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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.

No MeSH data available.


Relative mRNA levels (fold changes relative to the control) induced after treatment of A549 cells with benzo[a]pyrene, 1-nitropyrene and 3-nitrobenzanthrone. (A) CYP1A1, 4 h; (B) CYP1A1, 24 h; (C) CYP1B1, 4 h; (D) CYP1B1, 24 h; (E) NQO1, 4 h; (F) NQO1, 24 h; (G) POR, 4 h; (H) POR, 24 h. Data represent mean values ± standard deviation from two triplicates from two independent experiments (analyzed as n = 6). Asterisks denote significant (* p < 0.05) gene expression changes relative to the control. Control expression level of the respective gene is represented by an emboldened horizontal line.
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ijms-17-01393-f006: Relative mRNA levels (fold changes relative to the control) induced after treatment of A549 cells with benzo[a]pyrene, 1-nitropyrene and 3-nitrobenzanthrone. (A) CYP1A1, 4 h; (B) CYP1A1, 24 h; (C) CYP1B1, 4 h; (D) CYP1B1, 24 h; (E) NQO1, 4 h; (F) NQO1, 24 h; (G) POR, 4 h; (H) POR, 24 h. Data represent mean values ± standard deviation from two triplicates from two independent experiments (analyzed as n = 6). Asterisks denote significant (* p < 0.05) gene expression changes relative to the control. Control expression level of the respective gene is represented by an emboldened horizontal line.

Mentions: Expression levels of genes encoding metabolic activation enzymes are shown in Figure 6. As expected, B[a]P induced CYP1A1 expression in both tested doses and intervals (Figure 6A,B). Similarly, this compound induced CYP1B1 expression, although no significant effect was observed for the 4-h treatment with 0.1 μM B[a]P. Both tested nitro-PAHs required the 24-h treatment to exert consistent induction of CYP1A1 and CYP1B1 expression (Figure 6B,D), although 3-NBA (5 μM) induced CYP1A1 expression (Figure 6A) and 1-NP (1 μM) induced CYP1B1 expression (Figure 6C) after the 4-h treatment. However, the induction levels were weak compared to those after the B[a]P treatment. None of the tested compounds had significant effect on NQO1 and POR expression after the 4-h treatment (Figure 6E,G). In most cases, the longer treatment period increased expression of these mRNAs significantly (Figure 6F,H). For NQO1, no effects were observed for 1-NP at 10 μM (Figure 6F). Substantial differences in induction levels between compounds were not observed, but B[a]P tended to induce higher expression of NQO1. Highest levels of POR expression were induced after 1-NP treatment; for B[a]P and 3-NBA, only the lower test dose induced significant changes in gene expression (Figure 6H).


Toxic Effects of the Major Components of Diesel Exhaust in Human Alveolar Basal Epithelial Cells (A549)
Relative mRNA levels (fold changes relative to the control) induced after treatment of A549 cells with benzo[a]pyrene, 1-nitropyrene and 3-nitrobenzanthrone. (A) CYP1A1, 4 h; (B) CYP1A1, 24 h; (C) CYP1B1, 4 h; (D) CYP1B1, 24 h; (E) NQO1, 4 h; (F) NQO1, 24 h; (G) POR, 4 h; (H) POR, 24 h. Data represent mean values ± standard deviation from two triplicates from two independent experiments (analyzed as n = 6). Asterisks denote significant (* p < 0.05) gene expression changes relative to the control. Control expression level of the respective gene is represented by an emboldened horizontal line.
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Related In: Results  -  Collection

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ijms-17-01393-f006: Relative mRNA levels (fold changes relative to the control) induced after treatment of A549 cells with benzo[a]pyrene, 1-nitropyrene and 3-nitrobenzanthrone. (A) CYP1A1, 4 h; (B) CYP1A1, 24 h; (C) CYP1B1, 4 h; (D) CYP1B1, 24 h; (E) NQO1, 4 h; (F) NQO1, 24 h; (G) POR, 4 h; (H) POR, 24 h. Data represent mean values ± standard deviation from two triplicates from two independent experiments (analyzed as n = 6). Asterisks denote significant (* p < 0.05) gene expression changes relative to the control. Control expression level of the respective gene is represented by an emboldened horizontal line.
Mentions: Expression levels of genes encoding metabolic activation enzymes are shown in Figure 6. As expected, B[a]P induced CYP1A1 expression in both tested doses and intervals (Figure 6A,B). Similarly, this compound induced CYP1B1 expression, although no significant effect was observed for the 4-h treatment with 0.1 μM B[a]P. Both tested nitro-PAHs required the 24-h treatment to exert consistent induction of CYP1A1 and CYP1B1 expression (Figure 6B,D), although 3-NBA (5 μM) induced CYP1A1 expression (Figure 6A) and 1-NP (1 μM) induced CYP1B1 expression (Figure 6C) after the 4-h treatment. However, the induction levels were weak compared to those after the B[a]P treatment. None of the tested compounds had significant effect on NQO1 and POR expression after the 4-h treatment (Figure 6E,G). In most cases, the longer treatment period increased expression of these mRNAs significantly (Figure 6F,H). For NQO1, no effects were observed for 1-NP at 10 μM (Figure 6F). Substantial differences in induction levels between compounds were not observed, but B[a]P tended to induce higher expression of NQO1. Highest levels of POR expression were induced after 1-NP treatment; for B[a]P and 3-NBA, only the lower test dose induced significant changes in gene expression (Figure 6H).

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 &mu;M), 1-NP (1 and 10 &mu;M) and 3-NBA (0.5 and 5 &mu;M). 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.