[Micronutrient deficiencies in osteoporosis]. / Carenze di micronutrienti nell'osteoporosi

Osteoporosis is a disease characterized by a progressive reduction of bone mass and a simultaneous deterioration of skeletal microarchitecture leading to a loss of bone strength, resulting in bone fracture as consequence of even very low traumas. Osteoporosis has only recently been accorded growing clinical and pathological importance for its impact on health. This disease, thanks to considerable increases in life expectancy, is becoming more visible and is now treated either as a serious public health issue of socio-economic importance, and as a multifactorial disease. In fact, both in women and men, osteoporosis is often associated with e hypogonadism as well as with individual traits such as genetic constitution, cytokines, sex and race, which represent non-modifiable endogenous risk factors. In addition, modifiable exogenous risk factors related to lifestyle (e.g. smoking, alcohol consumption, diet) can lead to an acceleration in the genesis of osteoporosis. This article is intended to contribute to the knowledge of exogenous risk factors in osteoporosis, with special consideration to the role of micronutrient deficiencies.

[Selenium deficiency in celiac disease: risk of autoimmune thyroid diseases]. / Carenza di selenio nella malattia celiaca: rischio di tireopatie autoimmuni.

The essential trace mineral selenium is of fundamental importance to human health. It is incorporated in the proteome in the forms of the genetically encoded amino acids selenocysteine and selenomethionine, which are the characteristic components of selenoproteins (SeP) such as glutathione peroxidases (GPx), thioredoxin reductases and iodothyronine deiodinase families. Thyroid is especially sensitive to selenium deficiency, because SeP can modify thyreocytes function by acting as antioxidants and modifying redox status and thyroid hormone metabolism. SeP are also involved in apoptosis, cell growth and modification of the action of cell signalling systems and transcription factors. Some intestinal GPx modulate apoptosis by removing the cells affected by oxidative damage preserving tissue integrity. The malfunctioning of the GPx antioxidant system in intestinal mucosa can trigger a continuous cycle of reactive oxygen species and inflammation. Selenium deficiency is a risk factor, due to the malabsorption, in celiac disease (CD) because the inflammatory damage affects the small intestine; this deficiency can modulate SeP genes expression, with consequent reiteration of inflammation and increase of mucosal damage. In active CD, overexpression of interleukin-15 (IL-15) may increase activation of effector mechanisms of epithelial damage by stimulating T helper 1 cytokine proliferation and production and intraepithelial lymphocytes cytotoxicity by protecting these lymphocytes from apoptosis. Blocking IL-15 has the potential to provide new therapeutic tools to prevent both tissue damage and complication of CD such as autoimmune thyroid diseases (AITD) where IL-15 expression is also increases. In view of the role played by SeP in apoptosis inhibition, the presence of environmental factors such as selenium deficiency can be considered an important direct factor of thyroidal damage in development of AITD.

[Reproduction, endocrine disorders and celiac disease: risk factors of osteoporosis]. / Riproduzione, disordini endocrini e malattia celiaca: fattori di rischio di osteoporosi.

In genetically predisposed individuals, celiac disease (CD) is permanent intolerance to gluten. Besides the overt enteropathy, there are clinical and subclinical forms which appear later in life; target organs include liver, thyroid, skin and reproductive systems. CD interference is related to the different concurrent genetic-environmental factors, showing multifactorial nature. CD induces malabsorption with consequent deficiencies of micronutrients essential for organogenesis, spermatogenesis and bone structure, such as vitamin D and calcium. In fact, among extraintestinal manifestations of CD, osteoporosis deserves attention because it can be a sign of silent CD. In celiac patients' serum, cytochinic imbalance related to bone loss is present; in vitro these sera act on the osteoblastic activity. The IL-1b is also present in celiac patients' relatives, confirming the genetic predisposition to its etiopathogenesis which is also regulated by endocrine-environmental factors. In females, CD acts indirectly on the bone, determining early menopause and amenorrhea. Even frequent pregnancies and long periods of lactation can bring to bone loss; in such periods, silent CD can appear, suggesting the presence of endocrine-immunology factors. In celiac males, osteoporosis presence, besides calcium and vitamin D deficiencies, is associated to growth hormone deficit and hypogonadism, which is related to hyperprolactinemia, endocrine factors which affect the reproduction. Osteoporosis is relevant among the elderly and vitamin D and calcium supplementations are important to people diagnosed with CD later in life. Thus, to prevent damages such as osteoporosis, early CD screening among people with reproductive problems is necessary.

