Abstract
Exposure to xenobiotic such as benzo[a]pyrene (B[a]P) induces metabolic changes, which have a considerable impact on the cellular response. Nevertheless, we are just in the beginning to reach an understanding of these processes. In this study, a gas chromatography–mass spectrometry (GC–MS)-based metabolomics approach was applied to distinguish the metabolic changes that bladder epithelia cells undergo upon B[a]P exposure. To closely reflect the epithelia cell conditions in vivo, freshly isolated primary porcine urinary bladder epithelial cells (PUBEC) were utilized for the current study. An untargeted metabolomics approach was used to characterize the time- (6 h, 24 h, 48 h) and dose-dependent (0.5 µM, 5 µM, 10 µM B[a]P) changes in the metabolome of PUBEC upon B[a]P exposure, which led to the profiling of more than 200 metabolites that differed significantly between control and exposed samples. Multivariate analysis of the data highlighted that in the experimental setup/model used other than the exposure concentration, it is the exposure time which seems to be most important for distinguishing between different groups and hence may have a bigger role in B[a]P-mediated toxicity but may be specific for cell model used and hence requires further investigations. Further, enrichment and pathway analysis using MetaboAnalyst highlighted that exposure to B[a]P mainly alters the cellular amino acid metabolism. Particularly, 1-pyrroline-5-carboxylic acid (P5C), an intermediate of the cycling of the amino acid proline, was identified as a differentially altered metabolite at all concentrations and exposure times used in the experiment. An increase in the activity of proline dehydrogenase/proline oxidase (PRODH/POX), which oxidizes proline to P5C, was also observed, further supporting our metabolomic data. Our findings contribute to an improved knowledge about the reprogramming of metabolism which is a fundamental element of the cellular response to B[a]P and draw attention to the role of proline in this context.
Similar content being viewed by others
References
Boada LD, Henriquez-Hernandez LA, Navarro P et al (2015) Exposure to polycyclic aromatic hydrocarbons (PAHs) and bladder cancer: evaluation from a gene-environment perspective in a hospital-based case-control study in the Canary Islands (Spain). Int J Occup Environ Health 21(1):23–30. https://doi.org/10.1179/2049396714Y.0000000085
Chong J, Soufan O, Li C et al (2018) MetaboAnalyst 4.0: towards more transparent and integrative metabolomics analysis. Nucleic Acids Res 46(W1):W486–W494. https://doi.org/10.1093/nar/gky310
Czerniak B, Dinney C, McConkey D (2016) Origins of bladder cancer. Annu Rev Pathol 11(1):149–174. https://doi.org/10.1146/annurev-pathol-012513-104703
Domingo-Almenara X, Brezmes J, Vinaixa M et al (2016) eRah: a computational tool integrating spectral deconvolution and alignment with quantification and identification of metabolites in GC/MS-based metabolomics. Anal Chem 88(19):9821–9829. https://doi.org/10.1021/acs.analchem.6b02927
Donald SP, Sun XY, Hu CA et al (2001) Proline oxidase, encoded by p53-induced gene-6, catalyzes the generation of proline-dependent reactive oxygen species. Cancer Res 61(5):1810–1815
Forster K, Preuss R, Rossbach B, Bruning T, Angerer J, Simon P (2008) 3-Hydroxybenzo[a]pyrene in the urine of workers with occupational exposure to polycyclic aromatic hydrocarbons in different industries. Occup Environ Med 65(4):224–229. https://doi.org/10.1136/oem.2006.030809
Fӧllmann W, Guhe C (1994) A cell culture model of isolated porcine urinary bladder epithelial cells for genotoxicity studies. Toxicol In Vitro 8(4):763–765. https://doi.org/10.1016/0887-2333(94)90062-0
Guhe C, Degen GH, Schuhmacher US, Kiefer F, Follmann W (1996) Drug metabolizing enzyme activities in porcine urinary bladder epithelial cell cultures (PUBEC). Arch Toxicol 70(10):599–606. https://doi.org/10.1007/s002040050318
Horai H, Arita M, Kanaya S et al (2010) MassBank: a public repository for sharing mass spectral data for life sciences. J Mass Spectrom 45(7):703–714. https://doi.org/10.1002/jms.1777
Kiriluk KJ, Prasad SM, Patel AR, Steinberg GD, Smith ND (2012) Bladder cancer risk from occupational and environmental exposures. Urol Oncol 30(2):199–211. https://doi.org/10.1016/j.urolonc.2011.10.010
Kopka J, Schauer N, Krueger S (2005) GMD@CSB.DB: the Golm metabolome database. Bioinformatics 21(8):1635–1638. https://doi.org/10.1093/bioinformatics/bti236
Kowaloff EM, Phang JM, Granger AS, Downing SJ (1977) Regulation of proline oxidase activity by lactate. Proc Natl Acad Sci USA 74(12):5368–5371. https://doi.org/10.1073/pnas.74.12.5368
Lilienfeld AM (1964) The relationship of bladder cancer to smoking. Am J Public Health Nations Health 54(11):1864–1875. https://doi.org/10.2105/ajph.54.11.1864
