Effects of the genotoxic compounds, benzo[a]pyrene and cyclophosphamide on phase 1 and 2 activities in EpiDerm™ models.

The micronucleus assay in the 3D human reconstructed EpiDerm™ skin model (RSMN) is a promising new assay for evaluating genotoxicity of dermally applied chemicals. To complement the testing of metabolically activated chemicals, such as cyclophosphamide (CPA) and benzo[a]pyrene (B[a]P), we measured phase 1 (ethoxyresorufin O-deethylation (EROD) and testosterone metabolism) and 2 activities (UGTs and GSTs) in non-treated and genotoxin treated EpiDerm™ models in a study design which mimics the RSMN assay. The assay involved a three-dose dosing regimen over 72 h to take into account effects e.g. enzyme induction, which requires longer than the standard 2 dose 48-h assay. These studies demonstrated the presence of basal phase 1 and 2 activities of EpiDerm™ models. With the exception of GST, all of the activities measured did not reproducibly change over time. It was possible to measure enzyme induction using this assay design. EROD activity was significantly induced by B[a]P but not by CPA. CPA and B[a]P had little or no reproducible effects on GST and UGT activities. In conclusion, a number of metabolic enzyme activities were present in the EpiDerm™ skin model and at least the CYP1 family was inducible.

Oxidant generation and toxicity of size-fractionated ambient particles in human lung epithelial cells.

Exposure to ambient particulate matter (PM) is associated with respiratory and cardiovascular disease and lung cancer. In this study, we used size fractionated PM samples (3-7, 1.5-3, 0.95-1.5, 0.5-0.95, and <0.5 microm), collected at four contrasting locations (three urban sites, one remote background) in the UK with a Sierra-Andersen high volume cascade impactor. The H(2)O(2)-dependent oxidant generating capacity of the samples was determined by electron spin resonance with 5,5-dimethyl-1-pyrroline-N-oxide spin trapping. In A549 human lung epithelial cells, we determined the cytotoxicity of samples by LDH assay, and interleukin-8 (IL-8) release as an indicator of their inflammatory potency. Oxidative DNA damage was measured by the formamido-pyrimidine-glycosylase (fpg)-modified comet assay. Marked contrasts were observed for all endpoints. Remote background PM showed the lowest oxidant potential, was neither cytotoxic nor genotoxic and did not increase IL-8 release. For the other samples, effects were found to depend more on sampling location than on size fraction. PM collected at high-traffic locations generally showed the strongest oxidant capacity and toxicity. Significant correlations were observed between the oxidant generating potential and all toxicological endpoints investigated, which demonstrates that measurement of the oxidant generating potential by ESR represents a sensitive method to estimate the toxic potential of PM.

Dose dependence of oral tolerance to nickel.

The dose dependence of oral nickel tolerance was analyzed by comparing three different subsets of C57BL/6 mice: Ni(very low) mice were reared in a nickel-reduced environment, Ni(low) and Ni(high) mice were reared in a stainless steel-containing environment and the latter received oral NiCl(2) (10 mM). In spleen and feces, Ni(very low) mice exhibit significantly lower nickel concentrations than Ni(low) and Ni(high) mice. In contrast to Ni(very low) mice that can be sensitized with a single intradermal administration of NiCl(2) alone, Ni(low) mice can only be sensitized in the presence of an adjuvant and Ni(high) mice cannot be sensitized at all. This dose-dependent resistance to nickel sensitization (i.e. Ni(high) > Ni(low) > Ni(very low)) correlates with differences in the number and type of nickel-specific T regulatory (Treg) cells. Adoptive transfer studies into Ni(very low) recipients showed that Ni(very low) mice completely lack specific Treg cells whereas Ni(low) and Ni(high) mice harbor them, albeit their numbers and/or suppressive strength are much higher in Ni(high) than Ni(low) mice. The principal Treg subset in Ni(low) mice consists of CD4(+)CD25(+) cells, among which CD4(+)CD25(+)alpha(E)beta(7)(+) cells are the most effective. In Ni(high) mice, CD4(+)CD25(+) Treg cells co-exist with an ensemble of CD8(+) Treg and CD4(+)CD25(-) suppressor-inducer cells.

Formation of PAH-DNA adducts after in vivo and vitro exposure of rats and lung cells to different commercial carbon blacks.

OBJECTIVE: The current study was designed to test the possible release and bioavailability of polycyclic aromatic hydrocarbons (PAHs) from a set of commercial carbon blacks (CBs) as well as the ability of these PAHs to form bulky DNA adducts. METHODS: In four commercial CBs (Printex 90, Sterling V, N330, Lampblack 101), leaching of PAH was examined through (1) release of parent PAHs in saline with or without surfactant, and (2) PAH adducts in lung epithelial cells (A549) or in rat lungs after exposure to two CBs (Printex 90, Sterling V) for 13 weeks (50 mg/m(3)). In vitro experiments were done with original and extracted particles, as well as organic extracts of CB in DMSO. As positive controls, B[a]P (0.03 microM) and a mixture of 16 PAHs (0.1 microM) were used. RESULTS: No leaching of PAHs was measured in saline or surfactant-containing saline. In vitro incubations with CB particles (30-300 microg/cm(2)) revealed no adduct spots except for Sterling V. However, the spot was not concentration dependent and remains unidentified. Lung DNA from rats after inhalation of Printex 90 or Sterling V showed no spots related to PAH-DNA adduct formation compared to sham-exposed rats. CONCLUSION: The results suggest that PAHs are very tightly bound to these CBs. Only using organic extracts or particles of low-surface Sterling V, with high PAH content, PAHs may become available to form PAH-DNA adducts. However, the in vitro conditions showing this effect will not be encountered in vivo and renders this mechanism in particle-induced lung cancer at in vivo exposures highly unlikely.