D-Chiro-Inositol and Myo-Inositol Induce WAT/BAT Trans-Differentiation in Two Different Human Adipocyte Models (SGBS and LiSa-2).

White adipose tissue/brown adipose tissue trans-differentiation is one of the main study targets for therapies against obesity and metabolic diseases. In recent years, numerous molecules able to induce such trans-differentiation have been identified; however, their effect in obesity therapies has not been as expected. In the present study, we investigated whether myo-inositol and its stereoisomer D-chiro-inositol could be involved in the browning of white adipose tissue. Our preliminary results clearly indicate that both, at 60 µM concentration, induce the upregulation of uncoupling protein 1 mRNA expression, the main brown adipose tissue marker, and increase mitochondrial copy number as well as oxygen consumption ratio. These changes demonstrate an activation of cell metabolism. Therefore, our results show that human differentiated adipocytes (SGBS and LiSa-2), assume the features typical of brown adipose tissue after both treatments. Furthermore, in the cell lines examined, we proved that D-chiro-inositol and myo-Inositol induce an increase in the expression of estrogen receptor mRNAs, suggesting a possible modulation by these isomers. We also found an increase in the mRNA of peroxisome proliferator-activated receptor gamma, a very important target in lipid metabolism and metabolic diseases. Our results open new opportunities for the use of inositols in therapeutic strategies to counteract obesity and its metabolic complications.

Cow and Ewe Cheeses Made with Saffron: Characterization of Bioactive Compounds and Their Antiproliferative Effect in Cervical Adenocarcinoma (HeLa) and Breast Cancer (MDA-MB-231) Cells.

Saffron is a widespread consumed spice containing many phytochemicals. It is often used in dairy technologies to enhance color and flavor of cheeses, but it is also known for its several therapeutic effects, as well as its antiproliferative and anticancer properties. In this study High Performance Liquid Chromatography was used to characterize saffron bioactive compounds in cow and ewe cheeses made with saffron, and the antiproliferative effect of the crocin-rich extracts from cheeses was investigated on different cellular lines (CaCo2, MDA-MB-231 and HeLa) by MTT assay. Crocins were observed in all cheese samples, with the total content ranging between 0.54 and 30.57 mg trans-4-GG/100 g cheese, according to the different cheese making process. Picrocrocin was detected in no cheese (probably due to its degradation during cheese making), while safranal was detected only in one ewe cheese (mainly due to its high volatility). HeLa and MDA-MB-231 cells were sensitive to treatment with crocin-rich extracts from cheeses, while no effect was observed on CaCo2 cells. The chemical environment of the food matrix seems to have a great influence on the crocin antiproliferative effect: the crocin-rich extracts from cheese with both high residual N/protein and fat contents showed increased antiproliferative effect compared to pure crocin (trans-4-GG), but cheeses from different milk species (type of fats and proteins) could also play an important role in modulating crocin's antiproliferative effects.

Involvement of miR-190b in Xbp1 mRNA Splicing upon Tocotrienol Treatment.

We previously demonstrated that apoptosis induced by tocotrienols (γ and δT3) in HeLa cells is preceded by Ca2+ release from the endoplasmic reticulum. This event is eventually followed by the induction of specific calcium-dependent signals, leading to the expression and activation of the gene encoding for the IRE1α protein and, in turn, to the alternative splicing of the pro-apoptotic protein sXbp1 and other molecules involved in the unfolded protein response, the core pathway coping with EndoR stress. Here, we showed that treatment with T3s induces the expression of a specific set of miRNAs in HeLa cells. Data interrogation based on the intersection of this set of miRNAs with a set of genes previously differentially expressed after γT3 treatment provided a few miRNA candidates to be the effectors of EndoR-stress-induced apoptosis. To identify the best candidate to act as the effector of the Xbp1-mediated apoptotic response to γT3, we performed in silico analysis based on the evaluation of the highest ∆ in Gibbs energy of different mRNA-miRNA-Argonaute (AGO) protein complexes. The involvement of the best candidate identified in silico, miR-190b, in Xbp1 splicing was confirmed in vitro using T3-treated cells pre-incubated with the specific miRNA inhibitor, providing a preliminary indication of its role as an effector of EndoR-stress-induced apoptosis.

