Genotoxic effects of the fungicide thiophanate-methyl on Podarcis sicula assessed by micronucleus test, comet assay and chromosome analysis.

The increasing use of pesticides in modern agriculture has raised the need to evaluate their potential threat to animal and human health. In the present study, the genotoxic effects of environmentally relevant exposure to the fungicide thiophanate-methyl (TM) were assessed in the lizard Podarcis sicula (Reptilia, Lacertidae) using micronucleus test, chromosome aberration analysis and single-cell gel electrophoresis (comet) assay. The number of micronuclei increased significantly with exposure time in lizard specimens exposed to 1.5% TM for 30-40 days. In situ hybridization with the specific HindIII centromeric satellite was positive in 18.7% of the micronuclei observed, suggesting an aneugenic effect of TM during mitosis. DNA damage, evaluated by the comet assay, documented a significant gain in comet length in relation to exposure time that was paralleled by a reduction in head size. Finally, cytogenetic analysis showed a significant increase in chromosome aberrations in exposed animals compared with controls. Our data suggest that long-term TM exposure induces a genomic damage that is positively correlated to exposure time. If such genotoxic effects arise so clearly in an ectothermal vertebrate, such as P. sicula, prolonged exposure TM must be considered as a cytogenetic hazard.

Apoptosis and human placenta: expression of proteins belonging to different apoptotic pathways during pregnancy.

Apoptosis occurs during normal development and it is important for the right balance between the loss of old, non-functional cells and the formation of new cells in different organs and tissues. Apoptosis is triggered by different cell-type-specific signals which involve several pathways, such as mitochondrial and receptor-mediated pathways, resulting in caspase cascade activation. Several studies have suggested that apoptosis plays an important role in the normal development, remodelling and aging of the placenta. Moreover, it has been demonstrated that apoptosis increases as pregnancy progresses suggesting that it is a normal physiological phenomenon throughout gestation. In the last years, it has been hypothesized that the process known as syncytial fusion is directly or indirectly related to apoptotic events. In particular, it has been suggested that cytotrophoblast cells early express most important apoptotic proteins that translocate in the syncytiotrophoblast with the fusion. This suggests that apoptosis has a central role in the villous trophoblast turnover. Recently, another important involvement of apoptotic processes in human placenta has been demonstrated. In particular, the apoptosis, mainly through Fas-FasL or TRAIL-R-TRAIL signalling, may be a defence mechanism against rejection of the fetal allograft by maternal immune system. The whole data suggest that regulation of apoptotic events is important to allow a correct development, differentiation and function of the placenta throughout pregnancy and that an unbalance of this process leads to severe pathologies such as pre-eclampsia and intrauterine growth retardation. Therefore, due to its extensive proliferation and invasive properties, the placenta mimics a malignant tumor and represents an interesting model to evaluate those processes leading to carcinogenesis.

Morphology and distribution of chromaffin cells in the adrenal gland of Cordylidae (Reptilia, Sauria): a comparative study.

The distribution of the adrenaline and noradrenaline chromaffin cells in the adrenal glands of 10 members of the family Cordylidae have been examined. In the genus Gerrhosaurus, all the catecholamine cells lie on the surface of the adrenal gland, forming a continuous envelope of one or two layers of cells that mainly contain noradrenaline (NA). In the genus Platysaurus, the chromaffin envelope is intermittent. There are relatively large tracts of interspersed interrenal tissue containing some adrenaline cells (A). Islets of chromaffin cells are scattered between these interrenal tracts. In the genus Pseudocordylus and the genus Cordylus, the superficial chromaffin cells tend to gather into a multilayered dorsal mass, containing mainly NA cells. Inside the interrenal parenchyma, there are always numerous chromaffin islets, containing mainly A cells.

Effects of adrenaline administration on the interrenal gland of the newt, Triturus carnifex: evidence of intraadrenal paracrine interactions.

