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

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Abstract

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/m3). 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 μM) and a mixture of 16 PAHs (0.1 μM) were used. Results: No leaching of PAHs was measured in saline or surfactant-containing saline. In vitro incubations with CB particles (30–300 μg/cm2) 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.

Introduction

The formation of tumors by poorly soluble particles (PSPs) in rat lungs is generally considered as the chronic endpoint of persistent inflammation and cell proliferation during exposures leading to overload (Borm et al., 2004, Greim et al., 2001, Schins, 2002). Among the responsible mediators, reactive oxygen and nitrogen species (ROS and RNS) generated by inflammatory cells are considered to play a crucial role in genotoxicity, mutagenicity, and cell proliferation pathways (Knaapen et al., 2004). However, apart from this inflammation-associated DNA damage, an additional and potentially important mechanism of genotoxicity is contained in the fact that many particles, such as diesel exhaust particles (DEP) and carbon black (CB), can carry surface-adsorbed components into the peripheral lung. One important group of these compounds are polycyclic aromatic hydrocarbons (PAHs), among which some are established carcinogens, such as benzo[a]pyrene (B[a]P). Using organic extractions of ambient particulate matter (PM), it has been indicated that these compounds may play an important role in the possible mutagenicity and carcinogenicity of automobile exhaust (Alink et al., 1983, Binkova et al., 2003, Buschini et al., 2001, Dukovich et al., 1981).

The genotoxic and carcinogenic activity of PAH has usually been attributed to the formation of DNA adducts by reactive PAH metabolites (Hall and Grover, 1990). Although most DNA-reactive PAH metabolites are suggested to arise from biotransformation by cytochrome P450 enzymes, inflammatory cell-derived ROS (Borm et al., 1997) and quinones (Cavalieri et al., 2004) have been shown to play a role in the formation of DNA adducts by PAHs. In both cases, ROS either from inflammatory cells or generated by redox cycling are able to form adducts to PAHs leading to reactive metabolic intermediates that can react with DNA (Knaapen et al., 2004). However, it is still debated whether formation of DNA adducts from PAH metabolites plays a role at all in particle-induced lung tumor induction and whether adsorbed PAHs are bioavailable in the lung in vivo. Early studies showed that absorption of PAH onto organic particles clearly affects their retention in the lung and the elution of absorbed PAH also depends on particle size and surface area (Sun et al., 1984). Recent work by Gerde et al. (2001) in dogs showed for B[a]P-coated DEP particles that 36% of the B[a]P is rapidly released from DEP in the lung, but that its absorption rate is so high that all B[a]P enters the circulation without further metabolic transformation within the lung. The remaining fraction of the B[a]P (64%) was found not to be bioavailable and remained particle bound in the lung up to 5.6 months (Gerde et al., 2001). The role of PAH on carbon particles was further questioned by the findings of Dasenbrock et al. (1996), who found no increased tumor rate of DEP after coating particles with B[a]P (11 μg/mg), compared to 0.9 μg B[a]P/mg in control particles. In line with these observations, the tumorigenicity of native vs. organic-extracted DEP in rats appeared to be rather similar and with undetectable PAH–DNA adduct levels (Dasenbrock et al., 1996, Gallagher et al., 1994).

The current study was designed to test the bioavailability and acellular release of PAHs from a set of commercial carbon blacks that differ widely in their PAH content, and test the ability of these PAH-containing particles to form bulky DNA adducts in target lung epithelial cells in vitro and in vivo. The outcomes will contribute to current issues of hazard classification and risk assessment of CBs (Borm et al., 2004, Rausch et al., 2004), since they provide quantitative data for whether these specific PSPs and PAHs are bioavailable and may play a significant role in the formation of DNA adducts.

Section snippets

Chemicals and test materials

Dulbecco's modified Eagle's medium (DMEM), fetal calf serum (FCS), l-Glutamine, penicillin–streptomycin, Trypsin-EDTA, sodium pyruvate, β-NADH, 2,4-dinitrophenylhydrazine, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and dipalmytoyl phosphatidylcholine (DPPC) were obtained from Sigma-Aldrich (Taufkirchen, Germany). Hank's balanced salt solution (HBSS) was obtained from Life Technologies (Kahrlsruhe, Germany). 32P-ATP and Nuclease P1 were supplied by ICN (Indianapolis,

Leaching of PAH from carbon blacks in DPPC-containing suspensions

Our investigations showed no measurable release of PAHs from standard diesel or carbon blacks (1 mg/ml) in aqueous DPPC solutions containing 100–1000 μg/ml DPPC. An increase in DPPC concentration up to 10 mg/ml also showed no measurable PAH release. The same was true when increasing the amount of diesel up to 20 mg/ml before the extraction experiments. Based on the detection limit for HPLC determination of PAH, the amount of a single PAH that possibly leaches from diesel particles under these

Discussion

Our results using acellular, in vitro, and in vivo approaches demonstrate that under the conditions and sensitivity of the assays used, (i) PAHs when present on different carbon blacks do not leach into aqueous media with or without suppletion of surfactant and (ii) that PAHs are not bioavailable from three out of four tested commercial products that contain little PAHs. Some evidence suggests that in the carbon black sample with high PAH content (Sterling V) some PAHs are bioavailable for

Acknowledgments

The authors gratefully acknowledge the contribution of Dr. Jane Gallagher who provided the extracted DNA from animals exposed to different carbon blacks, and Dr. Nils Kruger (Degussa) and Dr Len Levy (University of Leicester) for acting as study monitors. This research was partially supported by the International Carbon Black Association (ICBA) and the DFG (SFB 503).

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