Supplementation with dairy matrices impacts on homocysteine levels and gut microbiota composition of hyperhomocysteinemic mice.

PURPOSE: Several studies highlighted a correlation between folic acid deficiency and high plasma homocysteine concentration, considered a risk factor for multifactorial diseases. Natural folates represent an emerging alternative strategy to supplementation with synthetic folic acid, whose effects are controversial. The present work was, therefore, performed in hyperhomocysteinemic mice to study the impact of supplementation with dairy matrices containing natural folates on plasma homocysteine levels and faecal microbiota composition. METHODS: Forty mice were divided into six groups, two of which fed control or folic acid deficient (FD) diets for 10 weeks. The remaining four groups were fed FD diet for the first 5 weeks and then shifted to a standard control diet containing synthetic folic acid (R) or a FD diet supplemented with folate-enriched fermented milk (FFM) produced by selected lactic acid bacteria, fermented milk (FM), or milk (M), for additional 5 weeks. RESULTS: Supplementation with dairy matrices restored homocysteine levels in FD mice, although impacting differently on hepatic S-adenosyl-methionine levels. In particular, FFM restored both homocysteine and S-adenosyl-methionine levels to the control conditions, in comparison with FM and M. Next generation sequencing analysis revealed that faecal microbiota of mice supplemented with FFM, FM and M were characterised by a higher richness of bacterial species in comparison with C, FD and R groups. Analysis of beta diversity highlighted that the three dairy matrices determined specific, significant variations of faecal microbiota composition, while hyperhomocysteinemia was not associated with significant changes. CONCLUSIONS: Overall, the results represent a promising starting point for the applicability of food matrices enriched in natural folates to manage hyperhomocysteinemia.

Protective Effect of Proanthocyanidins in a Rat Model of Mild Intestinal Inflammation and Impaired Intestinal Permeability Induced by LPS.

SCOPE: Intestinal dysfunction consists of a defective barrier function, which allows the influx of luminal endotoxins, thus causing intestinal inflammation. Proanthocyanidins are natural bioactive compounds that could modulate intestinal dysfunction. This study analyzes the protective effects of proanthocyanidins in a rat model of intestinal dysfunction. METHODS AND RESULTS: To investigate the preventive effects of both high dietary (75 mg kg-1 body weight) and pharmacological (375 mg kg-1 body weight) oral doses of proanthocyanidins (GSPE), rat intestinal dysfunction is induced with LPS (i.p.). In vivo intestinal permeability (ovalbumin [OVA] assay) and systemic inflammation and endotoxemia (TNF-α and LPS plasma levels) are assessed. Intestinal inflammation and oxidative stress are determined using myeloperoxidase (MPO), cyclooxygenase-2 (COX-2) activities, and reactive oxygen species (ROS) levels, respectively. Ileal gene expression of permeability/inflammatory genes is analyzed. LPS administration induces intestinal permeability, inflammation, and oxidative stress. GSPE normalizes in vivo OVA levels. In the small intestine, the GSPE treatment decreases MPO and COX-2 activities; modulates the ileum inflammatory and permeability proteins gene expression; and in the large intestine, prevents increase of ROS levels. CONCLUSIONS: Proanthocyanidins, at nutritional and pharmacological doses, prevents endotoxin-induced-intestinal inflammation, permeability, and oxidative stress in rats differentially in each intestinal section. Proanthocyanidins are nutritional-therapeutic novel candidates for preventing intestinal dysfunction.

Tocotrienols: A Family of Molecules with Specific Biological Activities.

