Supplemental role of the Ames mutation assay and gap junction intercellular communication in studies of possible carcinogenic compounds from diesel exhaust particles

Abstract

This study presents a new strategy for the carcinogenic evaluation of complex chemical mixtures based on genotoxic and nongenotoxic assays. We studied the ability of organic extracts of diesel exhaust particles (DEP) to induce point mutations in five different Salmonella typhimurium strains (Ames test) and to inhibit gap junction intercellular communication (GJIC) in rat liver epithelial cell lines. A crude extract of DEP was prepared by extraction with dichloromethane (DCM), and fractionated according to polarity into five fractions: aliphatic hydrocarbons, polycyclic aromatic hydrocarbons (PAH), nitro-PAH, dinitro-PAH, and polar compounds. Statistical experimental design, multivariate data analysis, and modeling were used to quantify the mutagenicity of individual and combined DEP fractions in the Ames assay. Quantitative determination of GJIC was carried out using a recently described combination of scrape loading and digital image analysis. Both assays responded to the DEP extract, but the responses were due to different fractions. The nitro-PAH fraction showed the strongest mutagenic potential, followed by the dinitro-PAH fraction. The effect on GJIC was due to the fraction containing the polar components, followed by the dinitro-PAH fraction. The extract was found to induce both basepair substitutions and frameshift mutations, through activation by bacterial nitroreductases. Hyperphosphorylation of connexin43, the major connexin in the epithelial cell lines, was less evident for DEP extract than for other communication inhibitors such as phorbol esters and growth factors, and consequently inhibitors of the protein kinase C (PKC) and mitogen-activated protein (MAP) kinase pathway were unable to counteract the inhibition by DEP extract. Since the Ames test is a well accepted method to screen for substances with genotoxic activity while inhibition of GJIC is associated with effect of tumor promoters and nongenotoxic carcinogens, it is not surprising but encouraging and interesting that the present data indicate that the two endpoints supplement each other as screening tests and in the evaluation of hazardous compounds in complex mixtures.

Mutations in the lungs of gpt delta transgenic mice following inhalation of diesel exhaust

“…The mutagenicity of DEP extracts has been evaluated in vitro using Salmonella typhimurium TA98 assay without an exogenous metabolic activation system (S9 mixture), in which frameshift mutations were found to predominate [Salmeen et al, 1984;Østby et al, 1997;Rivedal et al, 2003]. Although we demonstrated the mutagenicity of DE in vivo using Big Blue 1 rats [Sato et al, 2000], further studies on its pulmonary effects are required for an assessment of the health risks of air pollution.…”

Comparison of BTX Profiles and Their Mutagenicity Assessment at Two Sites of Agra, India

“…Several other researchers have also reported higher revertants plate −1 for TA100 in contrast to TA98 for organic extracts from diesel exhaust particles [56], biofuel combustion [57], and photocatalytic degradation of toluene [58]. Similar studies on mutagenicity assays have been conducted on exhaust from mobile sources like natural gas fueled truck [59], diesel passenger cars [60], and motorcycles exhaust [61].…”

Loss of gap junctional intercellular communication in rat lung epithelial cells exposed to carbon or silica-based nanoparticles

“…In fact, it was recently suggested that determination of GJIC could prove suitable as a standard screening assay for nongenotoxic carcinogens (Rivedal et al, 2003). Although acute cytotoxic action of NPs could be scanned in vitro in a multitude of available standard toxicity assays, test systems are required that allow for an estimate of long-term adverse effects owing to exposure to NPs.…”

“…Hence, a signaling cascade known to be a major modulator of GJIC in response to stress is affected. (ii) Diesel exhaust particles were demonstrated to strongly inhibit GJIC in rat liver epithelial cells (Rivedal et al, 2003) and quartz particles were demonstrated to attenuate GJIC in rat lung epithelial cells (Ale-Agha et al, 2009a). (iii) Soluble components of particulate matter include organic material but also metal ions that were shown to affect connexin levels in cardiac myocytes (Graff et al, 2004).…”