(182f) Understanding Mechanisms of Conazole Toxicity in Cultured Hepatocytes

The liver, being the major site of metabolism, plays a critical role in xenobiotic biotransformation and clearance. Quantifying the links between central hepatic and xenobiotic metabolism is critical to understanding the systemic response to xenobiotic exposure and overall better understanding the pathogenesis of toxicant-induced hepatic injury. Metabolic flux, the flow of carbon through metabolic networks, is a crucial indicator of cell physiology. Flux Balance analysis (FBA) has found widespread application to characterizing intracellular fluxes and describing cellular states. We are utilizing FBA along with pathway analysis to explore the effect of the conazoles (triadimefon and myclobutanil) on the interaction between central hepatic metabolism and detoxification pathways. Conazoles are a class of azole fungicides used in agriculture to prevent fungal growth in fruit vegetables and seeds, and in pharmaceuticals for the treatment of local and systemic fungal infections. Certain conazoles are found to be tumorigenic in rats and mice (1,2,3).

In this study, cultured primary hepatocytes are treated to varying doses (up to 1mM) of triadimefon and myclobutanil on a temporal basis to analyze interactions between xenobiotic transformation and central hepatic metabolism. The experimental system is as follows. Hepatocytes are plated at a density of one million cells per ml in a collagen sandwich configuration that mimics the organization of the liver sinusoid, allowing the cultured hepatocytes to maintain structural integrity and stable, differentiated functions for periods up to two months (4). The medium is changed daily for 3-6 days of stabilization. At this time, they are challenged with media containing conazoles. Following exposure, medium is collected daily for 3 days, and cells are sacrificed each day for viability and damage. Further, concentration of various metabolites (conazole, glucose, urea, albumin, amino acids, fatty acids, cholesterol etc.) in the media and supernatant are measured to determine the extracellular fluxes. Preliminary dosing experiments have shown that at 0.5 mM of triadimefon, cell viability is not affected but there is a steady drop in urea production from day 1 to day 3 as compared to the control. Urea production is an important marker of hepatic function, and a drop in urea production indicates active interaction of central metabolism with detoxification pathways. Further, at 1.0 mM of triadimefon, the drop in urea production was enhanced and was accompanied by cell death.

Using the extracellular measurements from the in vitro culture experiment, a developed metabolic network that depicts hepatic metabolism and conazole detoxification pathways and stoichiometric balances on each metabolite in a FBA optimization framework, intracellular fluxes under varying levels of conazoles can be evaluated. We use pathway analysis with Gibbs free energy changes to further constrain the undetermined system for flux evaluation (5,6). The extent to which metabolism of hepatocytes is sufficiently flexible as to allow these alterations as well as ones related to cellular energy production, nitrogen metabolism, etc. will be determined and quantified. For example, requirements of sulfur and glucose for conjugation reactions can lead to alterations in cysteine utilization and gluconeogenic pathways, respectively. Particularly, the pathways that respond the most between various treatments could elucidate the involvement of a particular subset responsible for the observed phenotype. Taken together, these findings are expected to have significant impact in our understanding of hepatocellular exposure.

References

1. Ward, W.O., Delker, D.A., Hester, S.D., Thai, S.F., Wolf, D.C., Allen, J.W. and Nesnow, S. (2006) Transcriptional profiles in liver from mice treated with hepatotumorigenic and nonhepatotumorigenic triazole conazole fungicides: Propiconazole, triadimefon, and myclobutanil. Toxicol Pathol, 34, 863-878.

2. Hester, S.D., Wolf, D.C., Nesnow, S. and Thai, S.F. (2006) Transcriptional profiles in liver from rats treated with tumorigenic and non-tumorigenic triazole conazole fungicides: Propiconazole, triadimefon, and myclobutanil. Toxicol Pathol, 34, 879-894.

3. Wolf, D.C., Allen, J.W., George, M.H., Hester, S.D., Sun, G., Moore, T., Thai, S.F., Delker, D., Winkfield, E., Leavitt, S. et al. (2006) Toxicity profiles in rats treated with tumorigenic and nontumorigenic triazole conazole fungicides: Propiconazole, triadimefon, and myclobutanil. Toxicol Pathol, 34, 895-902.

4. Dunn, J.C., Tompkins, R.G. and Yarmush, M.L. (1991) Long-term in vitro function of adult hepatocytes in a collagen sandwich configuration. Biotechnol Prog, 7, 237-245.

5. Nolan, R.P., Fenley, A.P., Lee, K. (2006) Identification of distributed metabolic objectives in the hypermetabolic liver by flux and energy balance analysis. Metab. Engg, 8(1), 30-45.

6. Llaneras, F., Picó, J. (2007). An interval approach for dealing with flux distributions and elementary modes activity patterns. J. of Theor. Biol. 246(2), 290-308.