Vitamin E is a generic term frequently used to group together eight different molecules, namely: α-, ß-, γ- and δ-tocopherol and the corresponding tocotrienols. The term tocopherol and eventually Vitamin E and its related activity was originally based on the capacity of countering foetal re-absorption in deficient rodents or the development of encephalomalacia in chickens. In humans, Vitamin E activity is generally considered to be solely related to the antioxidant properties of the tocolic chemical structure. In recent years, several reports have shown that specific activities exist for each different tocotrienol form. In this short review, tocotrienol ability to inhibit cancer cell growth and induce apoptosis thanks to specific mechanisms, not shared by tocopherols, such as the binding to Estrogen Receptor-ß (ERß) and the triggering of endoplasmic reticulum (EndoR) stress will be described. The neuroprotective activity will also be presented and discussed. We propose that available studies strongly indicate that specific forms of tocotrienols have a distinct mechanism and biological activity, significantly different from tocopherol and more specifically from α-tocopherol. We therefore suggest not pooling them together within the broad term "Vitamin E" on solely the basis of their putative antioxidant properties. This option implies obvious consequences in the assessment of dietary Vitamin E adequacy and, probably more importantly, on the possibility of evaluating a separate biological variable, determinant in the relationship between diet and health.

Tocotrienols induce endoplasmic reticulum stress and apoptosis in cervical cancer cells.

BACKGROUND: We have previously reported that γ- and δ-tocotrienols (γ- and δ-T3) induce gene expression and apoptosis in human breast cancer cells (MDA-MB-231 and MCF-7). This effect is mediated, at least in part, by a specific binding and activation of the estrogen receptor-ß (ERß). Transcriptomic data obtained within our previous studies, interrogated by different bioinformatic tools, suggested the existence of an alternative pathway, activated by specific T3 forms and leading to apoptosis, also in tumor cells not expressing ER. In order to confirm this hypothesis, we conducted a study in HeLa cells, a line of human cervical cancer cells void of any canonical ER form. RESULTS: Cells were synchronized by starvation and treated either with a T3-rich fraction from palm oil (10-20 µg/ml) or with purified α-, γ-, and δ-T3 (5-20 µg/ml). α-tocopherol (TOC) was utilized as a negative control. Apoptosis, accompanied by a significant expression of caspase 8, caspase 10, and caspase 12 was observed at 12 h from treatments. The interrogation of data obtained from transcriptomic platforms (NuGO Affymetrix Human Genechip NuGO_Hs1a520180), further confirmed by RT-PCR, suggested that the administration of γ- and δ-T3 associates with Ca2+ release. Data interrogation were confirmed in living cells; in fact, Ca-dependent signals were observed followed by the expression and activation of IRE-1α and of other molecules involved in the unfolded protein response, the core pathway coping with endoplasmic reticulum stress in eukaryotic cells, finally leading to apoptosis. CONCLUSIONS: Our study demonstrates that γ- and δ-T3 induce apoptosis also in tumor cells lacking of ERß by triggering signals originating from endoplasmic reticulum stress. Our observations suggest that tocotrienols could have a significant role in tumor cell physiology and a possible therapeutic potential.

Vitamin E δ-tocotrienol triggers endoplasmic reticulum stress-mediated apoptosis in human melanoma cells.

Malignant melanoma is the leading cause of death from skin cancer. Drug toxicity and resistance represent a serious challange for melanoma treatments. Evidence demonstrates that natural compounds may play a crucial role in cancer prevention, growth and progression. Vitamin E tocotrienols (TT) were shown to possess antitumor activity. Here, we analyzed the effects of δ-TT on melanoma cell growth and the involvement of the endoplasmic reticulum (ER) stress in this activity. The experiments were performed on human melanoma cell lines, BLM and A375. δ-TT exerted a significant proapoptotic effect on both cell lines, involving the intrinsic apoptosis pathway; importantly, this compound did not affect the viability of normal human melanocytes. In melanoma cells, δ-TT exerted its antitumor effect through activation of the PERK/p-eIF2α/ATF4/CHOP, IRE1α and caspase-4 ER stress-related branches. Salubrinal, an inhibitor of the ER stress, counteracted the cytotoxic activity of δ-TT. In vivo experiments performed in nude mice bearing A375 xenografts evidenced that δ-TT reduces tumor volume and tumor mass; importantly, tumor progression was significantly delayed by δ-TT treatment. In conclusion, δ-TT exerts a proapoptotic activity on melanoma cells, through activation of the ER stress-related pathways. δ-TT might represent an effective option for novel chemopreventive/therapeutic strategies for melanoma.

