Selection and evaluation of stable housekeeping genes for gene expression normalization in carbon nanoparticle-induced acute pulmonary inflammation in mice.

Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) is a highly specific and sensitive technique for the quantification of gene expression on the mRNA levels. But use of unconfirmed housekeeping genes (HKGs) could lead to misinterpretation of the expression of genes of interest (GOI). In this study, the stability and suitability of 11 frequently used housekeeping genes, namely 18S rRNA, ACTB, B2M, CYPA, GADPH, GUSB, HMBS, HPRT1, RPL13A, SDHA and TBP in 36 lung tissues isolated from either wild-type (WT) mice or p50 knock out (p50-/-) mice or p105 knock-out (p105-/-) mice which were treated with either carbon nanoparticle (CNP) or H(2)O or non-treated, have been validated by geNorm, NormFinder and BestKeeper programs. The expression levels of ACTB, GUSB and RPL13A were the most constant in lung tissues across three genotypes and three kinds of treatments. A set of three most stable genes is found sufficient to be used as housekeeping genes for lung tissues in studies of similar design.

Deducing in vivo toxicity of combustion-derived nanoparticles from a cell-free oxidative potency assay and metabolic activation of organic compounds.

BACKGROUND: The inhalation of combustion-derived nanoparticles (CDNPs) is believed to cause an oxidative stress response, which in turn may lead to pulmonary or even systemic inflammation. OBJECTIVE AND METHODS: In this study we assessed whether the in vivo inflammatory response--which is generally referred to as particle toxicity-of mice to CDNPs can be predicted in vitro by a cell-free ascorbate test for the surface reactivity or, more precisely, oxidative potency (OxPot) of particles. RESULTS: For six types of CDNPs with widely varying particle diameter (10-50 nm), organic content (OC; 1-20%), and specific Brunauer, Emmett, and Teller (BET) surface area (43-800 m2/g), OxPot correlated strongly with the in vivo inflammatory response (pulmonary polymorphonuclear neutrophil influx 24 hr after intratracheal particle instillation). However, for CDNPs with high organic content, OxPot could not explain the observed inflammatory response, possibly due to shielding of the OxPot of the carbon core of CDNPs by an organic coating. On the other hand, a pathway-specific gene expression screen indicated that, for particles rich in polycyclic aromatic hydrocarbon (PAHs), cytochrome P450 1A1 (CYP1A1) enzyme-mediated biotransformation of bio-available organics may generate oxidative stress and thus enhance the in vivo inflammatory response. CONCLUSION: The compensatory nature of both effects (shielding of carbon core and biotransformation of PAHs) results in a good correlation between inflammatory response and BET surface area for all CDNPs. Hence, the in vivo inflammatory response can either be predicted by BET surface area or by a simple quantitative model, based on in vitro OxPot and Cyp1a1 induction.

Reduced in vitro toxicity of fine particulate matter collected during the 2008 Summer Olympic Games in Beijing: the roles of chemical and biological components.

Beijing has implemented systematic air pollution control legislation to reduce particulate emissions and improve air quality during the 2008 Summer Olympics, but whether the toxicity of fine fraction of particles (PM(2.5)) would be changed remains unclear. In present study we compared in vitro biological responses of PM(2.5) collected before and during the Olympics and tried to reveal possible correlations between its chemical components and toxicological mechanism(s). We measured cytotoxicity, cytokines/chemokines, and related gene expressions in murine alveolar macrophages, MH-S, after treated with 20 PM(2.5) samples. Significant, dose-dependent effects on cell viability, cytokine/chemokine release and mRNA expressions were observed. The cytotoxicity caused at equal mass concentration of PM(2.5) was notably reduced (p<0.05) by control measures, and significant association was found for viability and elemental zinc in PM(2.5). Endotoxin content in PM(2.5) correlated with all of the eight detected cytokines/chemokines; elemental and organic carbon correlated with four; arsenic and chromium correlated with six and three, respectively; iron and barium showed associations with two; nickel, magnesium, potassium, and calcium showed associations with one. PM(2.5) toxicity in Beijing was substantially dependent on its chemical components, and lowering the levels of specific components in PM(2.5) during the 2008 Olympics resulted in reduced biological responses.