Sarah Lopez8/1/14McMartin Rotation Report

A. IntroductionDuring our lifetime we encounter various substances foreign to the body known as

xenobiotics. Xenobiotics are not necessarily harmful. For instance, during bacterial infection the medication prescribed will support treatment despite the fact that it is not man-made per say. This also doesn’t imply that xenobiotics are only formed via synthetic sources, but rather they can also be made naturally e.g. toxins molds secrete. To be excreted from the body, xenobiotics must be converted into more polar compounds via biotransformation.

Diethylene glycol (DEG) is a colorless, odorless liquid. Historically, its properties as an organic compound have made it a popular solvent for dissolving nonpolar solids in pharmaceuticals. However, it is also part of common consumer products such as antifreeze preparations, cosmetics, lubricants, brake fluids, wallpaper strippers, heating/cooling fuel and plasticizers.1 DEG is now closely regulated by FDA because exposure to a dose of 1 mg/kg may be lethal. Typically, it accumulates in kidney and liver tissue ultimately leading to kidney failure despite dialysis treatments.

DEG is converted to diglycolic acid (DGA) via a series of steps summarized in Figure 1.2 DGA has been found to be the toxic metabolite of DEG poisoning2 for many reasons. For instance, blocking metabolism of DEG by fomepizole is protective against renal failure.

Figure 1. DEG metabolic pathway

DGA is thought to cause mitochondrial dysfunction and ultimately lead to more reactive oxygen species (ROS).3The theory is that DGA indirectly affects mitochondrial enzymes by its changes in GSH (glutathione) metabolism. GSH is an important component of biotransformation. GSH is

conjugated to metabolites during a phase II reaction to increase hydrophillicity. GSH, thus, prepares metabolites for their excretion.

The goal of this work will be to study the role of GSH protection from DGA’s toxicity using HPT cells. It is expected with more treatment of DEG, GSH levels will be depleted. First, this will be examined in untreated cells as summarized here. Current treatment plans include fomepizole but if GSH is an important mechanism this new development can supplement the limited therapies available for DEG poisonings.

B. Methods

I. Digitonin fractionation4: First, preparation must be done to obtain a 40% glycerol solution (in PBS), a 6:1 silicone (Sigma 85419): mineral oil (Sigma M5904) mixture, and a 1.2 mg/ml digitonin solution. It is possible to create the 6:1 silicone: mineral oil mixture 1 day in advance and store at 4°C. For best accuracy, however, all solutions described were prepared fresh on day of use because over time solutions gain density as water from air is dissolved. Then, they are layered in the same order as listed above to make the bottom layer the glycerol solution, etc. The 6:1 oil mixture was centrifuged lightly to avoid any bubbling from the mineral oil. After layering, pre-centrifuge the eppendorf tube (no period specified but at least 1 minute) and check that the 3 layers have NOT mixed. If they have mixed, try a different oil ratio to attain the desired 3 layers. It was discovered that even 4:1 had some mixing “issues” Also, if cell suspension isn’t ready at this point place the 3-layered oil tubes in the refrigerator since separation is very temperature sensitive. Heating can lead to mixing of layers! A 500 µl sample (1 × 106 cells in PBS suspension) was added to the top oil layer and immediately centrifuged at 1400 rpm for 9 minutes at room temperature. The top layer was saved as the cytosolic fraction. The remaining layers were removed to isolate the pellet as the mitochondrial fraction. This pellet was resuspended in 200 µl PBS or 5% TCA for GSH/GSSG analysis. One modification from the work of L.H. Lash et al. was to prepare digitonin in PBS.

II. Enzyme assays: Lactate dehydrogenase (LDH) levels were measured using the Lactate Assay Kit (Biovision). α-ketoglutarate dehydrogenase (KGDH) activity was also measured using the α-Ketoglutarate Dehydrogenase Assay Kit (Biovision). The LDH and KGDH enzymes are expected to be a marker of their cellular origin (either the cytosol or mitochondria, respectively). Therefore, the ratio of LDH to KGDH activity, determined in the cytosolic fraction and pellet, confirmed success of digitonin fractionation. In order to use a more standard method, we later changed the mitochondrial enzyme to glutamate dehydrogenase (GDH). GDH activity protocols was done as described by Motherway et al.5

III. GSH: GSH-levels will be approximated using the Dugas lab protocol. However, the primary means desired to measure GSH amounts is a more specific analysis provided by High-performance liquid chromatography (HPLC). HPLC is a common technique since it can analyze small samples under large pressures and attain a high resolution to separate a mixture of thiols within a sample.

C. Results

Figure 2. LDH Assay standard curve is shown.

Table 1. LDH Concentration (u/ml)

100-fold cytosolic 0.5150-fold pellet 0.1450-fold whole cell 2.02

LDH activity is much higher in the cytosolic fraction than the pellet with nearly a 4-fold increase in LDH concentration. Each of the samples was diluted with PBS for proper fitting onto the standard curve. The whole cell sample is still not appropriately diluted and its value will require further testing.

Figure 3. KGDH Assay standard curve is shown. The data is not a true representation of the reaction since it was later discovered the NADH standard had evaporated, meaning concentrations used in the plot are not accurate.

Figure 4. KGDH activity is compared based on raw absorbance readings due to the errors in the standard curve. Digitonin fractionation was successful where the pellet is enriched in activity

beyond a 9 minute reaction time. Also, whole cells demonstrate little if no KGDH absorbance readings.

D. DiscussionHere we have applied digitonin fractionation to a unique problem of studying GSH metabolism because of its specificity to mitochondrial enzymes. Much of the work is in progress. The isolation procedure has been done repeatedly with mixed results. For simplicity, the assays are shown for an N=1 because a different extraction showed an opposite result. KGDH is a mitochondrial enzyme and should be enriched in the pellet as shown in this data. Likewise, LDH was enriched in the cytosolic fraction. Differences can occur based on length of time samples are exposed to digitonin oil layer, and ratio of mineral to silicone oils used. Other possible variables are cell density and temperature at which digitonin fractionation was done. Nonetheless, untreated cells have been shown to be separated in agreement with Table 1 and Figure 4. GSH work will be done in the future.

References1. California Poison Control System. http://www.calpoison.org/hcp/2012/callusvol10no4.html 2. Besenhofer, L.M., Adegboyega, P.M., Bartels, P., Filary, M.J., and Perala, A.W. (2010).

Toxicological Sciences 17: 25–35.3. Laundry, G.M., Martin, S., and McMartin, K.E. (2011). Toxicological Sciences 124: 35–44. 4. Lash, L.H., Visarius, T.M., Sall, J.M., Qian, W., and Tokarz, J.J. (1998). Toxicology 130: 1-15.5. Motherway, M., Tipton, K.F., McCarthy, A.D., Couée, I., and Irwin, J. (2002). Protocols in Protein

Science 1.4.15.