Difference between revisions of "Part:BBa K5477047"

Latest revision as of 13:20, 2 October 2024

Detoxification device against dioxin and PCBs

This system consists of two detoxification modules in yeast to create a device for detoxification of contaminants, including dioxins and polychlorinated biphenyls (PCBs). These modules, CYP1A1-pGAL1/10-POR and UDPD-pGAL1/10-UGT1A1, work in tandem under the control of the pGAL1/10 bidirectional promoter, which allows for the expression of the enzymes in opposite directions.

The CYP1A1-pGAL1/10-POR module catalyzes the oxidation of compounds like dioxin and PCBs, converting these toxic hydrophobic molecules into more reactive and soluble intermediates (2) (3). This reaction requires electrons supplied by cytochrome P450 oxidoreductase (POR), which transfers electrons from NADPH to CYP1A1. The UDPD-pGAL1/10-UGT1A1 module complements this system by facilitating phase II metabolism. UGT1A1 (UDP-glucuronosyltransferase 1A1) conjugates glucuronic acid to the oxidized intermediates produced by CYP1A1. However, for this reaction to proceed, UDP-glucuronic acid is needed, which is generated by UDP-glucose dehydrogenase (UDPD). UDPD converts UDP-glucose into UDP-glucuronic acid, providing UGT1A1 with the necessary substrate for the glucuronidation process (1).

Together, these two modules, regulated by the bidirectional pGAL1/10 promoter, form an integrated detoxification system that mimics both phase I (oxidation) and phase II (conjugation) of human metabolism. By expressing these systems in yeast, we can efficiently detoxify environmental pollutants such as dioxins, and PCBs, transforming them into less toxic, more water-soluble metabolites that can be readily excreted. The following composites were used to build this device: BBa_K5477037 and BBa_K5477036

Results

Objective: To evaluate the detoxification system by incubating it with specific contaminants and performing LC-MS analysis to determine whether new compounds are produced.

Methodology: The detoxification module CYP1A1-pGAL1/10-POR with UDPD-pGAL1/10-UGT1A1 was induced with galactose and incubated with PCB. Following overnight incubation, each detoxification system was centrifuged in Eppendorf tubes to separate the cells from the supernatant. The supernatant, henceforth referred to as the “media” samples, was collected. Absolute ethanol was added to the cell pellets, which were vortexed with micro glass beads to lyse the cells. The mixtures were centrifuged to separate cellular debris from the lysate, and the resulting supernatant was collected as the “pellet” samples.

All samples were filtered prior to being loaded into LC-MS glass vials.

Result: We did not have optimized LC-MS methods available for detecting highly hydrophobic compounds, even with the use of a Reverse Phase Column. Additionally, it took some time to identify ethanol as a suitable solvent for the system available in the department. Therefore, the results presented here are based on data generated from a single run of the samples on the LC-MS system. A previous run, where DMSO was used as a solvent, was excluded from the experiment due to the presence of multiple nonspecific peaks.

Here the system was incubated with 10 µM of PCB#3

Figure 1 Base Peak Chromatogram for C1A1 detox system zoomed in to 9.6 minutes to 10.8 minutes retention time. Brown peaks represent C1A1 + PCB pellet samples. Here we can see a peak at retention time 21.2 which can be considered to be unique.

Figure 3 Mass spectrum for Empty yeast system pellet sample zoomed at specific peak between 21.2 retention time When the mass spectra for the unique looking peak is compared to the mass spectra of the empty yeast cell pellet we find most similar peaks in both. However particular peaks in the C1A1 pellet sample that seem to be unique include ions of masses 107.0825+1 and 188.1259+1. We hypothesize this could be by products given by the enzyme on incubation. We wold in future like to anlayze this further.

Sequence and Features

References

1. Inui H, Itoh T, Yamamoto K, Ikushiro S, Sakaki T. Mammalian cytochrome P450-dependent metabolism of polychlorinated dibenzo-p-dioxins and coplanar polychlorinated biphenyls. Int J Mol Sci. 2014 Aug 13;15(8):14044-57. doi: 10.3390/ijms150814044. PMID: 25123135; PMCID: PMC4159838.

2. Grimm FA, Hu D, Kania-Korwel I, Lehmler HJ, Ludewig G, Hornbuckle KC, et al. Metabolism and metabolites of polychlorinated biphenyls. Crit Rev Toxicol. 2015 Mar;45(3):245–72.

3. Liu J, Tan Y, Song E, Song Y. A Critical Review of Polychlorinated Biphenyls Metabolism, Metabolites, and Their Correlation with Oxidative Stress. Chem Res Toxicol. 2020 Aug 17;33(8):2022–42.