Liu Y, Borchert GL, Donald SP, Diwan BA, Anver M, Phang JM (2009) Proline oxidase functions as a mitochondrial tumor suppressor in human cancers. Cancer Res 69(16):6414–6422. https://doi.org/10.1158/0008-5472.CAN-09-1223
Nemoto N, Sakurai J (1991) Proline is required for transcriptional control of the aromatic hydrocarbon-inducible P(1)450 gene in C57BL/6 mouse monolayer-cultured hepatocytes. Jpn J Cancer Res 82(8):901–908. https://doi.org/10.1111/j.1349-7006.1991.tb01919.x
Nemoto N, Sakurai J, Tazawa A, Ishikawa T (1989) Proline-dependent expression of aryl hydrocarbon hydroxylase in C57BL/6 mouse hepatocytes in primary culture. Cancer Res 49(21):5863–5869
Pandhare J, Dash S, Jones B, Villalta F, Dash C (2015) A novel role of proline oxidase in HIV-1 envelope glycoprotein-induced neuronal autophagy. J Biol Chem 290(42):25439–25451. https://doi.org/10.1074/jbc.M115.652776
Pei XH, Nakanishi Y, Takayama K, Bai F, Hara N (1999) Benzo[a]pyrene activates the human p53 gene through induction of nuclear factor kappaB activity. J Biol Chem 274(49):35240–35246. https://doi.org/10.1074/jbc.274.49.35240
Phang JM (1985) The regulatory functions of proline and pyrroline-5-carboxylic acid. Curr Top Cell Regul 25:91–132
Phang JM (2019) Proline metabolism in cell regulation and cancer biology: recent advances and hypotheses. Antioxid Redox Signal 30(4):635–649. https://doi.org/10.1089/ars.2017.7350
Phang JM, Downing SJ, Yeh GC (1980) Linkage of the HMP pathway to ATP generation by the proline cycle. Biochem Biophys Res Commun 93(2):462–470. https://doi.org/10.1016/0006-291x(80)91100-6
Phang JM, Liu W, Hancock C, Christian KJ (2012) The proline regulatory axis and cancer. Front Oncol 2:60. https://doi.org/10.3389/fonc.2012.00060
Phang JM, Liu W, Hancock CN, Fischer JW (2015) Proline metabolism and cancer: emerging links to glutamine and collagen. Curr Opin Clin Nutr Metab Care 18(1):71–77. https://doi.org/10.1097/MCO.0000000000000121
Plottner S, Selinski S, Bolt HM et al (2009) Distinct subtypes of urinary bladder epithelial cells with inducible and non-inducible cytochrome P450 1A1. Arch Toxicol 83(2):131–138. https://doi.org/10.1007/s00204-008-0329-3
Polyak K, Xia Y, Zweier JL, Kinzler KW, Vogelstein B (1997) A model for p53-induced apoptosis. Nature 389(6648):300–305. https://doi.org/10.1038/38525
Rink M, Crivelli JJ, Shariat SF, Chun FK, Messing EM, Soloway MS (2015) Smoking and bladder cancer: a systematic review of risk and outcomes. Eur Urol Focus 1(1):17–27. https://doi.org/10.1016/j.euf.2014.11.001
Rivera A, Maxwell SA (2005) The p53-induced gene-6 (proline oxidase) mediates apoptosis through a calcineurin-dependent pathway. J Biol Chem 280(32):29346–29354. https://doi.org/10.1074/jbc.M504852200
Roemer E, Stabbert R, Rustemeier K et al (2004) Chemical composition, cytotoxicity and mutagenicity of smoke from US commercial and reference cigarettes smoked under two sets of machine smoking conditions. Toxicology 195(1):31–52. https://doi.org/10.1016/j.tox.2003.08.006
Verma N, Bauerlein C, Pink M, Rettenmeier AW, Schmitz-Spanke S (2011) Proteome and phosphoproteome of primary cultured pig urothelial cells. Electrophoresis 32(24):3600–3611. https://doi.org/10.1002/elps.201100220
Verma N, Pink M, Petrat F, Rettenmeier AW, Schmitz-Spanke S (2012) Exposure of primary porcine urothelial cells to benzo(a)pyrene: in vitro uptake, intracellular concentration, and biological response. Arch Toxicol 86(12):1861–1871. https://doi.org/10.1007/s00204-012-0899-y
Verma N, Pink M, Rettenmeier AW, Schmitz-Spanke S (2013) Benzo[a]pyrene-mediated toxicity in primary pig bladder epithelial cells: a proteomic approach. J Proteomics 85:53–64. https://doi.org/10.1016/j.jprot.2013.04.016
Verma N, Pink M, Boland S, Rettenmeier AW, Schmitz-Spanke S (2017) Benzo[a]pyrene-induced metabolic shift from glycolysis to pentose phosphate pathway in the human bladder cancer cell line RT4. Sci Rep 7(1):9773. https://doi.org/10.1038/s41598-017-09936-1
Wolf A, Kutz A, Plottner S et al (2005) The effect of benzo(a)pyrene on porcine urinary bladder epithelial cells analyzed for the expression of selected genes and cellular toxicological endpoints. Toxicology 207(2):255–269. https://doi.org/10.1016/j.tox.2004.09.006
Zhang J, Pavlova NN, Thompson CB (2017) Cancer cell metabolism: the essential role of the nonessential amino acid, glutamine. EMBO J 36(10):1302–1315. https://doi.org/10.15252/embj.201696151
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
The authors declare no conflict of interests.
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Verma, N., Pink, M., Kersch, C. et al. Benzo[a]pyrene mediated time- and dose-dependent alteration in cellular metabolism of primary pig bladder cells with emphasis on proline cycling. Arch Toxicol 93, 2593–2602 (2019). https://doi.org/10.1007/s00204-019-02521-7
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00204-019-02521-7