be inhibitory, partially reverses the depressant effect of morphine. 3 Pre-treatment of guinea-pigs with 6-hydroxydopamine results in a slight supersensitivity of innervated longitudinal muscle preparations to dopamine and has no effect on morphine activity. 4 Naloxone antagonizes the depressant effect of morphine but not that of dopamine or apomorphine. 5 The response of theileum preparation to morphine is not affected by phentolamine or propranolol; the effect of dopamine, however, is abolished by alpha-adrenoceptor blockade.

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.

Development of tolerance to the antinociceptive effect of mescaline intraventricularly administered to rabbits.

Some effects of intraventricular injection of mescaline are examined in conscious rabbits. By means of electrical stimulation of the tooth pulp it is shown that an acute treatment with 70, 100, 150 mug/kg of mescaline elicits analgesia, the intensity of which is dose-dependent: with daily administration of 100 mug/kg for 5 days a complete tolerance develops to the antinociceptive effect. A tolerance also develops to the behavioral effects of mescaline after repeated administrations, with the exception of the stuporous state, a symptom which, on the contrary, is accentuated as the treatment proceeds. An EEG arousal is induced in the rabbit by acutely administered mescaline; the chronic treatment (100 mug/kg) makes the return of voltage to original levels progressively slower. Finally, the confrontation of certain of the mescaline-induced effects with those of morphine suggests some biochemical and neural patterns common to the 2 drugs.

Cross tolerance to antinociception elicited by intracerebroventricular administration of mescaline and morphine to rabbits, and EEG correlates.

Tolerance and cross tolerance to the antinociceptive effect of equipotent doses of morphine (10 mug/kg) and mescaline (100 mug/kg) are shown in the rabbit after their repeated intracerebroventricular administration. The recording of the electrical activity of different brain areas indicates that a partial tolerance also develops to the EEG effects in animals undergoing chronic treatment with mescaline. The comparison of certain of the mescaline-induced effects with those of morphine suggests that some biochemical and neural patterns are common to the 2 drugs.

Effects of Met-enkephalin on body temperature of normal and morphine-tolerant rats.

The endogenous opioid met-enkephalin intraventricularly adminstered to the rat at the dose of 100 microgram raised rectal temperature, whereas 400 microgram of the pentapeptide caused a diphasic effect, i.e., hypothermia followed by hyperthermia. Met-enkephalin was ineffective when administered i.p. The effects on temperature were substantially similar to those elicited, for both routes of administration, by morphine, which may either raise or lower rat temperature depending on the dose. More naloxone was required to antagonize thermic effects of met-enkephalin than morphine. Finally, there was a lack of effects on temperature for met-enkephalin centrally administered to morphine-tolerant animals, thus providing further evidence, in vivo, of cross tolerance between opiates and naturally occurring ligands of opiate 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.

Intestinal effect and analgesia: evidence for different involvement of opioid receptor subtypes in periaqueductal gray matter.

Periaqueductal gray matter (PAG) has been shown to be one of the sites in the central nervous system where microinjections of morphine strongly inhibit intestinal transit. To investigate the nature of opioid receptor populations involved in this central effect, selective opioid agonists, FK 33824 for mu, DALA for delta, dynorphin for kappa and tentatively beta-endorphin for epsilon, were microinjected in all PAG areas previously identified as morphine-sensitive for intestinal inhibition. The PAG-induced inhibition of intestinal transit appears to be mediated mainly by mu receptors and possibly by epsilon receptors. kappa and delta receptors seem not to be involved.

Toxicity and distribution of 2-methyl-4-chlorophenoxyacetic acid (MCPA) in developing chick embryos.

