Characterization of HuH6, Hep3B, HepG2 and HLE liver cancer cell lines by WNT/ß - catenin pathway, microRNA expression and protein expression profile.

Somatic mutations in the genes members of WNT/ß-catenin pathway, especially in CTNNB1 codifying for ß-catenin, have been found to play an important role in hepatocarcinogenesis. The purpose of this work is to characterize alterations of the WNT/ß-catenin signalling pathway, and to study the expression pattern of a panel of microRNAs and proteins potentially involved in the pathogenesis of liver cancer. In this respect, the molecular characterization of the most used liver cancer cell lines HuH6, Hep3B, HepG2, and HLE, could represent a useful tool to identify novel molecular markers for hepatic tumour. A significant modulation of FZD7, NLK, RHOU, SOX17, TCF7L2, TLE1, SLC9A3R1 and WNT10A transcripts was observed in all the four liver cancer cell lines. The analysis of selected microRNAs showed that miR-122a, miR-125a and miR-150 could be suitable candidates to discriminate tumoural versus normal human primary hepatocytes. Finally, Grb-2 protein expression resulted to be increased more than two-fold in liver cancer cell lines in comparison to normal human primary hepatocytes. These advances in the knowledge of molecular mechanisms involved in the pathogenesis of liver cancer may provide new potential biomarkers and molecular targets for the diagnosis and therapy.

Low dose exposure to HBCD, CB-153 or TCDD induces histopathological and hormonal effects and changes in brain protein and gene expression in juvenile female BALB/c mice.

Developmental health risks of chronical exposure to low doses of foodborne persistent organic pollutants (POP) are recognized but still largely uncharacterized. Juvenile female BALB/c mice exposed to either HBCD, CB-153 or TCDD at doses relevant to human dietary exposures (49.5 µg, 1.35 µg and 0.90 ng kg-1 bw-1 day-1, respectively) for 28 days displayed histopathological changes in liver (HBCD, CB-153, TCDD), thymus (HBCD, CB-153) and uterus (HBCD), reduced serum oestradiol 17ß (E2) levels (HBCD), increased serum testosterone (T) levels (CB-153) and an increased T/E2 ratio (HBCD). Proteomics analysis of brain provided molecular support for the HBCD-induced reduction in E2. Neural gene expression analysis, confirmed effects on 18 out of 30 genes previously found to be affected after exposure to higher doses to the same pollutants. Our findings indicate that exposure to POP at low doses is associated with subtle, but toxicological relevant effects on post-natal development in female mice.

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.

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.

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.

[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.

Crude oil spill in sea water: an assessment of the risk for bathers correlated to benzo(a)pyrene exposure.

In the spring of 1991, there was a shipwreck of the oil tanker "Haven" off the Ligurian coast of Italy. This resulted in the spillage of a very large amount of crude oil, some of which was burned off by fire. The accident caused several serious problems (sea and air pollution, damage to the marine fauna, risk of human exposure, etc.). In this context, an assessment was carried out at the Istituto Superior di Sanità with the aim of determining any possible risks to humans which might derive from bathing activities during the following summer season. The whole evaluation carried out after the accident demonstrated that the impacts induced were not serious enough to require bathing restrictions in the coastal areas involved. Assuming a benzo(a)pyrene (BaP) concentration in sea water of 1 microgram/m3 cancer risk is in the order of 10(-8) and in the case of 10-kg child, a 10(-6) risk level correspond to about 0.18 microgram/l of BaP in sea water.

Dietary exposure of juvenile female mice to polyhalogenated seafood contaminants (HBCD, BDE-47, PCB-153, TCDD): comparative assessment of effects in potential target tissues.

Fish represents source of nutrients and major dietary vehicle of lipophilic persistent contaminants. The study compared the effects of two legacy and two emerging fish pollutants (Hexabromocyclododecane HBCD; 2,2',4,4'-Tetrabromodiphenyl ether BDE-47; 2,2',4,4',5,5'-Hexachlorobiphenyl PCB-153; 2,3,7,8-Tetrachlorodibenzo-p-doxin TCDD) in juvenile female mice exposed through a salmon based rodent diet for 28 days (dietary doses: HBCD 199 mg/kg bw/day; BDE-47 450 µg/kg bw/day; PCB-153 195 µg/kg bw/day; TCDD 90 ng/kg bw/day). Dose levels were comparable to previously reported developmental Lowest Observed Adverse Effect Levels. None of the treatments elicited signs of overt toxicity, but HBCD increased relative liver weight. All compounds caused changes in liver, thymus and thyroid; spleen was affected by BDE-47 and PCB-153; no effects were seen in uterus and adrenals. Strongest effects in thyroid follicles were elicited by PCB-153, in thymus and liver by BDE-47. HBCD and BDE-47 induced liver fatty changes, but appeared to be less potent in the other tissues. HBCD, BDE-47 and TCDD increased serum testosterone levels and the testosterone/estradiol ratio, suggesting a potential involvement of pathways related to sex steroid biosynthesis and/or metabolism. The results support the role of toxicological studies on juvenile rodents in the hazard characterization of chemicals, due to endocrine and/or immune effects.