The existence of paracrine control of steroidogenic activity by adrenochromaffin cells in Triturus carnifex was investigated by in vivo adrenaline (A) administration. The effects were evaluated by examination of the ultrastructural morphological and morphometrical features of the tissues as well as the serum levels of aldosterone, noradrenaline (NA), and adrenaline. In March and July, adrenaline administration reduced aldosterone release (from 187.23 +/- 2.93 pg/ml to 32.28 +/- 1.85 pg/ml in March; from 314.60 +/- 1.34 pg/ml to 87.51 +/- 2.57 pg/ml in July) from steroidogenic cells. The cells showed clear signs of lowered activity: they appeared full of lipid, forming large droplets. Moreover, adrenaline administration decreased the mean total number of secretory granules in the chromaffin cells in July (from 7.74 +/- 0.74 granules/microm(2) to 5.14 +/- 1.55 granules/microm(2)). In this period T. carnifex chromaffin cells contain almost exclusively NA granules (NA: 7.42 +/- 0.86 granules/microm(2); A: 0.32 +/- 0.13 granules/microm(2)). Adrenaline administration reduced noradrenaline content (4.36 +/- 1.40 granules/microm(2)) in the chromaffin cells, enhancing noradrenaline secretion (from 640.19 +/- 1.65 pg/ml to 1030.16 +/- 3.03 pg/ml). In March, adrenaline administration did not affect the mean total number of secretory vesicles (from 7.24 +/- 0.18 granules/microm(2) to 7.25 +/- 1.97 granules/microm(2)). In this period the chromaffin cells contain both catecholamines, noradrenaline (3.88 +/- 0.13 granules/microm(2)), and adrenaline (3.36 +/- 0.05 granules/microm(2)), in almost equal quantities; adrenaline administration reduced adrenaline content (1.74 +/- 0.84 granules/microm(2)), increasing adrenaline release (from 681.27 +/- 1.83 pg/ml to 951.77 +/- 4.11 pg/ml). The results of this study indicate that adrenaline influences the steroidogenic cells, inhibiting aldosterone release. Adrenaline effects on the chromaffin cells (increase of noradrenaline or adrenaline secretion) vary according to the period of chromaffin cell functional cycle. The existence of intraadrenal paracrine interactions in T. carnifex is discussed.

Effects of noradrenaline administration on the interrenal gland of the newt, Triturus carnifex: evidence of intra-adrenal paracrine interactions.

The existence of paracrine control of steroidogenic activity by adrenochromaffin cells in Triturus carnifex was investigated by in vivo noradrenaline (NA) administration. The effects were evaluated by examination of the ultrastructural morphological and morphometrical features of the tissues as well as the serum levels of aldosterone, NA, and adrenaline (A). In March and July, NA administration increased aldosterone release (from 187.23 +/- 2.93 pg/ml to 878.31 +/- 6.13 pg/ml in March; from 314.60 +/- 1.34 pg/ml to 622.51 +/- 2.65 pg/ml in July) from steroidogenic cells. The cells showed clear signs of stimulation, as evidenced by a strong reduction of lipid content. Moreover, NA administration decreased the mean total number of secretory vesicles in the chromaffin cells in March (from 7.24 +/- 0.18 granules/micro2 to 5.57 +/- 1.88 granules/micro2) and July (from 7.74 +/- 0.74 granules/micro2 to 6.04 +/- 1.13 granules/micro2). In March, however, when T. carnifex chromaffin cells contain both catecholamines, NA (3.88 +/- 0.13 granules/micro2) and A (3.36 +/- 0.05 granules/micro2) in almost equal quantities, NA administration reduced A content (1.29 +/- 1.04 granules/micro2) in the chromaffin cells, enhancing adrenaline secretion (from 681.27 +/- 1.83 pg/ml to 1527.02 +/- 2.11 pg/ml). In July, when the chromaffin cells contain almost exclusively NA granules (NA: 7.42 +/- 0.86 granules/micro2; A: 0.32 +/- 0.13 granules/micro2), NA administration reduced the number of NA granules (5.45 +/- 1.10 granules/micro2), thereby increasing noradrenaline release from the chromaffin cells (from 640.19 +/- 1.65 pg/ml to 1217.0 +/- 1.14 pg/ml). The results of this study indicate that NA influences the steroidogenic cells, eliciting aldosterone release. Noradrenalin effects on the chromaffin cells, increase of NA or A secretion, according to the period of chromaffin cell functional cycle, may be direct and/or mediated through the steroidogenic cells. The existence of intra-adrenal paracrine interactions in T. carnifex is discussed.