Nanomolar Caffeic Acid Decreases Glucose Uptake and the Effects of High Glucose in Endothelial Cells.

Epidemiological studies suggest that moderate and prolonged consumption of coffee is associated with a reduced risk of developing type 2 diabetes but the molecular mechanisms underlying this effect are not known. In this study, we report the effects of physiological concentrations of caffeic acid, easily achievable by normal dietary habits, in endothelial cells cultured in 25 mM of glucose (high glucose, HG). In HG, the presence of 10 nM caffeic acid was associated with a decrease of glucose uptake but not to changes of GLUT-1 membrane localization or mRNA levels. Moreover, caffeic acid countered HG-induced loss of barrier integrity, reducing actin rearrangement and FITC-dextran passage. The decreased flux of glucose associated to caffeic acid affected HG induced apoptosis by down-regulating the expression of initiator (caspase 8 and 9) and effector caspases (caspase 7 and 3) and by increasing the levels of phosphorylated Bcl-2. We also observed that caffeic acid in HG condition was associated to a reduction of p65 subunit nuclear levels with respect to HG alone. NF-κB activation has been shown to lead to apoptosis in HG treated cells and the analysis of the expression of a panel of about 90 genes related to NF-κB signaling pathway revealed that caffeic acid significantly influenced gene expression changes induced by HG. In conclusion, our results suggest that caffeic acid, decreasing the metabolic stress induced by HG, allows the activation of survival mechanisms mediated by a different modulation of NF-κB-related signaling pathways and to the activation of anti-apoptotic proteins.

Sex hormones and macronutrient metabolism.

The biological differences between males and females are determined by a different set of genes and by a different reactivity to environmental stimuli, including the diet, in general. These differences are further emphasized and driven by the exposure to a different hormone flux throughout the life. These differences have not been taken into appropriate consideration by the scientific community. Nutritional sciences are not immune from this "bias" and when nutritional needs are concerned, females are considered only when pregnant, lactating or when their hormonal profile is returning back to "normal," i.e., to the male-like profile. The authors highlight some of the most evident differences in aspects of biology that are associated with nutrition. This review presents and describes available data addressing differences and similarities of the "reference man" vs. the "reference woman" in term of metabolic activity and nutritional needs. According to this assumption, available evidences of sex-associated differences of specific biochemical pathways involved in substrate metabolism are reported and discussed. The modulation by sexual hormones affecting glucose, amino acid and protein metabolism and the metabolization of nutritional fats and the distribution of fat depots, is considered targeting a tentative starting up background for a gender concerned nutritional science.

Vitamin C supplementation modulates gene expression in peripheral blood mononuclear cells specifically upon an inflammatory stimulus: a pilot study in healthy subjects.

