Formation of Covalent DNA Adducts by Enzymatically Activated Carcinogens and Drugs In Vitro and Their Determination by ³²P-postlabeling

The overall goal of this protocol is description of methods employing the reactions catalyzed by cytochrome p450 and additional biotransformation enzymes to investigate the potency of chemicals or drugs for their activation to metabolites forming covalent DNA adducts. This method can help to answer the key questions in the field of the development of cancer and its treatment. The main advantage of this technique is that it shows you a type of method to evaluate the potency of environmental chemicals or drugs to form covalent DNA adducts representing initiation of cancer development.

The implications of the technique determining the carcinogenic potency of chemicals. Environmental potence, toxicants, and drugs and also determines relatively simple methods for the formation of covalent DNA adducts formed by such compounds. Although this method can provide insight into initiation of the development of cancer and its treatment, it can also be applied to other scientific fields.

Such as studies of molecular mechanisms, of DNA damage, and oral studies on enzymatic activation and the toxification of chemicals. I first heard the idea for this method when I investigated processes involved in initiation phase of chemical carcinogenesis. First prepare the incubation mixture by adding all the compounds and the enzyme systems to a final volume of 0.75 milliliter at four degrees Celsius.

This is to incubate the carcinogen with the DNA in the presence of cytochrome p450. Then, add microsomes or pure recombinant p450 in supersome to the mixture. Next, add 7.5 microliters of 0.1 millimolar ellipticine drug dissolved in DMSO and 42.5 microliters of water to adjust the final volume to 0.75 milliliter.

Then, mix the components by vortexing the tube for five seconds. Immediately after, incubate the tube at 37 degrees celsius with the lid open for 30 to 60 minutes. Next, mix phenol or phenol chloroform in a one to one ratio with the incubation mixture in an Eppendorf Tube.

This will remove the protein contamination from the DNA in the incubation mixture. Then stir the mixture to form an emulsion. Then spin the mixture at 1600 times g for three minutes in ambient temperature.

Once the centrifugation is over use a pipette to transfer the upper water phase to a new polypropylene tube. Then discard the protein interface together with the organic phase. Next, mix the water phase with equivalent volume of phenol and chloroform in one to one ratio.

Then repeat the centrifugation. Next add equal volumes of chloroform to the water phase collected and again centrifuge. Then precipitate the DNA from the aqueous phase by adding two volumes of cold ethanol maintained at minus 20 degrees Celsius.

Then mix the solution well. To prepare for labeling the DNA adducts, mix the bicine buffer solution with radiolabeled ATP solution to label the DNA adducts. Add the labeling mixture to NP1 solution, or butanol enrichment mix.

Let the mixture stand at room temperature for 30 minutes. Then apply approximately 20 microliters at the entire sample on the PEI-cellulose TLC plate. After spotting the sample on PEI-cellulose TLC plate, leave the plate to develop in D1 direction to clean up the adducts.

Once the plate is dried up, leave the plate to develop in D3 and D4 directions. To develop in D3, use lithium formate buffer at pH 3.5 with urea. For D4 development, use Tris-HCl buffer with lithium chloride and urea.

In order to avoid any problems for developing in D4 direction, develop the plates in D5 direction as well. First, P32-postlabeling technique is utilized to study the efficiency of microsomal p450 mediated bioactivated ellipticine in forming DNA adducts. Results show formation of covalent DNA adducts in the deoxyguanosine of the calf thymus DNA in the presence of both rat and or human hepatic microsomes;however, no adducts are formed in the control group.

Next, the potential of peroxidase enzyme dependent activated ellipticine is studied. The image shows the formation of covalent DNA adducts in calf thymus DNA in the presence of horseradish peroxidase, bovine lactoperoxidase, and human myeloperoxidase. In fact, DNA adducts are also formed from the liver DNA when the rats are treated with 40 milligrams per kilogram body weight of ellipticine in vivo.

Interestingly, the human cytochrome p450 enzyme CYP3A4 also shows strong potential to efficiently oxidize ellipticine to metabolites such as 12-hydroxyellipticine and 13-hydroxyellipticine. These metabolites can easily bind to the DNA to form the adducts. Next, nuclease P1 and n-butanol enrichment methods are used to study the formation of DNA adducts upon activation of 3-nitrobenzanthrone with cytosolic reductase such as NQ01 from rat and human.

The results show formation of up to five DNA adducts from the calf thymus DNA obtained from the rat, human hepatic cytosol, and also mice liver. Once mastered, this technique can be done in two days if it is performed properly. By attempting this procedure it is important to remember the stability of the enzymatic systems used.

The enzyme activity may diminish if not stored on ice. After its development, this technique paved the way for researchers in the field of biochemistry, the biochemistry of chemical carcinogenesis to explore nature of phenomenon from the initiation processes of cancer development in organisms. After watching this video, you should have a good understanding of how to evaluate the potency of environmental chemicals or drugs to be enzymatically activated to metabolize genetic covalent DNA adducts.

This process represents high importance for development of cancer-based treatments. Don't forget that you are working with chemicals that can be extremely hazardous. Potential carcinogens, radioactive chemicals, for example phosphorus ATP.

Therefore precautions such as laboratory latex gloves and plexiglass sheets should always be taken while performing this procedure.