Release of aldosterone and catecholamines from the interrenal gland of Triturus carnifex in response to adrenocorticotropic hormone (ACTH) administration.

The influence of adrenocorticotropic hormone (ACTH) on the interrenal gland of Triturus carnifex was investigated by in vivo administration of synthetic ACTH. The effects were evaluated by examination of the ultrastructural morphological and morphometrical features of the tissues as well as the circulating serum levels of aldosterone, noradrenaline (NA), and adrenaline (A). In June and November, ACTH administration increased aldosterone release (from 281.50 +/- 1.60 pg/ml in carrier-injected newts to 597.02 +/- 3.35 pg/ml in June; from 187.45 +/- 1.34 pg/ml in carrier-injected animals to 651.00 +/- 3.61 pg/ml in November). The steroidogenic cells showed clear signs of stimulation, together with a reduction of lipid content in June and an increase of lipid content in November. Moreover, ACTH administration decreased the mean total number of secretory vesicles in the chromaffin cells in June (from 7.73 +/- 0.60 granules/microm2 in carrier-injected animals to 5.91 +/- 0.40 granules/microm2) and November (from 7.78 +/- 0.75 granules/microm2 in carrier-injected newts to 4.87 +/- 0.40 granules/microm2). In June, however, when T. carnifex chromaffin cells contain almost exclusively NA granules (NA: 7.42 +/- 0.86 granules/microm2; A: 0.32 +/- 0.13 granules/microm2), ACTH decreased NA content (5.52 +/- 0.32 granules/microm2) increasing NA release (from 639.82 +/- 3.30 pg/ml in carrier-injected to 880.55 +/- 4.52 pg/ml). In November, when both catecholamines, NA (3.92 +/- 0.34 granules/microm2) and A (3.84 +/- 0.33 granules/microm2), are present in the chromaffin cells, ACTH administration reduced A content (1.02 +/- 0.20 granules/microm2), enhancing adrenaline secretion (from 681.30 +/- 3.62 pg/ml in carrier-injected newts to 1,335.73 +/- 9.03 pg/ml). The results of this study indicate that ACTH influences the steroidogenic tissue, eliciting aldosterone release. The effects on the chromaffin tissue, increase of NA or A secretion, according to the period of chromaffin cell functional cycle, may be direct and/or mediated through the increase of aldosterone release. Finally, the lack of an increase of A content in the chromaffin cells, or A serum level, following ACTH administration in June might suggest an independence of PNMT enzyme on corticosteroids.

Immunohistochemical localization of NPY, VIP and 5-HT in the thyroid gland of the lizard, Podarcis sicula.

The thyroid gland of the lizard Podarcis sicula was immunohistochemically studied in adult male specimens using specific antibodies against NPY, VIP and 5-HT and the avidin-biotin peroxidase complex (ABC) procedure to localize the three peptides. Fine beaded VIP-immunoreactive nerve fibers ran between the follicles, and VIP-immunoreactivity was evenly distributed in the apical cytoplasm of follicular cells. NPY-immunoreactive fibers were found around the follicles, and, in the cells, immunoreactivity was localizated only in the cellular apices. Immunoreactivity to 5-HT was observed in the colloid, with a concentration in the follicular lumen exceeding that in the follicular cells. In fact, most follicles showed immunoreactivity in the cytoplasmic bridges formed between the apical portion of the follicular cells and the colloid.

Inhibin in the testis and adrenal gland of the male lacertid, Podarcis sicula Raf.: a light immunocytochemical study.