In order to study the effects of vitamin C supplementation on gene expression and compare its action between physiological and inflammatory conditions, a pilot study was set up utilizing microarray and qPCR technologies. Five healthy volunteers were supplemented with 1 g vitamin C (Redoxon(®)) per day for five consecutive days. Peripheral blood mononuclear cells (PBMNC) were isolated before and just after the last supplementation, and RNA was isolated for the Affymetrix gene 1.0 ST chip analysis. PBMNC were also, ex vivo, treated with LPS, and gene expression was quantified by means of a "Human NFkB Signaling" qPCR array. Only a very moderate effect on the baseline gene expression modulation was associated with vitamin C supplementation. However, in spite of the limited number of subjects analyzed, vitamin C supplementation resulted in a markedly different modulation of gene expression upon the inflammatory stimulus, specifically at the level of the MyD88-dependent pathway and of the anti-inflammatory cytokine IL-10 synthesis. This study suggests that vitamin C supplementation in healthy subjects, not selected according to a specific genetic profile, consuming an adequate amount of vitamin C, and having a satisfactory vitamin C plasma concentration at the baseline, does not result in a significant modification of gene expression profile. Under this satisfactory micronutrient status, supplementation of vitamin C is "buffered" within a homeostatic physiological equilibrium. Differently, following a second "hit" constituted of an inflammatory stimulus such as LPS, able to trigger a critical burst to the normal physiological state, the higher availability of ascorbic acid emerges, and results in a significant modulation of cell response.

Absorption, metabolism, and effects at transcriptome level of a standardized French oak wood extract, Robuvit, in healthy volunteers: pilot study.

The consumption of wine and spirits, traditionally aged in oak barrels, exposes humans to roburin ingestion. These molecules belong to a class of ellagitannins (ETs), and their only known source is oak wood. Very little is currently known about roburin bioavailability and biological activity. We reported for the first time human absorption of roburins from a French oak wood (Quercus robur) water extract (Robuvit) by measuring the increase of total phenols (from 0.63 ± 0.06 to 1.26 ± 0.18 µg GAE equiv/mL plasma) and the appearance of roburin metabolites (three different glucoronidate urolithins and ellagic acid), in plasma, after 5 days of supplementation. Robuvit supplementation induced also the increase of plasma antioxidant capacity from 1.8 ± 0.05 to 1.9 ± 0.01 nmol Trolox equiv/mL plasma. Moreover, utilizing a combined ex vivo cell culture approach, we assessed the effect of Q. robur metabolites (present in human serum after supplementation) on gene expression modulation, utilizing an Affymetrix array matrix, in endothelial, neuronal, and keratinocyte cell lines. The functional analysis reveals that Robuvit metabolites affect ribosome, cell cycle, and spliceosome pathways.

Tocotrienols activity in MCF-7 breast cancer cells: involvement of ERbeta signal transduction.

The term Vitamin E is utilized to describe eight molecules, subdivided into two groups, tocopherols and tocotrienols (TTs). It has been shown that specific TTs affect the growth of several lines of tumour cells, and that this activity is not shared by tocopherols. In agreement with these observations, a TTs-rich fraction from palm oil (PTRF) was reported to inhibit proliferation and induce apoptosis in several cancer cells. However, the molecular mechanism involved in TTs activity is still unclear. We have recently proposed that TTs pro-apoptotic activity involves estrogen receptor beta (ERbeta) signalling. In this study, we report that, in MCF-7 breast cancer cell, expressing both ERalpha and ERbeta, PTRF treatment increases ERbeta nuclear translocation, as demonstrated by immunofluorescence experiments and significantly inhibits ERalpha expression (-458.91-fold of change) and complete disappearing of the protein from the nucleus. Moreover, PTRF treatment induces ER-dependent genes expression (macrophage inhibitory cytokine-1, early growth response-1 and Cathepsin D) which is inhibited by the ER inhibitor, ICI 182.780, and induces DNA fragmentation. Finally, cDNA-array experiments suggest that the activation of specific pathways in cells treated with gamma-TT with respect to alpha-TT. Our data suggest a novel potential molecular mechanism for TTs activity.

Red wine metabolites modulate NF-kappaB, activator protein-1 and cAMP response element-binding proteins in human endothelial cells.

