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. 2016 Sep;90(9):2261-2273.
doi: 10.1007/s00204-015-1593-7. Epub 2015 Oct 5.

Inflammation-associated extracellular β-glucuronidase alters cellular responses to the chemical carcinogen benzo[a]pyrene

Q Shi  1 , G R Haenen  1 , L Maas  1 , V M Arlt  2   3 , D Spina  4 , Y Riffo Vasquez  4 , E Moonen  1 , C Veith  1 , F J Van Schooten  1 , R W L Godschalk  5
Affiliations

Inflammation-associated extracellular β-glucuronidase alters cellular responses to the chemical carcinogen benzo[a]pyrene

Q Shi et al. Arch Toxicol. 2016 Sep.

Abstract

Neutrophils infiltrate tissues during inflammation, and when activated, they release β-glucuronidase. Since inflammation is associated with carcinogenesis, we investigated how extracellular β-glucuronidase changed the in vitro cellular response to the chemical carcinogen benzo(a)pyrene (B[a]P). For this we exposed human liver (HepG2) and lung (A549) cells to B[a]P in the presence or absence of β-glucuronidase. β-Glucuronidase reduced B[a]P-induced expression of CYP1A1 and CYP1B1 at 6 h after exposure, which did not depend on β-glucuronidase activity, because the inhibitor D-saccharic acid 1,4-lactone monohydrate did not antagonize the effect of β-glucuronidase. On the other hand, the inhibitory effect of β-glucuronidase on CYP expression was dependent on signalling via the insulin-like growth factor receptor (IGF2R, a known receptor for β-glucuronidase), because co-incubation with the IGF2R inhibitor mannose-6-phosphate completely abolished the effect of β-glucuronidase. Extracellular β-glucuronidase also reduced the formation of several B[a]P metabolites and B[a]P-DNA adducts. Interestingly, at 24 h of exposure, β-glucuronidase significantly enhanced CYP expression, probably because β-glucuronidase de-glucuronidated B[a]P metabolites, which continued to trigger the aryl hydrocarbon receptor (Ah receptor) and induced expression of CYP1A1 (in both cell lines) and CYP1B1 (in A549 only). Consequently, significantly higher concentrations of B[a]P metabolites and DNA adducts were found in β-glucuronidase-treated cells at 24 h. DNA adduct levels peaked at 48 h in cells that were exposed to B[a]P and treated with β-glucuronidase. Overall, these data show that β-glucuronidase alters the cellular response to B[a]P and ultimately enhances B[a]P-induced DNA adduct levels.

Keywords: Benzo[a]pyrene; Carcinogen metabolism; Cytochrome P450 1A1; DNA adducts; IGF2R; Inflammation; β-Glucuronidase.

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Conflict of interest statement

The authors declare that there are no conflicts of interest in this study.

