Characterization of toxicological effects of a novel in vivo benzo[a]pyrene metabolite in colonic cells

Cancer of the colon and rectum is the third most common cancer form in the world. Where the majority of cases fall into the category of sporadic colorectal cancer (CRC), lifestyle and diet have been found to be important risk factors associated with CRC. Although underlying mechanisms remain unclear, a possible hypothesis is that carcinogens in the diet can cause DNA damage in the colon epithelial cells either directly or by producing reactive oxygen species (ROS) or that dietary components when further metabolised by the intestinal flora generate genotoxic metabolites. Several components like; polycyclic aromatic hydrocarbons (PAH), bile acids, N-nitroso compounds (NOC), heterocyclic amines (HCA), fecapentaenes and lipid peroxidation products have been proposed to increase the risk of CRC.

Exposure to benzo[a]pyrene (BP), a well known PAH, has in animal studies been shown to increase the levels of tumours in the colon. Epidemiological evidence has, on the other hand, not been convincing until recently when smoking, a well-known source of BP, was associated with increased risk of colon cancer. Due to methodological difficulties most studies on the characterisation of BP metabolites have been performed in vitro. We have been able to identify in vivo metabolites, in urine from germ-free rats, which have not previously been identified as being formed from BP.

We have performed mechanistic studies to reveal if one of the novel in vivo metabolites of BP, the anhydride of 7-oxobenz[d]anthracene-3,4dicarboxylic acid (ABADA) exhibits any toxicological activity in cultured colonic cells. ABADA was shown to have weak mutagenic activity in Salmonella typhimurium strain TA 102 (detects oxidative mutagens). The cytotoxicity of ABADA seen in HCT 116 cells appeared to be due to apoptosis, as caspase-3 activity and poly-ADP-ribose polymerase (PARP) cleavage was observed. Using the COMET assay, we demonstrated that ABADA is able to cause extensive DNA damage where oxidative damage was an important part. We also demonstrated that ABADA caused induction of ROS, lipid peroxidation, a rise in intracellular Ca2+ and a decrease in the mitochondrial membrane potential (AT.).

Our results indicate that ABADA has toxic effects in cultured colonic cells and may be contributing to the in vivo toxicity of BP. Preliminary attempts were also made to address a possible role of genotoxicity of the type induced by this PAH metabolite in adenoma formation in human patients. Results from this study showed that this type of genotoxic activity was present in human faecal water fractions. The possibility exists that metabolites such as ABADA could be contributing to this toxicity.

List of scientific papers

I. Yang Y, Griffiths WJ, Nordling M, Nygren J, Moller L, Bergman J, Liepinsh E, Otting G, Gustafsson JA, Rafter J, Sjovall J (2000). Ring opening of benzo[a]pyrene in the germ-free rat is a novel pathway for formation of potentially genotoxic metabolites. Biochemistry. 39(50): 15585-91.
https://pubmed.ncbi.nlm.nih.gov/11112546

II. Nordling MM, Nygren J, Bergman J, Sundberg K, Rafter JJ (2002). Toxicological characterization of a novel in vivo benzo[a]pyrene metabolite, 7-oxo-benz[d]anthracene-3,4-dicarboxylic acid anhydride. Chem Res Toxicol. 15(10): 1274-80.
https://pubmed.ncbi.nlm.nih.gov/12387625

III. Nordling M, Halliwell B, Rafter JJ (2004). Induction of reactive oxygen species and intracellualar calcium by the novel in vivo benzo[a]pyrene metabolite, 7-0xo-benz[d]anthracene-3,4-dicarboxylic acid anhydride. [Manuscript]

IV. Nordling MM, Glinghammar B, Karlsson PC, de Kok TM, Rafter JJ (2003). Effects on cell proliferation, activator protein-1 and genotoxicity by fecal water from patients with colorectal adenomas. Scand J Gastroenterol. 38(5): 549-55.
https://pubmed.ncbi.nlm.nih.gov/12795469