[Celiac disease and its endocrine and nutritional implications on male reproduction]. / Malattia celiaca e implicazioni endocrine e nutrizionali nella riproduzione maschile.

The problem of the interference of celiac disease (CD) with the male reproductive system is made evident both by the recognized adverse effects on female reproduction and by the multifactorial nature of the disease. It is important to consider CD as a multifactorial condition since its diverse effects can be modulated, besides gluten, by different concurrent genetic and environmental factors. The male CD patient has a greater risk of infertility and other reproductive disturbances, as well as a greater incidence of hypoandrogenism. In this paper the problems of CD associated to endocrine disorders and to deficiencies of micronutrients are discussed. Affected males show a picture of tissue resistance to androgens. Moreover, attention should be paid to increases of FSH and prolactin; these are not associated to infertility and/or impotence, but they may indicate an imbalance at hypothalamus-pituitary level, with general effects on health: an example is the increased risk of male osteoporosis in CD patients. Hormone alterations are reversible upon start of the gluten-free diet, emphasizing the importance of early diagnosis; this should be performed in the case of clinical suspicion, e.g., unexplained hypoandrogenism. As regards nutritional aspects, the folic acid deficiency of CD can affect rapidly proliferating tissues, such as the embryo and the seminiferous epithelium. More attention should be paid to deficiencies of fat-soluble vitamins, such as A and E, observed in CD. Vitamin A is important for Sertoli cell function as well as for early spermatogenetic phases. Vitamin E supports the correct differentiation and function of epidydimal epithelium, spermatid maturation and secretion of proteins by the prostate. Therefore, CD male patients should be considered as vulnerable subjects; thus, the detection of early biomarkers of andrological or endocrinological dysfunctions should trigger timely strategies for prevention and treatment.

[Possible animal models of endocrine, immune and reproductive complications of celiac disease]. / Possibili modelli animali delle complicanze endocrine, immunologiche e riproduttive della malattia celiaca.

This paper underlines the need of developing animal models to study the diverse complications of celiac disease (CD). CD is a multifactorial condition requiring both an exogenous element (gluten) and complex genetic factors; moreover, CD is associated to several endocrine, immune and reproductive diseases, whose onset may be influenced by other environmental factors as well. In particular, the intestinal absorption of exogenous factors may be important for the outcome of CD as well as of the associated diabetes and/or thyroiditis. Presently, there are no adequate animal models for the systemic complications of CD; in particular, there are no gene knock-out models. However, models are available as regards either gluten enteropathy, such as Irish Setter and Balb/c e BDF1 mouse strains, and endocrine-immune diseases associated with CD, such as BB rats and NOD mice. A deeper exploitation of the available models could provide important information on the factors modulating intestinal permeability, the pathogenesis of extraintestinal alterations and the interactions between gluten and other metabolic, nutritional and environmental factors. The elaboration of in vivo models requires a sound basis of knowledge at molecular level, as well as the modulation of the metabolic alteration through relevant exogenous factors, first of all the dietary assumption of gluten. Therefore, the availability of experimental models may provide significant advances on the prevention and treatment, allowing a complete analysis of affected organisms as well as the use of pharmacological and/or immune stimuli; more information may be also derived on the possible long-term effects of gluten traces in CD-affected subjects, thus reducing the need for lengthy clinical studies.

[Celiac disease. Risk factors for women in reproductive age]. / La malattia celiaca. Fattore di rischio per la donna in età fertile.