We have studied the effect of human serum, collected after red wine consumption (RWS), on TNF-alpha-dependent activation of transcription factors (NF-kappaB, activator protein-1 (AP-1) and cAMP response element-binding proteins) and on the expression of selected genes involved in cell adhesion or fibrinolysis processes in human primary endothelial cells (human umbilical vein endothelial cells (HUVEC)). Our data indicate that RWS containing RW metabolites, isolated after 40 min from an acute consume of wine (5 ml/kg body weight), induces nuclear translocation of NF-kappaB and AP-1 in the absence of any further stimulus. On the other hand, TNF-alpha treatment in the presence of RWS is associated with a delay in transcription factor activation and to a negative modulation on the expression of specific genes. Moreover, RWS stimulates c-jun binding to the tissue-type plasminogen activator cAMP responsive element consensus site modulating the expression of the specific gene downstream. These results confirm that RW metabolites affect the activity of different transcription factors playing an important preconditioning role in the modulation of the inflammatory pathway in endothelial cells. This is the first report on the effects of a complex food matrix, on the molecular mechanisms associated with inflammatory response in HUVEC cultured in condition that reproduces the physiological environment occurring in vivo.

A novel mechanism of natural vitamin E tocotrienol activity: involvement of ERbeta signal transduction.

Vitamin E is a generic term used to indicate all tocopherol (TOC) and tocotrienol (TT) derivates. In the last few years, several papers have shown that a TT-rich fraction (TTRF) extracted from palm oil inhibits proliferation and induces apoptosis in a large number of cancer cells. However, the molecular mechanism(s) involved in TT action is still unclear. In the present study, we proposed for the first time a novel mechanism for TT activity that involves estrogen receptor (ER) signaling. In silico simulations and in vitro binding analyses indicated a high affinity of TTs for ERbeta but not for ERalpha. In addition, in ERbeta-containing MDA-MB-231 breast cancer cells, we demonstrated that TTs increase the ERbeta translocation into the nucleus, which in turn activates estrogen-responsive genes (MIC-1, EGR-1 and cathepsin D), as demonstrated by cell preincubation with the ER inhibitor ICI-182,780. Finally, we observed that TT treatment is associated with alteration of cell morphology, DNA fragmentation, and caspase-3 activation. Altogether, these experiments elucidated the molecular mechanism underling gamma- and delta-TT effects.

The anti-inflammatory pharmacology of Pycnogenol in humans involves COX-2 and 5-LOX mRNA expression in leukocytes.

We investigated the effects of Pycnogenol supplementation on the arachidonic acid pathway in human polymorphonuclear leukocytes (PMNL) in response to an inflammatory stimulus. Pycnogenol is a standardised extract of French maritime pine bark consisting of procyanidins and polyphenolic monomers. Healthy volunteers aged 35 to 50 years were supplemented with 150 mg Pycnogenol a day for five days. Before and after the final day of supplementation, blood was drawn and PMNL were isolated. PMNL were primed with lipopolysaccharide (LPS) and stimulated with the receptor-mediated agonist formyl-methionyl-leucyl-phenylalanine (fMLP) to activate the arachidonic acid pathway and the biosynthesis of leukotrienes, thromboxane and prostaglandins. Pycnogenol supplementation inhibited 5-lipoxygenase (5-LOX) and cyclooxygenase-2 (COX-2) gene expression and phospholipase A2 (PLA2) activity. This effect was associated with a compensatory up-regulation of COX-1 gene expression. Interestingly, Pycnogenol suspended the interdependency between 5-LOX and 5-lipoxygenase activating protein (FLAP) expression. Pycnogenol supplementation reduced leukotriene production but did not leave prostaglandins unaltered, which we attribute to a decline of COX-2 activity in favour of COX-1. Here we show for the first time that Pycnogenol supplementation simultaneously inhibits COX-2 and 5-LOX gene expression and reduces leukotriene biosynthesis in human PMNL upon pro-inflammatory stimulation ex vivo.

Poly(ADP-ribosyl)ation of proteins and germ cell development in hyperthyroid rat testes.

