Energetic costs of pyrene metabolism in isolated hepatocytes of rainbow trout, Oncorhynchus mykiss

Abstract

The biotransformation of xenobiotics by fish can be important to their survival while living in contaminated environments. However, the energetic costs of such detoxification are unknown. This study examined the respiratory costs of pyrene biotransformation and excretion in isolated hepatocytes of adult rainbow trout, Oncorhynchus mykiss. Baseline oxygen consumption rates measured at an acclimation temperature of 7.5"C and during an acute temperature increase to 15•‹C were 10.1 kO.l and 22.6k0.4 ng O2 min-I mg cells-', respectively. Hepatoctyes exposed to 1, 5 and 10 vglml pyrene at 7.5"C exhibited concentration-dependent increases in oxygen consumption (ng O2 min-I mg cells-'): 18.8+0.7% (increase compared to acclimated baseline), 31 . I kO.8% and 40.6+0.7%, respectively. Exposure of cells to pyrene at 1 5OC also elevated oxygen consumption, however, the relationship with pyrene concentration was biphasic. Oxygen consumption rates of hepatocytes exposed to 1, 5 and 10 ~g l m l pyrene at 15OC increased l3.2+l.6%, 34.1 +I .7%, and 21.7+0.8%, respectively. The major metabolite identified was conjugated I-hydroxypyrene. At 7.50C1 increased pyrene metabolism correlated with increased hepatocyte respiration rates. At 15OC, however, pyrene metabolism reached a maximum at 5 pglml, suggesting saturation of detoxification enzymes, which correlated with maximum respiration rates at this concentration. Measures of respiration by isolated mitochondria indicated that changes in hepatocyte oxygen consumption were not through direct effects of pyrene or 1-OH pyrene on mitochondria. Furthermore, cytotoxicity tests via cell viability and LDH assays showed that the pyrene concentrations selected in this experiment did not significantly affect the integrity of the cells. Overall, this study indicates that significant respiratory iii costs may be accrued by teleost hepatocytes actively metabolizing and secreting xenobiotic compounds. DEDICATIONThis thesis is dedicated to the members of my family: my father (Gian), my mother (Joginder) and my brothers (Sukhjit and Sunny). ACKNOWLEDGMENTS

Functional and energetic characterization of P-gp-mediated doxorubicin transport in rainbow trout (Oncorhynchus mykiss) hepatocytes

“…Studies have examined energetic costs of cellular defense mechanisms by studying effects at the cellular and biochemical level; however, most studies have not been able to assess energetic costs associated with detoxification without also taking into account toxic effects which may affect energy use. Recently, work with trout hepatocytes has examined respiratory costs of pyrene exposure and biotransformation (Bains and Kennedy, 2004) and respiratory costs associated with P-glycoprotein (P-gp) transport activity of Rhodamine 123 (R123) (Bains and Kenndy, 2005). The aim of the current study was to further characterize the functional activity of P-gp, and specifically, to assess the energetic costs associated with its activity in the transport of the P-gp substrate doxorubicin (DOX).…”

“…Under these conditions, several changes in the cell may be observed. These changes may include induction and over expression of drug transporters, such as P-gp, and MRP (Albertus and Laine, 2001;Gottesman and Pastan, 1993;Bard, 2002), and enzymes involved in phase I and II detoxification systems such as CYP450 (Bard, 2002), or take the form of MDR supporting mechanisms such as acceleration of ATP generating processes such as cellular respiration (Bains and Kennedy, 2004;2005).…”

“…Phase II reactions require the use of enzymes such as glucuronosyl and sulfotransferases, which use energy for the activation of substrates, while others such as glutathione (GSH) S-transferase (GST) require energy for the synthesis of GSH (Klassen and Watkins, 1999). Several recent studies have examined the energetic costs of hepatic biotransformation and P-gp transport of xenobiotics in trout hepatocytes and have suggested that both incur higher energetic costs to organisms exposed to environmental toxins when compared to unexposed groups (Bains and Kennedy, 2004;Bains and Kennedy 2005). In 2004, Bains and Kennedy examined energetic costs associated with pyrene metabolism in isolated trout hepatocytes.…”

“…Isolation of hepatocytes was performed according to Bains and Kennedy (2004) which was slightly modified from Moon et aI., (1985). A ventral incision was made from the pectoral fins to the gills followed by an incision on each side behind each pectoral fin to exposing the liver.…”

“…Accumulation experiments were performed according to Bains and Kennedy (2004) In efflux experiments, cells were allowed to accumulate DOX as described above for 60 min, after which cell suspensions were centrifuged at 42 x 9 for 3 min. The supernatant was removed and cells washed twice with HSSB, resuspended in fresh HSSB, and incubated for various time periods (0, 5, 10, 15, 30, 60, 120 min).…”