Metabolic Activation of Benzo[ a]pyrene by Human Tissue Organoid Cultures

Abstract

Organoids are 3D cultures that to some extent reproduce the structure, composition and function of the mammalian tissues from which they derive, thereby creating in vitro systems with more in vivo-like characteristics than 2D monocultures. Here, the ability of human organoids derived from normal gastric, pancreas, liver, colon and kidney tissues to metabolise the environmental carcinogen benzo[a]pyrene (BaP) was investigated. While organoids from the different tissues showed varied cytotoxic responses to BaP, with gastric and colon organoids being the most susceptible, the xenobiotic-metabolising enzyme (XME) genes, CYP1A1 and NQO1, were highly upregulated in all organoid types, with kidney organoids having the highest levels. Furthermore, the presence of two key metabolites, BaP-t-7,8-dihydrodiol and BaP-tetrol-l-1, was detected in all organoid types, confirming their ability to metabolise BaP. BaP bioactivation was confirmed both by the activation of the DNA damage response pathway (induction of p-p53, pCHK2, p21 and γ-H2AX) and by DNA adduct formation. Overall, pancreatic and undifferentiated liver organoids formed the highest levels of DNA adducts. Colon organoids had the lowest responses in DNA adduct and metabolite formation, as well as XME expression. Additionally, high-throughput RT-qPCR explored differences in gene expression between organoid types after BaP treatment. The results demonstrate the potential usefulness of organoids for studying environmental carcinogenesis and genetic toxicology.

Keywords: 3D culture; CYP1A1; DNA adducts; DNA damage response; NQO1; RT-qPCR; benzo[a]pyrene; carcinogen; human tissue organoid.

Figure 1

Cell viability in human tissue organoids after BaP treatment. Organoids from normal human stomach (A) D95 and D88, pancreas (B) D39 and D44, kidney (C) D50 and D21, colon (D) D351 and D311, and liver (E) D4 undifferentiated and differentiated tissues were treated with various BaP concentrations (0–50 µM) for 48 h. Vehicle controls (0.5% DMSO) were included. Cell viability (% control) was measured using the CellTiter-Glo assay. Results are shown as mean ± SEM (n ≥ 3).

Figure 2

Relative gene expression of XMEs in human tissue organoids after BaP treatment. RT-qPCR and the 2^−ΔΔCT method were used to determine CYP1A1 and NQO1 expression in gastric (D95 and D88; (A,F)), pancreatic (D39 and D44; (B,G)), kidney (D50 and D21; (C,H), colon (D311 and D351; (D,I)) and liver undifferentiated and differentiated (D4; (E,J)) organoids treated with the indicated BaP concentrations for 48 h. Values were normalised to mRNA expression of the housekeeping gene GAPDH and are relative to the vehicle control (0.5% DMSO); for liver organoids the values are relative to the undifferentiated control. Results are shown as mean ± SD (n ≥ 3). Statistical analysis was performed by log2 transforming the data and a one sample t-test with Bonferroni correction against the control mean of 0: * p < 0.05; ** p < 0.01; *** p < 0.001 compared to untreated control; ^## p < 0.01; ^### p < 0.001 compared to undifferentiated liver control.

Figure 3

DDR in normal human tissue organoids after BaP treatment. Organoids from gastric D95 and D88; (A), pancreatic D39 and D44; (B), kidney D50 and D21; (C), colon D351 and D311; (D) and liver (D4 undifferentiated and differentiated; (E) tissues were treated with the indicated BaP concentrations for 48 h, and lysates were analysed by Western blotting. Various DDR proteins (p-p53, pCHK2, p21 and γ-H2AX) were detected and GAPDH was used as a loading control. cBoB + Cis (cBoB treated with 3.125 μM cisplatin) was used as a positive control. Representative blots are shown (n = 2).

Figure 4

BaP metabolite and DNA adduct levels in human tissue organoids after BaP treatment. Gastric D95 and D88; (A,F,K), pancreatic D39 and D44; (B,G,L), kidney D50 and D21; (C,H,M), colon (D311 and D351; (D,I,N) and liver undifferentiated and differentiated (D4; (E,J,O) organoids were treated with the indicated BaP concentrations for 48 h. Vehicle controls (0.5% DMSO) were included (not shown). The formation of BaP-t-7,8-dihydrodiol (A–E) and BaP-tetrol-l-1 (F–J) was determined by HPLC analysis. Metabolite levels are presented as peak area relative to phenacetine (arbitrary units). dG-N²-BPDE adduct formation was quantified using LC-ESI-MS/MS (K–O). Results are shown as mean ± SD (n ≥ 3).

Figure 5

Effects of BaP on gene expression related to xenobiotic metabolism, oxidative stress response, transcription, proliferation and cell cycle control, and apoptosis. Human tissue organoids were treated with the indicated BaP concentrations for 48 h. Gene expression changes were measured by HT RT-qPCR. Linear fold-changes for (A) NQO1, (B) UGT1A, (C) TXNRD1, (D) MDM2, (E) CDKN1A and (F) BBC3 are shown as mean ± SD (n = 3). Log2 values ±1 were considered biologically relevant (#), compared to the vehicle control (0.5% DMSO).

Figure 6

Effects of BaP on gene expression related to DNA damage response and repair. Human tissue organoids were treated with the indicated BaP concentrations for 48 h. Gene expression changes were measured by HT RT-qPCR. Linear fold-changes for (A) MGMT, (B) GADD45A, (C) DDB2 and (D) RRM2B are shown as mean ± SD (n = 3). Log2 values ±1 were considered biologically relevant (#), compared to the vehicle control (0.5% DMSO).