Toxic and genotoxic effects of oral administration of furan in mouse liver.

In this study, the effects induced in mouse liver by repeated oral exposure to furan were investigated. To this aim, the compound was given for 28 days by daily gavage to male B6C3F1 mice at 2, 4, 8 and 15 mg/kg body weight (b.w.)/day. Twenty-four hours after last administration, animals were sacrificed, liver was excised and the following parameters were evaluated: histological alterations, apoptosis, cell proliferation, polyploidy, overall DNA methylation, gene expression and DNA damage by the immunofluorescence detection of foci of phosphorylated histone H2AX (gamma-H2AX) and by alkaline comet assays, using both standard and modified protocols for the detection of DNA cross links. Liver DNA damage by comet assays was also evaluated in mice receiving furan as a single acute oral dose (15, 100 or 250 mg/kg b.w.). Microscopic analysis of liver sections indicated that repeated oral administration of furan was moderately toxic, producing mild histological alterations with necrotic figures, apoptosis and limited regenerative cell proliferation. The flow cytometric analysis of DNA content in single-cell suspensions of liver cells showed a statistically significant increase in polyploid (8N) cells at the highest dose. No treatment-related changes in overall DNA methylation, gamma-H2AX foci, DNA strand breaks and cross links were observed at the end of the 4-week exposure period. However, several genes involved in DNA damage response, beyond stress and liver toxicity, were over-expressed in mice treated with the highest furan dose (15 mg/kg b.w./day). Acute administration of furan induced evident liver toxicity at the highest dose (250 mg/kg b.w.), which was associated with a significant increase of DNA damage in the alkaline comet assay and with a distinct decrease in gamma-ray-induced DNA migration. Overall, the results obtained suggest that the contribution of genotoxicity to the mechanism of furan carcinogenicity in mouse liver should not be dismissed.

Effects of a low oral dose of diethylstilbestrol (DES) on reproductive tract development in F1 female CD-1 mice.

The synthetic estrogen diethylstilbestrol (DES) is a model to study the effects on female reproductive tract of endocrine disrupting chemicals interacting with estrogen receptors. Pregnant CD-1 mice were given daily by gavage 10microg/kg bw of DES (the lower range of therapeutic exposure) during gestational days 9-16, critical period for reproductive tract development. Parameters of sexual development were recorded after weaning and at sexual maturation. No signs of general toxicity were observed in dams. In DES-treated group, reduced litter weight during lactation and earlier vaginal patency was observed. Uterus weight was increased in F1 treated females at weaning. Histological analysis showed reduced endometrium thickness and increased polyovular follicles, irregular and oocytes with condensed chromatin in the ovary at sexual maturity. Prenatal DES oral administration induces subtle but significant effects on puberty onset, uterine and ovary morphology.

Lindane may modulate the female reproductive development through the interaction with ER-beta: an in vivo-in vitro approach.

Lindane (gamma-HCH) is a persistent environmental pollutant that may act as endocrine disrupter, affecting the nervous, immune and reproductive system, possibly through endocrine-mediated mechanisms. Since both estrogen receptors (ER-alpha and -beta) have shown to be target for endocrine disruption, we investigated the role of gamma-HCH on the development of female reproductive system. For an in vivo evaluation of gamma-HCH effects during prenatal period, pregnant CD1 mice were treated p.o. on gestational days 9-16 with 15 mg/kg bw/day of gamma-HCH and vehicle. The in vivo findings in treated F1 pups - in the absence of signs of systemic toxicity - included increase in the absolute and relative and absolute uterus weight revealed on post-natal day 22, earlier vaginal patency and reduced diameters of primary oocytes at fully sexual maturity. No effects on steroid hormone metabolism (aromatase, testosterone catabolism) were observed. Thus, gamma-HCH elicited subtle effects on female reproductive development likely mediated by ER-beta-mediated pathway(s), without a concurrent impairment of steroid hormone metabolism. Furthermore, to verify whether the endocrine interference of gamma-HCH is attributable to stimulation of ER-beta-mediated pathway(s), its effect has been evaluated in vitro on a cell line, LNCaP, expressing only functional ER-beta. In vitro treatments revealed a concentration-related effect on LNCaP cell viability and proliferation. Significantly, the contemporary addition of a pure anti-estrogen, the ER antagonist ICI 182,780, completely reversed gamma-HCH effects indicating an ER-beta-mediated action. Our findings indicate that gamma-HCH may act as endocrine disruptor during the female reproductive system development and ER-beta as a potential target for this compound and other endocrine disrupting chemicals as well.

The 65 kDa mannoprotein gene of Candida albicans encodes a putative beta-glucanase adhesin required for hyphal morphogenesis and experimental pathogenicity.

Mannoproteins are fungal cell wall components which play a main role in host-parasite relationship. Camp65p is a putative beta-glucanase mannoprotein of 65 kDa which has been characterized as a main target of human immune response against Candida albicans. However, nothing is known about its specific contribution to the biology and virulence of this fungus. We constructed CAMP65 knock-out mutants including null camp65/camp65 and CAMP65/camp65 heterozygous strains. The null strains had the same growth rate and morphology under yeast form as the wild-type strain but they were severely affected in hyphal morphogenesis both in vitro and in vivo. Hyphae formation was restored in revertant strains. The null mutants lost adherence to the plastic, and this was in keeping with the strong inhibition of fungal cell adherence to plastic exerted by anti-Camp65p antibodies. The null mutants were also significantly less virulent than the parental strains, and this loss of virulence was observed both in systemic and in mucosal C. albicans infection models. Nonetheless, the virulence in both infectious models was regained by the CAMP65 revertants. Thus, CAMP65 of C. albicans encodes a putative beta-glucanase, mannoprotein adhesin, which has a dual role (hyphal cell wall construction and virulence), accounting for the particular relevance of host immune response against this mannoprotein.