In the past coeliac disease, or intolerance to gluten, has been considered a rare disease in infancy, whose most important signs were chronic diarrhea with malabsorption and reduced growth. However, besides this classical form, there are a number of other clinical and subclinical forms which may appear even in the adult life and without any overt intestinal sign. The alterations may affect, e.g., the liver, thyroid, skin and the female and male reproductive system. The overall prevalence of the different forms of coeliac disease in Western Europe is at least 1:300. The aim of the present paper is to describe and evaluate the effects of coeliac disease on female reproduction. Such effects include delayed menarche, amenorrhea, infertility and early menopause. Epidemiological studies show that besides reduced fertility, affected women are at higher risk of reproductive problems such as pregnancy loss, low birthweight of offspring and reduced duration of breastfeeding. There are no adequate studies to evidentiate a possible increase of birth defects; nevertheless, coeliac disease induces malabsorption, with deficiencies of nutritional factors essential to prenatal development such as iron, folic acid and vitamin K. The mechanisms underlying the reproductive alterations are still awaiting clarification; however, an interaction among specific nutritional deficiencies, endocrine imbalances and immune disturbances is suspected. As for the other effects associated to the coeliac disease, the possible prevention or treatment of the reproductive effects is only the lifelong maintenance of a gluten-free diet.

A risk factor for female fertility and pregnancy: celiac disease.

Celiac disease is a genetically-based intolerance to gluten. In the past, celiac disease has been considered a rare disease of infancy characterized by chronic diarrhea and delayed growth. Besides the overt enteropathy, there are many other forms which appear later in life; target organs are not limited to the gut, but include liver, thyroid, skin and reproductive tract. It is now recognized that celiac disease is a relatively frequent disorder; the overall prevalence is at least 1:300 in Western Europe. Celiac disease may impair the reproductive life of affected women, eliciting delayed puberty, infertility, amenorrhea and precocious menopause. Clinical and epidemiological studies show that female patients with celiac disease are at higher risk of spontaneous abortions, low birth weight of the newborn and reduced duration of lactation. No adequate studies are available on the rate of birth defects in the progeny of affected women; however, celiac disease induces malabsorption and deficiency of factors essential for organogenesis, e.g. iron, folic acid and vitamin K. The overall evidence suggests that celiac disease patients can be a group particularly susceptible to reproductive toxicants; however, the pathogenesis of celiac disease-related reproductive disorders still awaits clarification. At present, like the other pathologies associated with celiac disease, the possible prevention or treatment of reproductive effects can only be achieved through a life-long maintenance of a gluten-free diet.

Problems in testing and risk assessment of endocrine disrupting chemicals with regard to developmental toxicology.

Endocrine disrupting chemicals (EDCs) may affect mammalian development either indirectly (by impairing implantation, placental development, lactation, etc.) or directly, altering the maturation of target tissues. Current regulatory tests for reproductive/developmental toxicity should be carefully evaluated with regard to risk assessment of EDCs, considering hazard identification (are relevant endpoints being assessed?) and dose-response assessment (are sensitive NOEL/dose-response curves being provided?). Many in vitro and in vivo assays for sex steroid disruption are available; provided that the metabolic capacities of the assays are defined, they could be integrated in a sensitive battery for early detection of steroid-disrupting potentials. The screening battery should address further regulatory in vivo tests (e.g. what specific parameters have to be investigated). As regards dose-response, qualitative differences may be observed between lower and higher exposures, showing primary hormone-related effects and frank embryotoxicity, respectively. Other problems concern (a) the identification of critical developmental windows, according to hormone concentrations and/or receptor levels in the developing target tissues; (b) the potential for interactions between chemicals with common mechanism/target (e.g. xenoestrogens); (c) most important, besides sex steroids more attention should be given to other mechanisms of endocrine disruption, e.g., thyroid effects, which can be highly relevant to prenatal and postnatal development.

[New aspects in the evaluation of risk factors for fetal development]. / Nuovi aspetti nella valutazione di fattori di rischio per lo sviluppo embrionale.

