Abstract
Human exposure to environmental carcinogens is often unavoidable. Many of these compounds initiate carcinogenesis by inducing mutagenic DNA damage. Some of these environmental carcinogens require metabolic activation to be able to damage DNA resulting in the formation of bulky DNA adducts. However, organ specificity of oncogenesis, in many cases, cannot be explained solely by DNA adduct formation in target tissue(s), as other cellular processes, including gene expression changes, also contribute to tumour development. Therefore, it is important to study the metabolic activation and/or detoxication of environmental carcinogens and the cellular changes they induce to understand which host factors contribute to their tissue specific carcinogenesis. Many studies of these mechanisms have been carried out in experimental animals, immortalised cell lines and primary cells; however, there can be inconsistencies between the translation of in vivo and in vitro findings. Human organoids are 3D cultures that to some extent reproduce the structure, composition and function of the organ they derive from; therefore, they offer the opportunity to investigate environmental carcinogens in an in vitro setting with more in vivo-like characteristics. Here, human organoids from normal gastric, pancreas, liver, colon and kidney tissues were used to study their ability to metabolise four well-characterised environmental carcinogens that have different target tissues: benzo[a]pyrene (BaP), aflatoxin B1 (AFB1), aristolochic acid I (AAI) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP).The aims of this study were to assess the potential of these tissue organoids to metabolically activate environmental carcinogens, and then to investigate carcinogen organotropism using this model system. For this purpose, organoids were treated with the selected carcinogens at a range of concentrations and different endpoints were measured. Cytotoxicity measurements were first used to select suitable concentrations of the agents for further experiments. Then metabolic activation was investigated by analysing the induction of xenobiotic-metabolising enzymes (XMEs) involved in the activation of the individual carcinogens. Bioactivation of the carcinogens was confirmed by investigating activation of the DNA damage response (DDR) pathway, and the formation of carcinogen-DNA adducts. In order to study tissue-specific responses, organoids from target and non-target tissues were selected for each compound. BaP, AFB1 and AAI treatment led to the induction of XMEs at various levels in the different tissue organoids. All carcinogens were activated by the organoids to some extent, as evidenced by DNA adduct formation and DDR protein induction. As PhIP induced the lowest levels of adducts, its reactive metabolite N-OH-PhIP was also tested on the organoids showing an increased level of adducts and therefore suggesting that the required XMEs may not be present at sufficient levels to activate PhIP. Additionally, in order to explore differences in gene expression between organoid types after BaP treatment, high-throughput RT-qPCR was used to study a panel of genes involved in cell cycle, DNA damage and repair, apoptosis, metabolism and stress responses. In general, the results of the organotropism study were inconclusive as the differences found between organoid types did not fully differentiate target from non-target organs. However, the results showed that tissue organoids are useful models in the study of environmental carcinogens and genetic toxicology.
| Date of Award | 1 Aug 2022 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | David Phillips (Supervisor), Volker Arlt (Supervisor) & Jill Kucab (Supervisor) |