Enzo Gilardi

DEVELOPMENTAL EXPOSURE TO CHLORPYRIFOS INDUCES ALTERATIONS IN THYROID AND THYROID HORMONE LEVELS WITHOUT OTHER TOXICITY SIGNS IN CD1 MICE

Organophosphorus insecticides, as Chlorpyrifos (CPF), are widely used in agriculture and against household pests; these compounds receive an increasing consideration as potential endocrine disrupters. The aim of the present study was to examine the potential short- and long-term effects of CPF on thyroid and adrenal glands in CD1 mice following exposure at dose levels not inducing brain acetyl cholinesterase (AchE) inhibition, during gestational and/or postnatal vulnerable phases. Pregnant dams were treated with 0, 3, 6 mg/kg bw/day of CPF on gestational days 15-18. After delivery, pups were treated subcutaneously on postnatal days (PND) 11-14 with: 0, 1, 3 mg/kg bw/day of CPF. Serum thyroxin (T4), thyroid and adrenals histology and histomorphometry were evaluated in dams and in F1 mice. In dams at 6 mg/kg, decreased T4 levels and increased cell height in thyroid were observed, and adrenal histology showed a slightly increased vacuolization in the X-zone. In the F1, short-term morphological modifications (reduced follicular size at PND 2) and long-term morphological (increased necrotic follicular cells) and biochemical alterations (reduced serum T4 levels) were found at PND 150 with an apparent higher vulnerability of males. For the first time these results indicate that CPF exposure at dose levels not inducing brain AchE inhibition causes thyroid alterations in dams and in F1 CD1 mice. Thyroid may be a sensitive target to CPF developmental exposure possibly leading to long-term effects on thyroid function. Because thyroid plays a pivotal role in mammalian development, these findings can be relevant to humans.

Control mechanisms of peripheral enkephalin hydrolysis in mammalian plasma.

The protection of the adrenal-released enkephalins from enzyme hydrolysis by endogenous plasma components was studied in laboratory animals and in man. The results indicate that mechanisms active in protecting leu-enkephalin from hydrolysis are present in the plasma of all species examined. The protection seems to be due to two groups of substances, possibly of peptidic nature. The amount of protection given by these substances seems to be sufficient to play a significant role in controlling the physiological levels of leu-enkephalin released into the bloodstream.

Reproductive toxicity and thyroid effects in Sprague Dawley rats exposed to low doses of ethylenethiourea

Ethylenethiourea (ETU) is the common metabolite of the widely used ethylenebisdithiocarbamate fungicides. It is identified as Endocrine Disruptor given its ability to interfere with thyroid hormone biosynthesis by inhibiting thyroid peroxidase activity. As far as we know, no studies have been performed to assess potential effects of ETU exposure at low dose levels, i.e. below the established LOAEL and NOAEL, during critical phases of development. Therefore, the aim of the present study was to verify the short- and long-term effects on thyroid function, reproduction and development of oral exposure to ETU levels comparable to and lower than LOAEL/NOAEL in rats. Sixty dams were treated daily by gavage during pregnancy and lactation with 0, 0.1, 0.3, 1.0 mg/kg bw per day of ETU. F1 generation was similarly treated from weaning to sexual maturity. Thyroid biomarkers were analyzed in dams and in offspring. Reproductive biomarkers were analyzed in F1 rats. For the first time this study has demonstrated reproductive toxicity and hypothyroidism at a lower than LOAEL dose exposure in pregnant dams and F1 generation. Our data suggest that even low doses of ETU can interfere with thyroid homeostasis and reproductive hormone profile if exposure starts in critical stages of development.

Hydrolysis and association of leucine enkephalin to lymphomic and erythroleukaemic cell lines--II.

The relationship between the hydrolysis of labelled leucine enkephalin and the association of its radioactive label to the cells of lymphomic and erythroleukaemic cell lines have been studied using intact cells and resealed membranes obtained from these cells as models. Hydrolysis by cell enzymes and its effect on association have been analysed using protease inhibitors and non-hydrolysable enkephalin analogues. Results obtained confirm that hydrolysis of the pentapeptide is a prerequisite for association of the radiolabel to cells. The same results provide evidence of marked differences between enkephalin hydrolysis by whole cells and hydrolysis measured in the presence of resealed membranes, suggesting the existence in intact cells of proteolytic enzymes other than those bound to the membranes. The lack of reversibility of association and the intracellular localization of the radioactive label suggest that the association measured is prevailingly caused by internalization of a hydrolysis fragment, and not by binding to receptors. In order to determine the nature of the active fragment, association was measured in the presence of all four labelled N-terminal hydrolysis fragments of leu-enkephalin under conditions of nearly-total inhibition of proteolytic enzymes. Under these conditions, the label carried by Tyr, but not that carried by the other N-terminal fragments, was associated with cells. Free Tyr, furthermore, inhibits the association to cells of both labelled Tyr and leu-enkephalin. Data summarized above are consistent with the hypothesis that the radioactive label is taken up by the cells as Tyr, freed from the parent peptide by cell-related enzymes. The same data tend to exclude that a relevant fraction of the intact pentapeptide is bound to membrane receptors or that the radioactive label is carried into the cell by a N-terminal fragment other than Tyr.