The toxicity of the herbicide Erbitox E30, a commercial formulation of 2-methyl-4-chlorophenoxyacetic acid (MCPA) containing 28% MCPA as sodium-potassium salt and 72% of unknown ingredients, was tested on chick embryos. Sterile aqueous solutions of MCPA were injected into the air chamber at doses of 0, 1.5, 3.0, 6.0, 9.0, or 10.5 mg/egg on day 0 or on day 4 of incubation. The mortality rate for the embryos treated on day 0 of incubation was high in the first 5 days, low from 5-12 days and again increased by 15 days. The 15-day LD50 was 4.4 mg/egg (95% C.I. 3.7-5.3 mg/egg). HPLC analysis of albumen and yolk showed that concentrations of MCPA in the albumen were detectable at 5 min, highest at 7 days and markedly diminished by 14 days of incubation; a significantly lower concentration of MCPA was found in the yolk throughout the incubation period, except at 14 days when the yolk concentration was 4 times higher than the albumen concentration. At 15 days of incubation, MCPA was evenly distributed in the tissues of the embryo. MCPA was more toxic to 4-day embryos; concentrations above 6.0 mg/egg were lethal to all embryos within the first week of incubation. The 15-day LD50 for treatment on day 4 of incubation was 2.8 mg/egg (95% C.I. 2.5-3.2 mg/egg). The liver was affected by treatment with MCPA, being green in treated embryos. However, histological examination revealed few changes in the liver parenchyma.

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.

Interactions of manganese with human brain glutathione-S-transferase.

Chronic exposure to manganese-laden dusts induces, in humans and lower primates, neurological disorders with clinicopathological features that resemble idiopathic Parkinson's disease. As many authors have suggested, manganese neurotoxicity could be related to the capability of this metal to increase catechol autoxidation in catecholaminergic neurons, therefore increasing the formation of toxic compounds such as peroxides, superoxides, free radicals, and semi-orthoquinones. Oxidative stresses and consequent neuronal damage could then occur if physiological scavenger mechanisms fail in their detoxifying action. We here report that manganese chloride weakly inhibits, in a dose-dependent way by a reversible competitive mechanism, human brain glutathione-S-transferases possibly suggesting that manganese intoxication could cause intraneuronal accumulation of cytotoxic compounds. We also report that both 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, a neurotoxin known to induce in man Parkinson-like syndromes, and one of its metabolites 1-methyl-4-phenylpyridinium failed to decrease glutathione-S-transferase activity.

Isoelectrofocusing on flat bed for determining and separating beta-endorphin.

Camel synthetic beta-endorphin focused in three bands by isoelectrofocusing on 1 mm polyacrylamide thin gel. All the bands have opioid activity, measured on guinea pig ileum, and radioimmunologically react with beta-endorphin antiserum. Since beta-endorphin from rat pituitary gland, particularly from the neurointermediate lobe, also focused in several bands, we hypothesized that the camel peptide occurs in different conformations. A quick, simple technique based on histoelectrofocusing is proposed as a good approach to separating and measuring beta-endorphin from rat pituitary lobes. The method gives very high recovery.

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.

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.

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.

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.

[Senile liver and its repercussions on cerebral activity]. / Fegato senile e sua ripercussione sull'attività cerebrale.

On the basis of previous personal data regarding glycidic, lipidic and protein metabolism in groups of young-old-cirrhotic and hypoglycaemic subjects, a correlative study has been carried out for the purpose of highlighting dependence situations between reduced cerebral activity in the elderly and liver-dependent metabolic changes. Specifically as regards protein metabolism, it is shown that all amino acids dependent on the senescence process (glutammic acid, cystine, thyrosine, histidine, threonine, serine) are related to those involved in altered consciousness states (hepatic and hypoglycaemic coma). It is thus possible that in the elderly too there may be some correlation between liver function deficiency and senile encephalopathy.

[Use of discriminating function in the study of lipid metabolism in normal subjects of various ages]. / Uso della funzione discriminante nello studio del metabolismo lipidico in soggetti normali di età diversa

Analysis of the discriminating function was applied to 15 plasma lipid parameters: palmitic, palmitoleic, stearic, oleic and linoleic acids, total NEFA, total lipids, triglycerides, phospholipids, total cholesterol, alpha and beta lipoproteic cholesterol, and lipoproteic alpha: beta, cholesterol: triglycerides and cholesterol: phospholipids ratios, in 84 healthy, normosomic subjects, divided into young, presenile and aged. Highly significant discrimination was obtained between all three groups. The coefficients of discrimination of all the variables employed in individual comparisons are illustrated. A brief account is given of the importance of biohumoral data in the detection of aging, and of the theoretical and practical utility of an investigation of this kind.