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Toxic Effects of the Major Components of Diesel Exhaust in Human Alveolar Basal Epithelial Cells (A549)

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ABSTRACT

We investigated the toxicity of benzo[a]pyrene (B[a]P), 1-nitropyrene (1-NP) and 3-nitrobenzanthrone (3-NBA) in A549 cells. Cells were treated for 4 h and 24 h with: B[a]P (0.1 and 1 μM), 1-NP (1 and 10 μM) and 3-NBA (0.5 and 5 μM). Bulky DNA adducts, lipid peroxidation, DNA and protein oxidation and mRNA expression of CYP1A1, CYP1B1, NQO1, POR, AKR1C2 and COX2 were analyzed. Bulky DNA adducts were induced after both treatment periods; the effect of 1-NP was weak. 3-NBA induced high levels of bulky DNA adducts even after 4-h treatment, suggesting rapid metabolic activation. Oxidative DNA damage was not affected. 1-NP caused protein oxidation and weak induction of lipid peroxidation after 4-h incubation. 3-NBA induced lipid peroxidation after 24-h treatment. Unlike B[a]P, induction of the aryl hydrocarbon receptor, measured as mRNA expression levels of CYP1A1 and CYP1B1, was low after treatment with polycyclic aromatic hydrocarbon (PAH) nitro-derivatives. All test compounds induced mRNA expression of NQO1, POR, and AKR1C2 after 24-h treatment. AKR1C2 expression indicates involvement of processes associated with reactive oxygen species generation. This was supported further by COX2 expression induced by 24-h treatment with 1-NP. In summary, 3-NBA was the most potent genotoxicant, whereas 1-NP exhibited the strongest oxidative properties.

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Related in: MedlinePlus

Autoradiographs of TLC maps of 32P-labeled DNA digests after incubation of A549 cells with various concentrations of benzo[a]pyrene, 1-nitropyrene and 3-nitrobenzanthrone for 4 h and 24 h. Control panels depict analyses of A549 cells treated withdimethylsulfoxide. DNA (5 µg) was analyzed using the nuclease P1 method of sensitivity enhancement. Screen-enhanced autoradiography was performed at −80 °C for 24 h.
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ijms-17-01393-f001: Autoradiographs of TLC maps of 32P-labeled DNA digests after incubation of A549 cells with various concentrations of benzo[a]pyrene, 1-nitropyrene and 3-nitrobenzanthrone for 4 h and 24 h. Control panels depict analyses of A549 cells treated withdimethylsulfoxide. DNA (5 µg) was analyzed using the nuclease P1 method of sensitivity enhancement. Screen-enhanced autoradiography was performed at −80 °C for 24 h.

Mentions: Four-hour treatment of A549 cells with 0.1 and 1 µM of B[a]P induced one major adduct spot whereas, after 24-h treatment with 1 µM of B[a]P, three additional minor spots representing three other adduct types were detected (Figure 1). With the exception of the 10-µM concentration after 24-h incubation (when multiple DNA adduct spots were visible on autoradiographs), 1-NP induced weak adduct spots. In contrast, 0.5 µM and 5 µM 3-NBA induced 3–4 strong adduct spots after 4-h and 24-h treatment (Figure 1). Levels of bulky DNA adducts after treatment with 3-NBA (5 μM) exceeded 275 adducts/108 nucleotides (Figure 2A). After 24-h treatment with 1 μM B[a]P, levels of DNA adducts reached 175 adducts/108 nucleotides, whereas the 0.1 μM concentration of B[a]P induced much lower levels of DNA adducts (≈10 adducts/108 nucleotides). Interestingly, although levels of DNA adducts induced by 3-NBA after 24-h treatment were high, we observed almost 50% reduction in their levels after incubation with 3-NBA (0.5 μM). Levels of DNA adducts induced by 1-NP were low even after 24-h incubation with the compound, when the higher test dose (10 μM) induced almost 30 adducts/108 nucleotides (Figure 2B).