Familial thyroxine-binding globulin deficiency detected in a pilot screening program for congenital hypothyroidism

During a pilot screening program for congenital hypothyroidism, performed in Italy over a three years period on 38,000 newborns, seven cases (1/5,400) of thyroxine-binding globulin (TBG) deficiency, have been detected. None of these infants was affected by any pathology or had been treated with drugs which could explain TBG deficiency as an acquired condition. Familial studies pointed out that the transmission of the defect is consistent or compatible with X-chromosome linkage.

Iodine nutritional status and thyroid effects of exposure to ethylenebisdithiocarbamates

Introduction: Italy is still characterized by a mild iodine deficiency and is among the most intensive users of chemical products for agriculture in Europe. The aim of this study was i) to evaluate thyroid effects of exposure to mancozeb, a fungicide widely used in agriculture, in a sample of Italian grapevine workers, and ii) to verify whether the iodine intake may modulate the risk of thyroid disruption due to the mancozeb metabolite ethylenthiourea (ETU). Methods: One hundred seventy‐seven occupationally exposed male workers (29 from Chianti, a mild iodine deficient area, and 148 from Bolzano an iodine sufficient province) and 74 non‐occupationally exposed male controls (34 from Chianti and 40 from Bolzano) were enrolled in the study. Serum biomarkers of thyroid function, as well as urinary iodine and ETU concentrations were assessed. Moreover all the recruited subjects underwent clinical examination and thyroid ultrasound. Results: Multivariate comparisons showed lower mean serum levels of FT4 in Chianti‐workers as compared to Bolzano‐workers. Moreover, an increased urinary iodine excretion (>250 &mgr;g/L) was more frequently found among more exposed workers (ETU>20 &mgr;g/L) than among less exposed ones and this effect was more pronounced in Chianti‐ than in Bolzano‐workers. Chianti‐workers also showed a significantly higher frequency of very low thyroid volume (≤6.0 ml) as compared to controls. Conclusions: These findings showed a mild thyroid disrupting effect due to occupational exposure to mancozeb, more pronounced in workers residing in an area characterized by a mild to moderate iodine deficiency as compared to workers residing in an area covered by a long‐lasting iodine prophylaxis program. HighlightsThyroid is vulnerable to endocrine disrupting effects due to environmental exposures.Mancozeb is a fungicide widely used in agriculture worldwide.Mancozeb is broken down into ethylenthiourea (ETU) which has anti‐thyroid activity.Iodine intake may modulate thyroid disruption due to occupational exposure to ETU.

Specificity of proteolysis inhibitors in rabbit plasma.

Hydrolysis and inhibition of hydrolysis of leucine enkephalin in Oryctolagus plasma were studied by kinetics and chromatographic techniques. By data obtained, in this species, enkephalins are degraded by the same enzymes active in other mammals: aminopeptidases, dipeptidylaminopeptidases, and dipeptidylcarboxypeptidases. At variance with data obtained in other species, where enkephalins are hydrolyzed mostly by aminopeptidases, in Oryctolagus Leu-enkephalin hydrolysis is mainly due to dipeptidylcarboxypeptidases, whereas aminopeptidases contribution is the minimum of all three enzyme groups. Comparative analyses performed in the presence and in the absence of plasma inhibitors indicate that the ability of these substances to reduce substratum hydrolysis is very limited. On the contrary, the specific hydrolysis pattern evidenced appears to originate primarily from selective inhibition of the three groups of enzymes. Results obtained appear consistent with a role of plasma inhibitors in tuning hydrolysis to specific substrata, without appreciably modifying the amount of the substratum degraded.

Enkephalin-degrading dipeptidylcarboxypeptidases in human and Cavia porcellus plasma

1. The dipeptidylcarboxypeptidases that degrade leucine enkephalin in human and guinea pig plasma were studied by kinetic and chromatographic techniques. 2. The extremely rapid degradation of enkephalins in Cavia plasma seems to be caused by both increased activity of enzymes and reduced role of inhibitors. 3. The increased role of dipeptidylcarboxypeptidases in Cavia as compared to Homo appears prevalently caused by the presence in the former species of a considerable number of very active enzymes. 4. The sum of these data indicates the existence of noticeable intraspecific differences either in peptide-degrading enzymes present in plasma, or in plasma peptides, or in both.