Design, Synthesis and Binding Affinity Evaluation of Cytochrome P450 1B1 Targeted Chelators

Background: Cytochrome P450 1B1 (CYP1B1) is specifically expressed in a variety of tumors which makes it a promise imaging target of tumor. Objective: We aimed to design and synthesize CYP1B1 targeted chelators for the potential application in positron emission tomography (PET) imaging of tumor. Methods: 1,4,7-triazacyclononane-1,4-diiacetic acid (NODA) was connected to the CYP1B1 selective inhibitor we developed before through polyethylene glycol (PEG) linkers with different lengths. The inhibitory activities of chelators 6a-c against CYP1 family were evaluated by 7-ethoxyresorufin o-deethylation (EROD) assay. The manual docking between the chelators and the CYP1B1 are conducted subsequently. To determine the binding affinities of 6a-c to CYP1B1 in cells, we further performed a competition study at the cell level. Results: Among three chelators, 6a with the shortest linker showed the best inhibitory activity against CYP1B1. In the following molecular simulation study, protein-inhibitor complex of 6a showed the nearest F-heme distance which is consistent with the results of enzymatic assay. Finally, the cell based competitive assay proved the binding affinity of 6a-c to CYP1B1 enzyme. Conclusion: We designed and synthesized a series of chelators which can bind to CYP1B1 enzyme in cancer cells.To our knowledge, this work is the first attempt to construct CYP1B1 targeted chelators for radiolabeling and we hope it will prompt the application of CYP1B1 imaging in tumor detection.

Enzymatic, Morphological and Genotoxic Effects of Benzo[a]pyrene in Rainbow Trout (Oncorhynchus Mykiss)

Fish have defense systems that are capable of repairing damages caused by xenobiotics like benzo[a]pyrene (BaP), so the aims of this study were to identify BaP toxicity in melanomacrophages (MMs) cytoskeleton, evaluate the melanin area in MMs and analyze genotoxicity. Rainbow trout juveniles (n = 24) were split in 48h and 7d treatments that received 2 mg/kg of BaP. After the experiment, blood samples were collected and liver was removed, to proceed with the analysis: EROD activity, MMs melanin area quantification, melanosomes movements, and a genotoxicity test. The results revealed increased in EROD activity after 48h and 7d BaP exposure. The group 7d displayed a reduction in MMs pigmented area, melanosomes aggregation, in addition to an increased frequency of micronuclei. By means of the EROD assay, it was possible to confirm the activation of BaP biotransformation system. The impairment of the melanosomes’ movements possibly by an inactivation of the protein responsible for the pigment dispersion consequently affects the melanin area and thus might negatively impact the MMs detoxification capacity. In addition to this cytotoxicity, the increased frequency of micronucleus might also indicate the genotoxicity of BaP in this important fish species.

Alternaria alternata Toxins Synergistically Activate the Aryl Hydrocarbon Receptor Pathway In Vitro

Alternaria molds simultaneously produce a large variety of mycotoxins, of which several were previously reported to induce enzymes of phase I metabolism through aryl hydrocarbon receptor activation. Thus, we investigated the potential of naturally occurring Alternaria toxin mixtures to induce Cytochrome P450 (CYP) 1A1/1A2/1B1 activity. Two variants of an extract from cultured Alternaria alternata, as well as the toxins alternariol (AOH), alternariol monomethyl ether (AME), altertoxin I (ATX-I), and altertoxin II (ATX-II), were tested singularly and in binary mixtures applying the 7-ethoxy-resorufin-O-deethylase (EROD) assay in MCF-7 breast cancer cells. Sub-cytotoxic concentrations of the two toxin mixtures, as well as ATX-I, ATX-II and AOH, exhibited dose-dependent enhancements of CYP 1 activity. ATX-I and ATX-II interacted synergistically in this respect, demonstrating the two perylene quinones as major contributors to the extract’s potential. Binary mixtures between AOH and the two altertoxins respectively exhibited concentration-dependent antagonistic as well as synergistic combinatory effects. Notably, AME showed no efficacy towards EROD enzyme activity or impact on other toxins’ efficacy. Hence, this study provides insights into synergistic and other combinatory effects of Alternaria toxins in natural co-occurrence scenarios in the context of AhR signalling pathway activation in breast cancer cells.

3. Comparing the Induction of CYP1A in American Eel (Anguilla rostrata) and Rainbow Trout (Oncorhynchus mykiss) by the Dioxin‐like Compound 2,3,7,8‐ tetrachlorodibenzo‐p‐dioxin (TCDD)

The American eel population native to Lake Ontario has declined by 98% since 1985, and the associated fishery in Ontario has been rendered uneconomical. By recognizing the American eel as a “species of concern” under Canada’s Species at Risk Act, the federal government has indicated that conservation of this population is a priority. Conservation is a difficult task, however, considering the underlying mechanism of decline remains unknown. One hypothesized mechanism states that dietary bioaccumulation of dioxin‐like compounds (DLCs) by the female and transfer of these environmental contaminants to her eggs precludes proper development of eel embryos and larvae. These developmental effects have been well characterized in the rainbow trout, and include craniofacial malformations, edema, hemorrhaging, increased mortality, and up‐regulation or induction of the CYP1A enzyme. Therefore, increased CYP1A concentrations may be used as a biomarker for potential toxicity, and a quantitative means of comparing the sensitivity of different organisms to DLCs and their effects. My project uses immunohistochemistry (IHC) and the EROD assay to quantify CYP1A induction in American eel exposed to DLCs, and compares this sensitivity to that of rainbow trout, a reference species. Preliminary EROD results suggest no significant difference between eels and rainbow trout, and results of IHC analysis are currently under examination. If these data suggest an increased sensitivity of the American eel to DLCs, this would substantiate the hypothesis that chemical accumulation in adult eels and exposure to eggs during oogenesis may threaten the viability of embryos and larvae, which would precipitate the catastrophic population decline.