Toxic Effects of the Major Components of Diesel Exhaust in Human Alveolar Basal Epithelial Cells (A549)
Autoradiographs of TLC maps of 32P-labeled DNA digests after incubation of A549 cells with various concentrations of benzo[a]pyrene, 1-nitropyrene and 3-nitrobenzanthrone for 4 h and 24 h. Control panels depict analyses of A549 cells treated withdimethylsulfoxide. DNA (5 µg) was analyzed using the nuclease P1 method of sensitivity enhancement. Screen-enhanced autoradiography was performed at −80 °C for 24 h.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5037673&req=5

ijms-17-01393-f001: Autoradiographs of TLC maps of 32P-labeled DNA digests after incubation of A549 cells with various concentrations of benzo[a]pyrene, 1-nitropyrene and 3-nitrobenzanthrone for 4 h and 24 h. Control panels depict analyses of A549 cells treated withdimethylsulfoxide. DNA (5 µg) was analyzed using the nuclease P1 method of sensitivity enhancement. Screen-enhanced autoradiography was performed at −80 °C for 24 h.
Mentions: Four-hour treatment of A549 cells with 0.1 and 1 µM of B[a]P induced one major adduct spot whereas, after 24-h treatment with 1 µM of B[a]P, three additional minor spots representing three other adduct types were detected (Figure 1). With the exception of the 10-µM concentration after 24-h incubation (when multiple DNA adduct spots were visible on autoradiographs), 1-NP induced weak adduct spots. In contrast, 0.5 µM and 5 µM 3-NBA induced 3–4 strong adduct spots after 4-h and 24-h treatment (Figure 1). Levels of bulky DNA adducts after treatment with 3-NBA (5 μM) exceeded 275 adducts/108 nucleotides (Figure 2A). After 24-h treatment with 1 μM B[a]P, levels of DNA adducts reached 175 adducts/108 nucleotides, whereas the 0.1 μM concentration of B[a]P induced much lower levels of DNA adducts (≈10 adducts/108 nucleotides). Interestingly, although levels of DNA adducts induced by 3-NBA after 24-h treatment were high, we observed almost 50% reduction in their levels after incubation with 3-NBA (0.5 μM). Levels of DNA adducts induced by 1-NP were low even after 24-h incubation with the compound, when the higher test dose (10 μM) induced almost 30 adducts/108 nucleotides (Figure 2B).

View Article: PubMed Central - PubMed

ABSTRACT

We investigated the toxicity of benzo[a]pyrene (B[a]P), 1-nitropyrene (1-NP) and 3-nitrobenzanthrone (3-NBA) in A549 cells. Cells were treated for 4 h and 24 h with: B[a]P (0.1 and 1 μM), 1-NP (1 and 10 μM) and 3-NBA (0.5 and 5 μM). Bulky DNA adducts, lipid peroxidation, DNA and protein oxidation and mRNA expression of CYP1A1, CYP1B1, NQO1, POR, AKR1C2 and COX2 were analyzed. Bulky DNA adducts were induced after both treatment periods; the effect of 1-NP was weak. 3-NBA induced high levels of bulky DNA adducts even after 4-h treatment, suggesting rapid metabolic activation. Oxidative DNA damage was not affected. 1-NP caused protein oxidation and weak induction of lipid peroxidation after 4-h incubation. 3-NBA induced lipid peroxidation after 24-h treatment. Unlike B[a]P, induction of the aryl hydrocarbon receptor, measured as mRNA expression levels of CYP1A1 and CYP1B1, was low after treatment with polycyclic aromatic hydrocarbon (PAH) nitro-derivatives. All test compounds induced mRNA expression of NQO1, POR, and AKR1C2 after 24-h treatment. AKR1C2 expression indicates involvement of processes associated with reactive oxygen species generation. This was supported further by COX2 expression induced by 24-h treatment with 1-NP. In summary, 3-NBA was the most potent genotoxicant, whereas 1-NP exhibited the strongest oxidative properties.

No MeSH data available.


Related in: MedlinePlus