Figures

Fig. 1
Fig. 1
Critical steps of B[a]P activation and UDP-glucuronosyltransferases (UGTs) detoxification. B[a]P is metabolized to hydroxylated B[a]P including B[a]P-7,8-diol and these B[a]P metabolites are further detoxified by UGTs. β-Glucuronidases are able to hydrolyse glucuronidated B[a]P metabolites and therefore increase the amount of B[a]P-7,8-diol, consequently leading to more BPDE and DNA adducts formation
Fig. 2
Fig. 2
RT-qPCR analysis of gene expression CYP1A1 in both A549 (a) and HepG2 (b) cells after exposure to β-glucuronidase and/or B[a]P. Cells were exposed to 1 μM B[a]P with or without 4 U/ml β-glucuronidase and harvested after the times indicated. Cells exposed to DMSO and sodium acetate buffer were used as a vehicle control. All values are given as the mean ± SEM (n = 4 per data point). (*p < 0.05; **p < 0.01; ***p < 0.001)
Fig. 3
Fig. 3
RT-qPCR analysis of gene expression CYP1B in both A549 (a) and HepG2 cells (b) after exposure to β-glucuronidase and/or B[a]P. Cells were exposed to 1 μM B[a]P with or without 4 U/ml β-glucuronidase and harvested after the times indicated. Cells exposed to DMSO and sodium acetate buffer were used as a vehicle control. All values are given as the mean ± SEM (n = 4 per data point). (*p < 0.05; **p < 0.01; ***p < 0.001)
Fig. 4
Fig. 4
RT-qPCR analysis of gene expression UGT1A6 in A549 (a) and HepG2 (b) cells after exposure to β-glucuronidase and/or B[a]P. Cells were exposed to 1 μM B[a]P with or without 4 U/ml β-glucuronidase and harvested after the times indicated. Cells exposed to DMSO and sodium acetate buffer were used as a vehicle control. All values are given as the mean ± SEM (n = 4 per data point). (*p < 0.05; **p < 0.01; ***p < 0.001)
Fig. 5
Fig. 5
HPLC fluorescence analysis of B[a]P-7,8-diol, B[a]P-9,10-diol, 3-OH-B[a]P and B[a]P in HepG2 cells (left column) and A549 cells (right column) after exposure to β-glucuronidase and/or B[a]P. Cells were exposed to 1 μM B[a]P with or without 4 U/ml β-glucuronidase and cell medium was harvested after the time indicated. Cells exposed to DMSO and sodium acetate buffer were used as a vehicle control. All values are given as the mean ± SEM (n = 3 per data point). Filled circle without β-glucuronidase, open circle with β-glucuronidase
Fig. 6
Fig. 6
32P-Postlabelling analysis of DNA adducts level in HepG2 cells and A549 cells after exposure to B[a]P with or without β-glucuronidase. a HepG2 cells were exposed to 1 μM B[a]P with or without 10 μg/ml β-glucuronidase and harvest after the times indicated. b A549 cells were exposed to 1 μM B[a]P with or without 4 U/ml β-glucuronidase and harvest after the times indicated. Data are expressed as number of B[a]P–DNA adducts per 107 nucleotides (n = 5 for HepG2 cells and n = 4 for A549 cells, mean ± SEM) (*p < 0.05; ***p < 0.001). Representative chromatograms obtained by 32P-postlabelling in HepG2 cells (c) and A549 cells (d). The adduct spot (arrow) that migrated during 2D-TLC to the same position as the major DNA adduct in a BPDE-DNA adduct standard was quantitated in all samples. Before phosphorimaging of the TLC plates the origin located at the bottom left-hand corner was excised
Fig. 7
Fig. 7
4MUgIA assay was applied to assess the possibility of B[a]P to bind to β-glucuronidase. A total volume of 140 μl contained 0.1 M sodium acetate buffer (pH 5.5), different concentration of 4MUgIA (e.g. 500, 250, 100, 50, 10 and 1 μM), and 4 U/ml β-glucuronidase with 1 μl of 200 μM B[a]P or 1 μl DMSO. The measurement of fluorescence [Relative Fluorescence Unit (RFU)] was performed for 10 h at 37 °C. B[a]P-treated samples were compared with control at each concentration, respectively. (*p < 0.05; **p < 0.01; ***p < 0.001)
Fig. 8
Fig. 8
a 4MUgIA assay was applied to assess the inhibition of β-glucuronidase by d-saccharic acid 1,4-lactone monohydrate. A total volume of 140 μl contained 0.1 M sodium acetate buffer (pH 5.5), 2 mM 4MUgIA, 4 U/ml β-glucuronidase and different concentration of d-saccharic acid 1,4-lactone monohydrate (10 μM, 100 and 500 μM). 100 μM of d-saccharic acid 1,4-lactone monohydrate was used in the following incubation. b A549 cells were exposed to 1 μM B[a]P with or without β-glucuronidase, d-saccharic acid 1,4-lactone monohydrate for 6 and 24 h. Cells exposed to 1 μM B[a]P and sodium acetate buffer was used as a control. All values are given as the mean ± SEM (n = 4 per data point) (*p < 0.05; **p < 0.01; ***p < 0.001)
Fig. 9
Fig. 9
RT-qPCR analysis of gene expression CYP1A1 in A549 cells after exposure to β-glucuronidase and B[a]P with the IGF2R inhibitor M6P. Cells were exposed to 1 μM B[a]P with or without 4 U/ml β-glucuronidase and with or without 100 μM M6P. Cells were harvested after the times indicated. Cells exposed to B[a]P and sodium acetate buffer were used as vehicle control. All values are given as the mean ± SEM (n = 4 per data point) (*p < 0.05; **p < 0.01; ***p < 0.001)
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