Much knowledge concerning the effects of xenobiotics on prenatal development derive from experimental studies, which are generally performed on laboratory animals according to standardized protocols. Conventional in vivo studies should be integrated by other models within a scientifically-based risk assessment strategy. The paper reviews a few in vitro and/or in vivo approaches: identification of critical effects through the characterization of the pathogenesis as well as the use of dose-response relationships; alternative models to identify reproductive risks from single substances and/or mixtures present in the environment; interactions between in vitro and in vivo studies in the risk assessment of solvents: embryonic metabolism in teratogenicity; interactions between experimental and epidemiological studies to understand the pathogenesis of embryolethality; risk assessment of the effects of prenatal exposure to ionizing radiations.

In vivo studies on possible adverse effects on reproduction of the fungicide methyl thiophanate.

The fungicide methyl thiophanate (MT), widely used to control some of the most common fungal diseases in crops, is metabolized in animals into benzimidazole compounds, including the well-known reproductive toxicant carbendazim. However, standard toxicological tests did not indicate that MT may cause testicular toxicity and/or embryotoxicity, which are typical effects of many benzimidazoles. In the present study some aspects of the MT potential for reproductive toxicity have been assayed by means of two non-conventional models. Following the oral administration of 700 and 1000 mg kg(-1) body wt. for five consecutive days, short-term testicular toxicity was examined in the B6C3F1 mouse through specific parameters (sperm head count, specific enzyme activities, histopathology on days 3-35 post-dosing). In spite of the high doses administered, none of the testicular parameters examined, including histopathology, showed significant alterations as compared to controls at any time post-dosing. Pregnant CD rat dams were administered orally the limit dose of 650 mg kg(-1) body wt. day(-1) during preimplantation (gestational day or GD 2-5) or peri-implantation (GD 6-9) phases; embryos and adnexa were evaluated morphologically on GD 12 as a window for the early observation of embryotoxicity. Evident maternal toxicity was present in both treated groups, whereas only marginal reductions of the growth of embryos and adnexa were observed. A full understanding of MT toxicology will need more quantitative data on metabolism, including plasma kinetics and dosimetry of carbendazim at the relevant targets. Nevertheless, the absence of any clear-cut effect on a number of specific endpoints may provide reassurance that no further testing of MT is needed with regard to testicular toxicity or embryotoxicity.

Developmental toxicity of carbendazim: comparison of no-observed-adverse-effect level and benchmark dose approach.

The benchmark dose (BD) approach has been applied to foetal data from four gavage segment II studies (rat studies 1 and 2, rabbit study, hamster study) on the teratogenic benzimidazole carbendazim. Nineteen parameters were assessed using the log-normal model as a practical tool to derive BDs; good model fitting was observed for all except two parameters. Data were evaluated on a 'per-implant/foetus' basis; BDs were derived from response rate increases of 1, 5, and 10%. The values were compared to the lowest-observed-adverse-effect levels (LOAELs) and no-observed-adverse effect levels (NOAELs) obtained by Fisher's exact test on a 'per-implant/foetus' basis. Frank effects observed only at the top dose and/or small sample size tended to increase the 95% confidence limits and this influenced the determination of BD. Generally, the BD approach provided slightly more conservative estimates than NOAEL; overall, BD01 and BD05 were similar to NOAEL, or even lower for several parameters. The LOAEL in most cases was similar to BD10. Reference doses obtained by dividing BD01 by a 10 or 100 uncertainty factor, corresponded to residual risks of 10(-5) or below. For two critical parameters (hydrocephalus in rat study 1 and resorption rate in the rabbit study) a NOAEL could not be found, whereas a BD was always determined.

[Effects of chemical mixtures on embryonic development: examples of experimental models and problems in risk assessment]. / Effetti tossici di miscele chimiche sullo sviluppo prenatale: esempi di modelli sperimentali e problemi relativi alla valutazione del rischio.

Toxicological risk deriving from the exposure to mixtures of toxic substances, the study of possible interactions among them and their mechanisms of action are of special interest in prenatal toxicology. In fact, embryo is a dynamic complex system whose gradual development substantially modulates the extent and type of damages to which it may be sensitive, through specific, critical periods of sensitivity. In this paper, a number of types of interactions among toxic substances which show the same mechanisms of action and/or the same target site, are analysed. Besides, pharmacokinetic interactions among teratogenic agents and substances modulating their metabolism, need specific evaluations because of the wide variability of possible events. In conclusion, risk assessment in prenatal toxicology has to put greater attention to the various types of effect and pharmacokinetic interaction since they might result in an increasing risk at low doses.