The effect of increased serum levels of thyroid hormone (triiodothyronine, T3) on young rat testis spermatogenesis was studied by analysing molecular and morphological parameters. Hyperthyroidism was induced by either T3-treatment or 2- and 10-day cold exposure. The poly(ADP-ribosyl)ation of proteins catalysed by poly(ADP-ribose) polymerase, which is particularly active at specific stages of rat spermatogenesis, was analysed as molecular index of DNA damage and cell stress. Poly(ADP-ribose) polymerase activity rose after both T3-treatment and 2- and 10-day cold exposure, with a trend of 10-day cold-exposed rats towards control values. In all hyperthyroid rats poly(ADP-ribose) turnover, as a contribution of both poly(ADP-ribose) polymerase and poly(ADP-ribose) glycohydrolase), was enhanced with respect to euthyroid animals. Poly(ADP-ribosyl)ation of proteins occurred with long and branched polymers suggesting an increased involvement of the modification system in DNA repair. Morphological changes of germ tissue were observed in hyperthyroid rats, mainly a high reduction of mature cells in the seminiferous tubule, and evidence of germ cell apoptosis was obtained by TUNEL method. In control animals germ cell apoptosis was within physiological levels. Conversely, in hyperthyroid rats a dramatic increase in the number of TUNEL-positive cells (some spermatogonia and numerous primary spermatocytes) was found, even though the increase was lower in 10-day than in 2-day cold-exposed animals.

Spermatogenesis, epididymis morphology and plasma sex steroid secretion in the male lizard Podarcis sicula exposed to diuron.

The present study investigates the effects of diuron, a substituted urea-based herbicide, in the male lizard Podarcis sicula utilizing quantitative and qualitative morphological features of the reproductive system and endocrinological analysis. Besides the control group, lizards were divided into three groups ([a-c]) (n=6/group) and placed for 3 weeks in terraria on polluted soil substrate sprayed with 3.75 L/ha of herbicide Toterbane 50F (50% diuron). Each terrarium was supplemented either with drinking water contaminated by herbicide (i.e. 1.08 microg/mL of diuron; group [a]), or with food contaminated by herbicide (i.e. 5.4 mg of diuron; group [b]), or with drinking water and food contaminated as described above (group [c]). None of the animals exposed to the contaminant showed any signs of general toxicity or death during the course of the experiments. Severe testicular effects are evidenced in all herbicide-treated groups, although, such effects are of a greater magnitude in lizards exposed to contaminated water (groups [a] and [c]). The main degenerative changes observed include: (1) a significant decrease in the mean gonadosomatic index of 55% in group [a] (P<0.001), 21% in group [b] (P<0.01) and 34% in group [c] (P<0.001) compared with control group; (2) a significant shrinking (P<0.001) of seminiferous tubule diameter (more than 60% of the control) in groups [a] and [c], and about 18% in group [b] (P<0.01); (3) a significant decrease in the crude numbers of spermatogonia of 92% in group [a] (P<0.001), 27% in group [b] (P<0.01) and 62% in group [c] (P<0.001) compared with control group. A complete loss of meiotic and mature germ cells in groups [a] and [c], and a reduction of primary spermatocytes, secondary spermatocytes and spermatids (more than 27% of the control) and a decrease of spermatozoa (more than 90% of the control) in group [b]; and (4) an hypertrophy of interstitial connective tissue which contains numerous lymphocytes, neutrophils and monocytes. The decrease and/or loss of germ cells seems to be related to an induction of inflammation (necrosis) rather than to apoptotic processes. Indeed, this hypothesis is supported by a TUNEL-assay, which failed to reveal any apoptotic cells either in the seminiferous epithelium or in the interstitial space in the testis of all exposed groups. Also the epididymis appears affected by diuron exposure. In particular, in experimental groups [a] and [c] it is regressed with abundant connective tissue and low epithelial cells without secretory granules, whereas in group [b] it appears partially regressed, with some secretory granules still present. At the same time, an impairment of the plasma sex-hormone levels is observed in treated lizards, as evidenced by RIA analysis. Testosterone values significantly decreased by 43% in group [a] (P<0.001), 34% in group [b] (P<0.01) and 52% in group [c] compared with control group. Instead, 17beta-estradiol plasma content is undetectable in all diuron-exposed lizards. Taken together, the results presented here indicate that diuron exposure resulted in direct male reproductive toxicity and reveal that this lizard is suitable as a laboratory reptile species for toxicological investigations.