Environment and women's reproductive health.

BACKGROUND: There is significant evidence that continuous and prolonged exposure to several endocrine disrupting chemicals (EDC) is a risk factor for reduced fertility and fecundity in women. There is also evidence that ED exposure has trans-generational effects. In this systematic review, we evaluate the evidence for an association between EDC exposure and women's reproductive health. METHODS: Studies were found by searching the PubMed database for articles published up to 2010. Associations between ED exposure and women's reproductive health reported in the PubMed database are summarized and classified as fertility and fecundity, pregnancy outcomes, transgenerational exposure and effects. RESULTS: Epidemiological studies on EDCs are not always consistent, in part due to limitations imposed by practical constraints. In order to make progress in this field, we recommend taking advantage of biomonitoring and biobanks, including the development of appropriate biomarkers, and taking into greater consideration modulating factors such as genetic polymorphisms and dietary habits. Further human studies are warranted with particular focus on impaired fertility/fecundity associated with currently widespread ED (e.g. bisphenol A, phthalates and polybrominated flame retardants). CONCLUSIONS: A detailed appraisal of compounds specifically related to adverse reproductive outcomes is very important for prevention and risk-communication strategies. Besides research needs, the current evidence is sufficient to prompt precautionary actions to protect women's reproductive health.

Effects of the food contaminant semicarbazide following oral administration in juvenile Sprague-Dawley rats.

Semicarbazide (SEM) is an azodicarbonamide by-product present in glass jar packaged foods including babyfoods, in bleaching steps and flour treatment. Experimental data showed SEM acting as osteolathyrogen agent, but few toxicological data are available in susceptible life-stages. This study aimed to evaluate effects of SEM oral administration for 28 days at 0, 40, 75, 140 mg/kg bw day during the juvenile period in Sprague-Dawley rats. Histopatological examinations of: epiphyseal cartilage - potential target of SEM lathyrogen action - testes, ovary, uterus, thyroid, thymus, spleen, adrenals, representative of the main developing organs relevant to juvenile toxicity, and neurobehavioural tests in males, were performed. Mortality at high and mid dose levels and significantly decreased body weight gain were observed in males even at the lowest dose. Lack of mineralization in cartilage at all dose levels was present. Marked alterations of spontaneous motor and exploratory behaviours were evident even at 40 mg/kg. Histological alterations were observed in all tissues; thyroid and ovary effects were present also at 40 mg/kg. The present study indicate that the NOAEL in juvenile rats is lower than 40 mg/kg for SEM oral administration. SEM administration during juvenile period exerted pleiotropic effects and further studies are suggested to elucidate mechanisms.

Impact of endocrine disruptor chemicals in gynaecology.

The potential hazardous effects that estrogen- and androgen-like chemicals may have both on wildlife and human health have attracted much attention from the scientific community. Endocrine disruptors (EDCs) are chemicals that have the capacity to interfere with normal signalling systems. EDCs may mimic, block or modulate the synthesis, release, transport, metabolism and binding or elimination of natural hormones. Even though potential EDCs may be present in the environment at only very low levels, they may still cause harmful effects, especially when several different compounds act on one target. EDCs include persistent pollutants, agrochemicals and widespread industrial compounds. Not all EDCs are man-made compounds; many plants produce substances (phytoestrogens) that can have different endocrine effects either adverse or beneficial in certain circumstances. Natural substances such as sex hormones from urban or farm wastes can become concentrated in industrial, agricultural and urban areas; thus, such wastes may be considered potential 'EDCs' for humans and/or wildlife. Much attention has focussed on changing trends in male reproductive parameters in relation to EDC exposure; however, studies on the female reproductive system have been less comprehensive. We have focussed this article on four major aspects of female reproductive health: fertility and fecundability, endometriosis, precocious puberty and breast and endometrial cancer.

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.