Long-term effects of lonidamine on mouse testes.

Lonidamine (LND) [1-(2,4-dichlorobenzyl)-1H-indazole-3-carboxylic acid] is a well-known antispermatogenic drug. The aim of this study was to identify its possible long-term sequelae on the reproductive system of mice as compared with rats, where most data have been obtained until now. Sexually mature CD1 male mice were administered a single dose of LND (200 mg/kg bw by gavage) and killed 24 and 48 h, 6 days and 2, 4 and 8 weeks after the treatment. Testes were collected, weighed and (1) fixed in Bouin's solution for histological analysis or (2) reduced to monocellular suspensions and ethanol fixed to undergo flow cytometry (FCM) DNA content analysis. No effect on body weight and/or food consumption was observed in the treated group in comparison with the control group. Testicular weight was significantly reduced 24 h after the treatment. Reduced seminiferous epithelium with a progressive lack of intercellular cohesion and marked depletion of spermatids, infiltration of granulocytes, desquamation into the tubular lumen and increased intertubular spaces were present by 24 h after the treatment and persisted to a marked degree at 48 h, 6 days and 2 and 4 weeks up to a marked degeneration of tubular structures with absence of spermatogenesis. The same effects, albeit with a moderate severity, were still present 8 weeks after the treatment. As also detected by FCM, primary spermatocytes appeared to be the main cellular target. Sertoli and Leydig cells were remarkably spared. The histological findings are consistent with those previously observed in rats and point out that testicular damage may persist for several weeks after a single-dose administration. Findings are discussed in comparison with testicular toxicity elicited by other xenobiotics.

Histological and histomorphometric alterations in thyroid and adrenals of CD rat pups exposed in utero to methyl thiophanate.

Pregnant CD rats were treated with an initial dose of 0, 310 or 560 mg/kg bw per day of the fungicide methyl thiophanate (MT) on gestational days 10-14, corresponding to formation of thyroid and adrenal primordia; newborns were sacrificed on postnatal days (PNDs) 10 and 23. No apparent maternal toxicity and no effects on litter size, viability or weight gain were present. Delayed ear pinna detachment and eye opening were present at top dose level. Thyroid histology showed increased irregular nuclei and/or mitoses (PND 10-both doses), cells with necrotic or hydropic changes (PND 23-top dose). The adrenal cortex showed increased karyomegaly and hydropic degeneration (PND 23-both doses). Thyroid histomorphometry showed reduced follicular density, moderately increased follicular cell height and number of nuclei/follicle (PND 10-top dose and PND 23-both doses), suggesting retarded follicular maturation. The adrenal cortex relative area was slightly decreased (PND 10-top dose and PND 23-both doses).MT may act as weak endocrine disrupter, suggesting that attention should be paid to delayed endocrine alterations elicited by agrochemicals.

Long-lasting effects of lindane on mouse spermatogenesis induced by in utero exposure.

Long-lasting effects on mouse spermatogenesis induced by prenatal exposure to the insecticide lindane have been investigated by conventional reproductive endpoints complemented by the flow cytometric (FCM) DNA content analysis of testis cells and by the Sperm Chromatin Structure Assay (SCSA). Two lindane dose levels, 15 and 25 mg/kg bw, and diethylstilboestrol (DES, 10 microg/kg bw) as positive control, were administered daily by gavage to pregnant CD1 mice on gestation days (GD) 9-16. Reproductive endpoints were evaluated on F1 male mice on postnatal day (PND) 60; additionally, animals treated with lindane 25 mg/kg per day and DES were examined on PND 100 to evaluate the possible reversibility of the effects. On PND 60, lindane and DES caused a reduction in the sperm head count and concentration, with recovery in older lindane 25 mg/kg per day animals (PND 100). By contrast, the DES group exhibited a greater reduction in the sperm head count on PND 100 than on PND 60. Changes in biochemical parameters in the testes, lactate dehydrogenase-C(4) (LDH-C(4)), and sorbitol dehydrogenase (SDH) activities, were also observed in adult treated F1 mice. Furthermore on PND 60, the FCM analysis revealed changes in the pattern of testicular germ cell distribution, especially in the haploid subcompartment, in the lindane 25 mg/kg per day group. A dose-dependent increase in chromatin abnormalities of the epididymal sperm was also shown by SCSA. These changes recovered on PND 100. Preliminary qualitative examination did not reveal any significant difference in the structure of testicular tissue; however, there were suggestions of a moderate increase in number and size of Leydig cells in both DES- and lindane-treated animals. The partial reversibility of these effects and the lack of structural modification of the testicular tissue as evidenced by histopathologic assessment suggest a functional impairment of sperm production and maturation, possibly associated with changes induced by lindane on factors affecting intratesticular steroidogenesis.