Claudio Vismara

Impact of tire debris on in vitro and in vivo systems

BackgroundIt is estimated that over 80% of respirable particulate matter (PM10) in cities comes from road transport and that tire and brake wear are responsible for the 3–7% emission of it. Data on the indicators of environmental impact of tire debris (TD), originated from the tire abrasion on roads, are extremely scarce, even though TD contains chemicals (zinc and organic compounds) which can be released in the environment.MethodsTD particle morphology was analysed with SEM, TEM and FIB instruments. TD eluates and TD organic extracts were tested at dilution series on human cell lines and Xenopus laevis embryos. 50 and 100 g/L TD were used for the eluates obtained after 24 h at pH 3 and the quantity of zinc present was measured with a ICP-AES. Eluates diluted to 1%, 10%, 50% in culture media and undiluted were used on X. laevis embryos in the FETAX test. HepG2 cells were exposed for 24 h to 0.05 – 50 μg/ml of zinc salt while A549 cells were exposed for 24, 48 and 72 h to 10, 50, 60, or 75 μg/ml of TD extract. X. laevis embryos were exposed to 50, 80, 100, or 120 μg/ml TD extract.ResultsThe solution of undiluted 50 g/L TD produced 80.2% mortality (p < 0.01) in X. laevis embryos and this toxic effect was three times greater than that produced by 100 g/L TD. Zn accumulation in HepG2 cells was evident after 4 h exposure. A549 cells exposed to TD organic extract for 72 h presented a modified morphology, a decrease in cell proliferation and an increase in DNA damage as shown by comet assay. The dose 80 μg/ml of TD extract produced 14.6% mortality in X. laevis embryos and 15.9% mortality at 120 μg/ml. Treatment with 80, 100, or 120 μg/ml TD organic extract increased from 14.8% to 37.8% malformed larvae percentages compared to 5.6% in the control.ConclusionSince the amount of Zn leached from TD is related to pH, aggregation of particles and elution process, the quantity of TD present in the environment has to be taken into account. Moreover the atmospheric conditions, which may deeply influence the particle properties, have to be considered. The TD organic fraction was toxic for cells and organisms. Thus, because of its chemical components, TD may have a potential environmental impact and has to be further investigated.

Axial-skeletal defects caused by Carbaryl in Xenopus laevis embryos.

Embryotoxic effects of Carbaryl (CB), a widely used carbamate insecticide, was evaluated by modified Frog Embryo Teratogenesis Assay-Xenopus (FETAX), coupled with a histopathological screening of the survived larvae. X. laevis embryos were exposed to 1, 2, 4, 8, 16 and 24 mg/L CB from stage 8 to stage 47. From an estimated LC50 of 20.28 mg/L and TC50 of 8.43 mg/L a TI of 2.41 was derived, indicating that CB is to be considered teratogenic for X. laevis embryos. The most characteristic terata, classified as abnormal tail flexure, involved a significant percentage of larvae from 1 mg/L CB onward, reaching 100% at 24 mg/L CB. Histopathological screening revealed tail musculature and notochord as the main targets for CB. Skeletal muscle lesions consisted of myotomes reduced in size, showing myocytes with disorganized contractile systems and irregular myosepta, coupled with disarranged myocyte apexes. Notochords from CB exposed larvae appeared wavy or bent, with irregular connective sheaths and histologically characterized by protrusions of fibrous matrix and inclusions of ectopic cell masses. This axial-skeletal damage was hypothesized to be related both to the inhibition of acetylcholinesterase, with consequent muscular tetanic spasms, and to disorders in the organization of the connective tissue matrix surrounding the notochord.

Rabbit teratology study with 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Abstract 2,3,7,8,-Tetrachlorodibenzo-p-dioxin was given orally to pregnant rabbits on the 6th–15th days of gestation, in dosages of 0 (control), 0.1, 0.25, 0.5, and 1 μg/kg/day. Severe maternal toxicity and embryotoxicity were evident at the 0.25 μg/kg/day dose and higher. Kidney anomalies were observed in treated groups.

The use of in vitro fertilization in the Frog Embryo Teratogenesis Assay in Xenopus (FETAX) and its applications to ecotoxicology

The Frog Embryo Teratogenesis Assay in Xenopus (FETAX) is a powerful assay for the presence of developmental toxicants in the environment that uses Xenopus embryos. We have applied the test to evaluate a water purification system by testing and comparing the input and the output waters.