Inhibin is a glycoproteic hormone mostly produced by the gonads. Through a feedback at the pituitary level, it selectively inhibits the release of follicle-stimulating hormone. In mammals, inhibin has been found also in some extragonadal tissues such as placenta, pituitary, adrenal, spleen, kidney, brain and spinal cord. At present, no information is available about the existence of inhibin in reptiles. The aim of the present work is to localise, through immunocytochemical methods, the sites of inhibin production in male lizards during the main phases of the reproductive cycle: the culmination phase (April-June), the early regressive phase (early July), the maximal regressive phase (August) and the winter stasis (January). In the testis, immunostaining is mainly localised in the Leydig cells during the early regressive phase, while it is observed in the Sertoli cells during the maximal regressive phase. In the epididymis, the immunostaining is present only during the reproductive period at the level of secreting cells and inside its ducts. In the adrenal gland, after immunostaining, both chromaffin and steroidogenetic tissues are inhibin-positive during the whole spermatogenetic cycle, though with variable intensity throughout the year: cross-reaction appears more evident from January to April (winter stasis and culmination phase) and weaker in June. However, in captive animals, the reaction persists in chromaffin cells, but disappears in steroidogenetic cells. The functional meaning of the presence of inhibin as a factor in the local regulation of spermatogenesis is discussed.

Effects of 4-nonylphenol on proliferation of AGS gastric cells.

OBJECTIVES: Nonylphenol (NP) is present ubiquitously in both aquatic and terrestrial environments. This compound is considered an important endocrine disruptor and its toxic/oestrogenic activity has been investigated in a number of in vitro and in vivo studies. Human exposure to NP may occur by cutaneous absorption, ingestion of contaminated food or water and inhalation. Moreover, while the cytotoxic effects of NP are known and studied, its effects on cell death and related mechanisms are not known. Our group decided to investigate NP effects on a gastric epithelial cell line (AGS), in particular NP effects on AGS cell cycle and apoptosis. MATERIALS AND METHODS: Cell cycle was analysed by flow cytometry, p21 and p27 induction, and apoptosis was analysed by flow cytometry and annexin-V assays, Fas, Fas-L, caspase 8 and caspase 3 activation. RESULTS: We have demonstrated that NP affected cell cycle and apoptosis in a time- and dose-dependent manner, reaching the most notable effect at concentration of 10(-7)m, for 48 h. Flow cytometry revealed that treatment with 10(-7) m NP led to accumulation of cells at G(2) /M transition and increased percentage population of apoptotic cells. Results have shown that NP at concentration 10(-7) m induced marked increase in Fas and Fas-L expression. Moreover, 10(7) m NP induced activation of caspases 8 and 3, but not activation of caspase 9. CONCLUSIONS: The results reported suggest that gastric cells can be useful as screening population for appropriate study of developmental toxicity.

Annual cycle of the chromaffin cells of Triturus cristatus.

Morphology of the chromaffin cells of Triturus cristatus during a complete annual cycle has been investigated. General ultrastructural characteristics are similar for all chromaffin cells, including numerous small mitochondria, well-developed Golgi apparatus and rough endoplasmic reticulum with short cisternae. The primary difference among cells is the type of the chromaffin granules they posses. These are of two kinds: adrenalin (A) and noradrenalin granules (NA). Both types are simultaneously present in the chromaffin cells but with different ratios during the year. During December-January and May-August, NA granules largely prevail, while in September-November and February-April, A and NA granules are present in about equal quantities. The total quantity of catecholamine granules, however, is relatively constant throughout the year. These findings suggest that T. cristatus has a single type of chromaffin cell, the granule content of which varies according to different functional states. The catecholamines are apparently discharged by exocytosis.

Effects of ketamine on the catecholamine containing cells of the adrenal gland of the lizard Podarcis s. sicula Raf.

The authors studied the effects of ketamine on the morphology of the adrenal gland of Podarcis s. sicula Raf.. Low doses of the drug failed to anesthetize the lizards and did not cause significant changes in the morphology of the adrenal cells. Higher doses of ketamine caused deep anesthesia and marked depletion of catecholamine granules. The results support the theory that ketamine acts on the peripheral autonomic nervous system and sympathetic ganglia.

The endocrine pancreas of Podarcis s. sicula Raf.: an immunocytochemical study at light and electron microscopic levels.