Evaluation of the placenta: suggestions for a greater role in developmental toxicology.

Both in human and in rat, two types of placenta are present: the yolk sac (YS) and the chorioallantoic placenta. Histiotrophy, alpha-fetoprotein synthesis and blood cell formation occur in YS of both species. Besides, the midgut, primordial germ cells and possibly immunological structures originate from the YS tissue. The specialised cells of the chorioallantoic placenta attach the embryo to the uterus and form the vascular connections necessary for the nutrient transport. The placenta redirects maternal endocrine, immune and metabolic functions to conceptus advantage. These complex activities are sensitive to direct toxicity. Indirect effects on the placental functions might be elicited by immunomodulators and endocrine disrupters. Some experimental models could be utilised to identify possible toxic effects on placenta. Among the in vitro models the rodent giant yolk sac culture may be used to study the transport of materials, morphological and/or biochemical alterations and biotransformation activity of the visceral YS epithelium. Other in vitro approaches utilise human derived trophoblastic cells and tissues to investigate implantation and perimplantation toxicology. Besides specific studies, in vivo reproductive toxicity tests could pay more attention to the evaluation of placental tissues. Nowadays, some physiologically based pharmacokinetic models for developmental toxicity are also available to describe the disposition of toxic substances and their metabolites during pregnancy in rodents. Thus, more detailed studies on the embryo-foetal placenta may provide an important tool to understand developmental toxicity mechanisms, with particular regard to embryolethality and delayed development.

Effects observed on gestational day 13 in rat embryos exposed to albendazole.

Albendazole (ABZ) was utilized as a model to investigate the pathogenesis of benzimidazole-induced abnormalities. Pregnant Sprague-Dawley rats were treated po with 0, 10, 20, and 30 mg/kg on gestational days (GD) 10 to 12. The embryos were examined on GD 13, as a window for observing the origin of alterations detected at term. Embryolethality and growth reduction showed dose-related increases at the three dose levels. At 10 mg/kg, an increased developmental delay of limb buds and a less than 5% incidence of embryos with abnormal head or shape were detected. At 20 and 30 mg/kg, > 20% of embryos showed morphologic alterations involving mainly shape abnormalities and the development of forelimb buds, branchial bars, eye, and telencephalon; closure of neuropores was unaffected. Dose-response relationships for morphologic alterations showed steeper slopes than for growth reduction and embryolethality.

Prenatal risks deriving from environmental chemicals.

Hundreds of environmental chemicals affect prenatal development in experimental animals. However, only methylmercury and PCBs have been connected with such effects in humans during localized outbreaks of high exposure. In addition growth and development might also be affected by long-term intake of lead, fluorides or PCBs. Several factors may explain the discrepancy between human and animal data: the actual exposure of the population is below threshold levels, unspecific or delayed effects can be difficult to identify, etc. When experimental data are used to assess the hazards for the conceptus, due consideration should be given to actual ability of the study to detect effects. Thus, the limitations in statistical power, the relevance of the parameters considered and low-dose extrapolation should be taken into account. Finally, understanding toxicokinetics and biological mechanisms is needed to perform interspecies comparisons. Three examples of environmental chemicals showing different prenatal hazards are presented: thiabendazole, a benzimidazole compound with a moderate teratogenic potential, but which could represent a good model for biological extrapolation; nitrofen, a diphenyl ether herbicide which may pose a significant hazard, because of its high potential, toxicokinetics, and specific, hormone-like, teratogenic mechanisms; PCBs, well-known, global, cumulative pollutants which are not teratogenic in the laboratory animals, but may affect the human conceptus at high intake levels.

Presence of tobramycin in kidneys and placentae of Sprague-Dawley rat fetuses and newborns following in utero exposure.