Lethality, teratogenicity and growth inhibition of heptanol in Xenopus assayed by a modified frog embryo teratogenesis assay-Xenopus (FETAX) procedure

The frog embryo teratogenesis assay-Xenopus (FETAX), a powerful test for the presence of developmental toxicants, has been modified mainly by performing an in vitro fertilization and increasing the exposure time to 112 h. The modified assay (modFETAX) that presents several advantages over the original FETAX methodology has been validated by the use of ZnSO4, a standard teratogen for FETAX. The modFETAX has been applied to evaluate the 1-heptanol effects on mortality, malformation and growth inhibition. The results indicate that heptanol causes a significant growth inhibition of Xenopus tadpoles and that LC50 and TC50 at 120 h are, respectively, 1.49 and 0.37 mM; the resulting teratogenic index (TI50) of 4.03 suggests that heptanol is a strong teratogen.

Embryotoxic effects of 2,3,7,8 tetrachlorodibenzo-p-dioxin administered to female rats before mating

2,3,7,8-Tetrachlorodibenzo-p-dioxin was administered to female rats by gavage for 2 consecutive weeks at daily doses of 0, 0.125, 0.5, and 2 micrograms/kg. After treatment females were caged with untreated males. All dams were killed on Day 21 of gestation. Their reproductive statuses were recorded and live fetuses were examined for external, visceral, and skeletal malformations. At 0.125 micrograms/kg no effects were observed on both maternal and fetal toxicity; 0.5 micrograms/kg reduced maternal weight gain and increased postimplantation loss. At 2 micrograms/kg the following features became evident: reduction of both maternal weight gain and ovulation rate, increase of pre- and postimplantation loss, and fetal growth retardation. Also, malformed fetuses were observed at this dose level.

TERATOLOGIC EVALUATION OF p-DICHLOROBENZENE IN THE RAT

p-Dichlorobenzene (p-DCB) is a significant environmental chemical largely used as a moth repellent, space deodorant and fungicide. Long term rodents studies did not demonstrate carcinogenic potential after inhalation exposure levels up to 500 ppm. Teratogenic study in rats exposed to atmospheric concentrations of 75,200 or 500 ppm did not reveal embryotoxic, fetotoxic or teratogenic effects; furthermore p-DCB was not teratogenic or fetotoxic in rabbits are exposure levels up to 800 ppm by inhalation. The purpose of this study was to assess the teratogenic potential of p-DCB by a different route from that of inhalation, allowing higher levels of exposition. Pregnant rats were exposed p-DCB by gavage.

Teratogenesis study of dioxane in rats.

The industrial solvent dioxane (1,4-diethylene dioxide) was evaluated for teratogenic potential in Sprague-Dawley rats. The compound was administered on days 6-15 of gestation by gavage (0, 0.25, 0.5 and 1.0 ml/kg/day). A slight maternal toxicity, as evidenced by reduced weight gain, was observed with 1.0 ml/kg. Animals were killed and subjected to uterine examination on day 21 of pregnancy. There were no differences between control and dioxane-treated groups in implantation numbers, live fetuses, postimplantation loss or major malformations. Embryotoxicity, manifested by reduced fetal weight, occurred only at the highest dose level.

Effects of cadmium, lead and copper on rat preimplantation embryos

The development of preimplantation mammalian embryos may be impaired by environmental chemicals. Metals and their salts are known to induce congenital malformations, resorptions, intrauterine death and developmental retardations in the laboratory species on which have been tested. The effects of these chemicals on preimplantation mammalian embryos have been little investigated; copper inserted into the uterine lumen prevents pregnancy in the rat, rabbit, and hamster and copper salts are lethal for mouse blastocysts cultured in vitro. Inorganic lead causes a delay of the first divisions in the 48h mouse embryos and interferes with the viability and the outgrowth of mouse blastocysts cultured in vitro. The present study was undertaken to investigate the effects of Cd, Pb, and Cu on the preimplantation development of the rat embryo.

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin administered to pregnant rats during the preimplantation period.

Abstract 2,3,7,8-Tetrachlorodibenzo- p -dioxin (TCDD) was given by gavage to pregnant rats on the 1st–3rd days of gestation in dosages of 0 (control), 0.125, 0.5, and 2 μg/kg/day. The treatment did not increase pre- and postimplantation losses. Mean fetal weight was reduced at the 0.5 and 2 μg/kg dose levels.