Impairment of spermatogenesis and enhancement of testicular germ cell apoptosis induced by exogenous all-trans-retinoic acid in adult lizard Podarcis sicula.

In mammals, retinoic acid is involved in the regulation of testicular function by interaction with two families of nuclear receptors, retinoic acid receptor (RAR) and retinoid X receptor (RXR). Among RAR isoforms, the testicular cells of the lizard were found to express only RARalpha (3.7 kb) and RARbeta (3.4 kb) mRNAs, as reported here. In this study, the effects of exogenous all-trans-retinoic acid (atRA) on spermatogenesis of a non-mammalian seasonal reproducer were investigated. Daily intraperitoneal injections of atRA or atRA plus testosterone (atRA+T) were given for 2 weeks to adult males of the lizard Podarcis sicula. In animals treated with atRA, the seminiferous tubules were markedly reduced in cross-area. The seminiferous epithelium collapse was responsible for a sensible reduction in the number of germ cells and disruption in normal epithelial organization. In comparison, in atRA+T-treated lizards the loss of germinal cells was significantly less. The loss of germ cells observed in both experimental groups results from an induction of apoptotic process, as revealed by TUNEL analysis. Although low in number, apoptotic germ cells were also observed in the control groups (saline- and T-treated lizard), where the main germ cells undergoing apoptosis are primary spermatocytes (most frequently) and some spermatogonia. In conclusion, it is shown here that retinoic acid has deleterious effects on lizard spermatogenesis, causing a severe depletion of seminiferous epithelium, probably via induction of apoptotic processes. These effects are not completely inhibited by simultaneous administration of testosterone, although this hormone, once injected, is able to stimulate spermatogenesis and protect germinal cells from apoptotic cell death.

Molecular cloning and characterization of the clock gene period2 in the testis of lizard Podarcis sicula and its expression during seasonal reproductive cycle.

Clock genes are known to oscillate with circadian rhythmicity in the central clock structure, the suprachiasmatic nucleus of the hypothalamus, and also in peripheral tissues. Reproduction is a peripheral activity that is strongly influenced by a circadian clock in many organisms. Most mammals that exhibit a seasonal cycle are able to decode the daily changes in light across the year and to translate these in hormonal signals that regulate reproductive cycles. Expression of many clock genes has been revealed in mouse testis, although transcription of these genes seems to be constitutive in 24 h, suggesting that these genes may play in the testis a different role with regard to the central clockwork function. The seasonal breeding lizard Podarcis sicula represents an attractive model for studying some developmental and differentiation phenomena, such as gonadal maturation, since in the adult male the testis shows a spring full activity and a complete summer regression. Experimental data seem to suggest that in lizard the environmental factors, as photoperiod and temperature, affect the endogenous elements, although the interaction mechanisms are unknown. It is known that temperature signals have a direct influence on clock processes such as transcription, translation, protein phosphorylation and degradation. In addition, most data show that the expression of circadian clock genes, such as period2, is affected by length of photoperiod. In this way, the core clockwork may also decode seasonal information. Here we report the cloning, sequencing and bioinformatic analysis of period2 gene, isolated from the testis of lizard P. sicula, and its expression both in the testis and in other tissues during the different phases of the seasonal cycle. RT-PCR assays enlighten the presence of transcript in testis, brain, heart, liver and kidney in all the phases analysed. Moreover, real time quantitative PCR assays detect a peak of per2 testicular expression during gonadal regression. Our preliminary results cannot clearly demonstrate the involvement of per2 gene in seasonal reproductive cycle of male lizard P. sicula, but its presence in the testis may suggest a role of this gene during spermatogenesis. Besides, our work can provide numerous starting points to clarify the role of per2 during seasonal reproductive cycle.