The maternal-fetal transfer of tobramycin (TBM) was investigated in the rat by means of a microbiological assay, to assess the presence and amount in the kidneys and placentae of fetuses at gestational day (GD) 20, in the kidneys of newborns 6 and 11 days after the end of treatment and in the kidneys of the dams. In the qualitative assay, pregnant rats were injected i.p. with 0, 30, 60 mg/kg b.w. of TBM on GD 10-19. A group of dams treated in parallel with 30 mg/kg b.w. was utilized for a microbiological semiquantitative assay. All litters contained some fetuses showing no detectable TBM accumulation in either kidney or in placentae: at 30 mg/kg/b.w. accumulation appeared more prevalent in placenta than in the kidneys of the corresponding fetuses, as confirmed also by the semiquantitative assay. Some newborns (about 6%) of both groups showed detectable renal TBM residues on the 6th and on the 11th day after the end of treatment. The frequency of newborns showing residues was not obviously related to the dose or the day of sampling, and the concentrations of TBM found were comparable to those observed in fetuses. It is possible that they represent a particularly sensitive subgroup.

Tobramycin-induced changes in renal histology of fetal and newborn Sprague-Dawley rats.

Effects on renal development were studied using tobramycin (TBM) as a model compound. Pregnant Sprague-Dawley rats were injected i.p. with TBM at 30 or 60 mg/kg body weight/day on gestational days (GD) 10-19. Kidneys from dams and conceptuses were examined on GD 20 and on postnatal day (PD) 9. The dosing regimen caused in dams moderate proximal tubular alterations and increased concentrations in serum creatinine. Fetal kidneys showed granularity and swelling of proximal tubule cells at the 30 mg/kg dose, poor glomerular differentiation at the 60 mg/kg dose, increased glomerular density at both doses, and no changes on macroscopic examination at either dose. In newborns were observed a moderate developmental delay and tubular lesions at the higher dose, and dose-related increases of glomerular density and relative medullary area at both doses. All findings were more pronounced in males. A maturational disruption of the tubular structures possibly leading to increased glomerular density was attributed to TBM exposure during renal organogenesis in the rat.

Pre-natal (segment II) toxicity study of cinnamic aldehyde in the Sprague-Dawley rat.

Cinnamic aldehyde (CA) was administered by gavage to Sprague-Dawley rats on days 7-17 of pregnancy at doses of 5,25 or 250 mg/kg body weight/day. Significantly lower weight gain of the dams was observed at the two higher dose levels. No significant dose-related increase of abnormalities was observed: the incidence of poor cranial ossification was significantly increased in all treated groups, while reduced ossification of the tympanic bulla was increased at 25 or 250 mg/kg/day. Significant increases of the incidences of dilated pelvis/reduced papilla in the kidney, dilated ureters and greater than or equal to 2 abnormal sternebrae per foetus were detected in the 2-mg/kg group, which had the highest overall prevalence of minor abnormalities. Since significant increases in the incidences of reduced cranial ossification, dilated ureters and renal variants were observed at 5 mg/kg, a dose at which there was no detectable maternal toxicity, it is suggested that the foetus might be slightly more sensitive than the adult to the action of CA.

Hematotoxic effects in the rat of a toluene dinitro derivative after short-term exposure.

3,5-Dinitro-4-chloro-alpha,alpha,alpha-trifluorotoluene (DNCTT) is an intermediate in the synthesis of dinitroaniline herbicides and was involved in an episode of ground water pollution in 1977. The compound presents a high environmental persistence, which may have possible implications concerning public health. In one experiment male Sprague-Dawley rats were administered DNCTT for 3 days at a dose level of 150 mg/kg body wt by oral gavage. Groups of rats were sacrificed up to 10 days after the end of the administration, at 2-day intervals. Methemoglobin was increased up to Day 7; white blood cells were also increased both in peripheral blood and in bone marrow smears. Spleen relative weights were observed to increase slightly at Days 7 and 10; microscopic examination revealed marked congestion with an increased density of the spleen's white pulp. In a similar scheduled experiment, but at a dose level of 300 mg/kg body wt, the bone marrow white cell series were not affected initially, but were affected after 3 days at the end of the administration. DNCTT has a definite effect on white cells.