The endocrine pancreas of Podarcis s. sicula Raf. was investigated at light and electron microscopic levels using immunocytochemical methods. It was constituted by endocrine islets, which were concentrated more in the splenic than in the duodenal region, and never formed large clusters. At the light and electron microscope, with the peroxidase-antiperoxidase reaction (PAP) and the colloidal gold technique, four cell types were identified: glucagon-positive A-cells, insulin-positive B-cells, somatostatin-positive D-cells and pancreatic polypeptide-positive F- or PP-cells. F- or PP-cells showed a different regional distribution, being absent or very rare in the splenic lobe of the pancreas and numerous in the duodenal lobe, where they were seldom found in the islets, but were scattered in the exocrine parenchyma. The functional meaning of F-cells is discussed. The E.M. observations were carried out at the CIRUB (Centro Interdipartimentale di Ricerca sulle Ultrastrutture Biologiche) of the University of Naples.

The endocrine pancreas of Triturus cristatus: an immunocytochemical study.

The endocrine pancreas of Triturus cristatus carnifex was studied with the aid of immunocytochemical methods, showing cells immunoreactive to anti-insulin serum (B cells), a small population of cells immunoreactive to anti-glucagon serum only (A cells), rare cells positive to anti-PP serum only (PP or F cells), and a larger population of cells immunoreactive both to anti-glucagon and to anti-PP sera. B cells lied in the core of the islet, while the A/PP cells were located at the periphery, forming digitations extending into the exocrine parenchyma. D cells were present in small number in the islet while they were more numerous scattered in the exocrine parenchyma. A/PP cells as well as D cells showed one or two long cytoplasmic extensions often in contact with blood vessels.

Endocrinologic effects of the anaesthetic propofol.

Because of the similarities of the adrenal glands of mammals and of the lizard Podarcis s. sicula, the latter has already been the subject of various studies on the effects of Propofol and other anaesthetics. Because a relationship between the activities of the thyroid and adrenal glands of this species has been demonstrated, the authors administered Propofol to a species of lizard to investigate its effects on the thyroid gland. Propofol inhibited thyroid activity, promoted steroid synthesis, and caused the contemporaneous appearance of both adrenaline and noradrenaline granules in the cytoplasm of the chromaffin cells. These results suggest that inhibition of the activity of the thyroid gland is secondary to the action of Propofol on the adrenal gland.

Possible relationship between the activity of the adrenal gland and the subcommissural organ in the lizard Lacerta s. sicula Raf. Effects of ACTH Administration during winter.

In order to study the possible functional relationship between the adrenal gland and the subcommissural organ (SCO) in the lizard Lacerta s. sicula Raf., ACTH was administered to some specimens of this species in January when both the adrenal gland and the subcommissural organ have a very low activity. In comparison to untreated controls, the adrenals of animals treated with ACTH showed clear signs of stimulation, presenting enlarged blood vessels, very few lipid droplets, numerous polymorphic mitochondria and abundant tubular smooth endoplasmic reticulum. In addition, a distinct increase in secretory material was observed in the subcommissural cells of specimens treated with ACTH. These cells showed large cisternae of the rough endoplasmic reticulum filled with granular material in the basal region, numerous secretory granules of two types in the paical region and a reduced number of microvilli on the free cell surface. These findings, together with the results of preceding studies, lead the authors to the consideration that steroid hormones might play a role in the regulation of the secretory activity of the SCO.

An immunocytochemical study of the endocrine pancreas in three genera of lacertids.

The comparative morphology of the endocrine pancreas was studied in 11 species of lacertids. Four major cell types were identified immunocytochemically in the endocrine pancreas: glucagon-immunoreactive A-cells, insulin-immunoreactive B-cells, somatostatin-(SRIF)-immunoreactive D-cells, and pancreatic polypeptide(PP)-immunoreactive F-cells. Different distributions of the four cell types were seen in the endocrine tissue within the exocrine parenchyma. F-cells were rare or absent in the splenic lobe and abundant in the duodenal lobe, in which they were usually widespread in the exocrine parenchyma and rarer in the islets. The other three cell types were always present in the islets. The central core consisted of B- and A-cells, with B-cells predominating. The peripheral mantle was formed by A-cells and less abundant D-cells. Rare D-cells were also found in the central core. D- and F-cells showed projections often closely associated with capillaries. The observed arrangements in islets and isolated cells may represent an endocrine network that, in addition to systemic actions, may regulate exocrine function in a paracrine fashion.

