Claudio Ricciardi

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.

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 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.

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.

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.

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.

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.

Significance of the Minor Alterations of the Axial Skeleton in Rat Foetuses. A Short Review

Abstract The toxicological significance of different kinds of minor alterations in the axial skeleton of foetal rat is still a matter of debate. These changes can be better evaluated by means of the double staining technique for cartilage and calcified tissue, which helps to distinguish between deficient calcification and actual structural changes. Sternebral alterations are a frequent finding in prenatal toxicity studies, while in humans they are seldom observed. However, very few data exist for the rat with regard to their persistence and/or consequences in postnatal life. Abnormalities of the vertebral bodies might be functionally important, but comparatively little attention has been given to them in prenatal toxicology. By contrast, the little significance of wavy ribs, potentially reversible alterations, has been extensively studied. The transiency of extra ribs in the rat might be a species‐specific feature, but they have been reported to cause health problems in humans. It is important to recognize a dose‐dependent trend both for specific minor changes and for general parameters (e.g., sternebral alterations). Finally, the range of prevalences in historical controls may be an insensitive parameter, due to the wide variability between different control groups. This range could be better replaced by the average prevalence ± Standard Deviation.

Subchronic oral toxicity of 4-chloro-α,α,α-trifluorotoluene in sprague-dawley rats

Abstract The subchronic oral toxicity of 4-chloro-α,α,α-trifluorotoluene (CTT) was assessed in Sprague Dawley rats. Four groups of six male and six female rats were treated daily for 28 days, by gavage, with doses of 0, 10, 100 and 1000 mg CTT/kg body weight using olive oil as a vehicle. No clinical signs were observed, other than salivation in the high-dose group in the last week. The males of this group showed a significant decrease in body-weight gain without a concurrent decrease in food consumption. In males, there were significant dose-dependent increases in blood cholesterol and triglycerides, suggestive of alterations in lipid metabolism. The females showed only a small dose-related increase in serum lactate dehydrogenase. Specific histological alterations were found in the males given 1000 mg/kg/day, namely hyaline droplet nephrosis, along with a significant increase in relative kidney weight, and an increase in lipid vacuoles in the adrenal cortex. Slight nephrosis was also observed in males given 100 mg/kg. Both male and female rats showed a significant increase in relative liver weight at a dose of 1000 mg CTT/kg. CTT appears to have a low subchronic oral toxicity. Neither pathological nor biochemical alterations were found at 10 mg/kg body weight/day and this can be defined as the no-observable-effect level (NOEL).

Histological Alterations in Gestational Day 13 Rat Embryos from Albendazole-Treated Dams

Gestational day (GD) 13 rat embryos from dams treated p. o. with albendazole (ABZ) on GD 10–12 were examined as an experimental model for the pathogenesis of embryotoxicity induced by benzimidazole compounds. Dosage levels were 0, 10, 20 and 30 mg/kg b. w. of ABZ. At 10 mg/kg b. w., most embryos appeared macroscopically normal. However, the histological examination of a representative sample showed subtle alterations including discrete necrotic foci in the lateral and rostral telencephalon, delayed differentiation of lens, increased wavyness of the notochord caudad to the forelimb bud and absent or poorly defined apical ectodermal ridge in the forelimb bud. At 20 and 30 mg/kg b. w. a sharp, dose‐related increase in embryolethality, developmental delays and macroscopic abnormalities were accompanied histologically by marked necrotic alterations of a number of embryonic tissues. The telencephalon and neural tube were severely affected, showing also a regeneration attempt through the disordered formation of tubules. Besides neuroectoderm, necrosis and tissue depletion were mostly evident in neural crest and mesoderm‐derived structures, such as branchial bars, limb buds and paraxial mesoderm. Lesions in the somites showed a head‐to‐tail increasing gradient; the sclerotomes were consistently more affected than the dermatomes. Wavyness of the notochord cephalad to the forelimb bud and fragmented/abnormal nuclei in the erythroblasts were also noted. Examination of embryos during in vivo organogenesis may be a useful tool for investigating the pathogenesis of embryotoxicity.