Immunocytochemical localization of substance P in the adrenal gland of Podarcis sicula (Reptilia, Lacertidae): evidence for its involvement in the modulation of adrenal activity.

The occurrence of substance P (SP) immunoreactivity was investigated in the adrenal gland of the lizard Podarcis sicula by ABC immunocytochemical technique: SP-immunoreactivity was present in both adrenaline and noradrenaline cells, in ganglion cells and nerve fibers in the connective capsule surrounding the gland. The involvement of substance P in the modulation of pituitary-interrenal axis was studied in vivo by intraperitoneal injections of SP. The effects were estimated by means of the morphological and morphometrical features of the tissues, as well as the plasma levels of adrenocorticotropic hormone (ACTH), corticosterone and catecholamines, adrenaline and noradrenaline. Substance P (0.07 mg/100 g body wt) decreased ACTH plasma levels and raised corticosterone release from steroidogenic tissue, that showed clear signs of stimulation. In the chromaffin tissue, the decrease in the number of noradrenaline cells, and the increase in the number of adrenaline cells, lowered numeric noradrenaline/adrenaline cell ratio. Moreover, an increase in adrenaline plasma level and a decrease in noradrenaline plasma level were found. The results suggest that (1) also in Reptiles as in other Vertebrates, SP may affect pituitary-adrenal axis activity, and (2) the chromaffin cells may be involved in the paracrine control of steroidogenic activity.

Vasoactive intestinal peptide stimulation modulates the expression of Bcl-2 family members in the adrenal gland of the lizard Podarcis sicula.

The adrenal gland of the lizard Podarcis sicula is formed by a dorsal ribbon of chromaffin cells, generally defined as medullary tissue, arranged along a central part of steroidogenic cells considered as cortical tissue. These two tissues produce catecholamines and steroids as part of the hypothalamo-hypophyseal-adrenal gland axis. Recent studies have demonstrated that Podarcis sicula adrenal gland is not only under hypothalamo-hypophyseal axis control but that several peptides may influence the physiological activity of the gland; among these, vasoactive intestinal peptide is able to enhance strongly both catecholamine and steroid hormone production. The aim of the present study was to verify whether vasoactive intestinal peptide administration could become deleterious. For this reason, we monitored the pattern of expression of two members of the Bcl-2 family, Bcl-2 and Bax, in control and vasoactive intestinal peptide treated specimens. Furthermore, we also tested if peptide treatment induces apoptosis by TUNEL assay.

Shift from noradrenaline to adrenaline production in the adrenal gland of the lizard, Podarcis sicula, after stimulation with vasoactive intestinal peptide (VIP).

The aim of this study was to investigate the distribution and function of VIP in the adrenal gland of the lizard, Podarcis sicula. We have shown by immunohistochemistry that VIP fibers were localized exclusively around clusters of chromaffin cells in the dorsal ribbon of the lizard adrenal gland. Moreover, a strong positivity for this peptide was observed within ganglial cells and within most chromaffin cells of the gland. To investigate the effects of VIP on the adrenal gland, we have treated lizards with several doses of this peptide and we have shown that injections of exogenous VIP increased plasma levels of catecholamines and corticosteroids, but not of ACTH. This probably suggests a direct effect of VIP on the control of adrenal hormone secretion without the involvement of the hypothalamo-hypophyseal axis. Our results also establish that the increased levels of the hormones were modulated in a time- and dose-dependent manner. Therefore, our morphological studies showed a clear increased function of steroidogenic cells. In the medullary region, VIP administration induced not only a functional enhancement of adrenaline release from adrenergic cells, but also a shift of noradrenaline cells to adrenaline ones.