Doxorubicin (DOX) and daunorubicin (DAUN) are effective anticancer drugs; however, considerable interpatient variability exists in their pharmacokinetics. This may be caused by altered metabolism by nonsynonymous single-nucleotide polymorphisms (ns-SNPs) in genes encoding aldo-keto reductases (AKRs) and carbonyl reductases. This study examined the effect of 27 ns-SNPs, in eight human genes, on the in vitro metabolism of both drugs to their major metabolites, doxorubicinol and daunorubicinol. Kinetic assays measured metabolite levels by high-performance liquid chromatography separation with fluorescence detection using purified, histidine-tagged, human wild-type, and variant enzymes. Maximal rate of activity (V max ), substrate affinity (K m ), turnover rate (k cat ), and catalytic efficiency (k cat /K m ) were determined. With DAUN as substrate, variants for three genes exhibited significant differences in these parameters compared with their wild-type counterparts: the A106T, R170C, and P180S variants significantly reduced metabolism compared with the AKR1C3 wild-type (V max , 23-47% decrease; k cat , 22-47%; k cat /K m , 38 -44%); the L311V variant of AKR1C4 significantly decreased V max (47% lower) and k cat and k cat /K m (both 43% lower); and the A142T variant of AKR7A2 significantly affected all kinetic parameters (V max and k cat , 61% decrease; K m , 156% increase; k cat /K m , 85% decrease). With DOX, the R170C and P180S variants of AKR1C3 showed significantly reduced V max (41-44% decrease), k cat (39 -45%), and k cat /K m (52-69%), whereas the A142T variant significantly altered all kinetic parameters for AKR7A2 (V max , 41% decrease; k cat , 44% decrease; K m , 47% increase; k cat /K m , 60% decrease). These findings suggest that ns-SNPs in human AKR1C3, AKR1C4, and AKR7A2 significantly decrease the in vitro metabolism of DOX and DAUN.

The role of metabolism in daunorubicin (DAUN)-and doxorubicin (DOX)-associated toxicity in cancer patients is dependent on whether the parent drugs or major metabolites, doxorubicinol (DOXol) and daunorubicinol (DAUNol), are the more toxic species. Therefore, we examined whether an association exists between cytotoxicity and the metabolism of these drugs in cell lines from nine different tissues. Cytotoxicity studies using MTT [3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide] cell viability assays revealed that four cell lines [HepG2 (liver), HCT-15 (colon), NCI-H460 (lung), and A-498 (kidney)] were more tolerant to DAUN and DOX than the five remaining cell lines [H9c2 (heart), PC-3 (prostate), OVCAR-4 (ovary), PANC-1 (pancreas), and MCF-7 (breast)], based on significantly higher LC 50 values at incubation times of 6, 24, and 48 hours. Each cell line was also assessed for its efficiency at metabolizing DAUN and DOX. The four drug-tolerant cell lines converted DAUN/DOX to DAUNol/DOXol more rapidly than the five drug-sensitive cell lines. We also determined whether exposure to DAUN or DOX induced an increase in metabolic activity among any of these nine different cell types. All nine cell types showed a significant increase in their ability to metabolize DAUN or DOX in response to pre-exposure to the drug. Western blot analyses demonstrated that the increased metabolic activity toward DAUN and DOX correlated with a greater abundance of eight aldo-keto and two carbonyl reductases following exposure to either drug. Overall, our findings indicate an inverse relationship between cytotoxicity and DAUN or DOX metabolism in these nine cell lines.

ABSTRACT:Carbonyl reductases (CBRs) are a group of metabolic enzymes belonging to the short-chain dehydrogenase family with NADPHdependent oxidoreductase activity. These enzymes are known to metabolize the anthracyclines doxorubicin (DOX) and daunorubicin (DAUN). Both DOX and DAUN are highly effective in cancer therapy; however, there is considerable interpatient variability in adverse effects seen in patients undergoing treatment with these drugs. This may be attributed to altered metabolism associated with nonsynonymous single nucleotide polymorphisms (ns-SNPs) in the genes encoding for CBRs. In this study, we examine the effect of the V88I and P131S mutations in the human CBR1 gene on the metabolism of anthracyclines to their respective major metabolites, doxorubicinol and daunorubicinol. Kinetic studies using purified, histidine-tagged, recombinant enzymes in a high-performance liquid chromatography-fluorescence assay demonstrated that the V88I mutation leads to a significantly reduced maximal rate of activity (V max ) (2090 ؎ 112 and 257 ؎ 11 nmol/min ⅐ mg of purified protein for DAUN and DOX, respectively) compared with that for the wild-type (3430 ؎ 241 and 364 ؎ 37 nmol/min ⅐ mg of purified protein for DAUN and DOX, respectively). In the case of the P131S mutation, a significant increase in substrate affinity (K m ) was observed for DAUN only (89 ؎ 13 M) compared with that for the wild-type (51 ؎ 13 M). In the presence of either anthracycline, both variants exhibited a 20 to 40% decrease in catalytic efficiency (k cat /K m ) compared with that for the wild-type enzyme. Therefore, the ns-SNPs generating both these mutations may alter bioavailability of these anthracyclines in cancer patients and should be examined in clinical studies as potential biomarkers for DAUN-and DOX-induced adverse effects.

Doxorubicin (DOX) and daunorubicin (DAUN) are anthracycline anticancer agents; however, considerable interpatient variability exists in their pharmacokinetics. This interpatient variability is attributed in part to altered metabolism by nonsynonymous single-nucleotide polymorphisms (ns-SNPs) in genes encoding the carbonyl reductases. This study examines the effect of seven naturally occurring ns-SNPs in the CBR3 gene on in vitro metabolism of anthracyclines to doxorubicinol and daunorubicinol. Kinetic assays measure metabolite levels by high-performance liquid chromatography separation with fluorescence detection by use of purified, histidine-tagged, human CBR3 wild type and variant enzymes. The V224M, C4Y, and V93I variants resulted in significantly reduced maximal reaction velocity (V max ) for both anthracyclines compared with the wildtype enzyme, whereas the M235L variant had significantly reduced V max for DOX only. Significant increases in substrate affinity were found for the V244M variant with DAUN, as well as the C4Y and V93I variants with DOX. The catalytic efficiency values for the V244M, C4Y, and V93I variants were significantly lower than the wild type for DAUN and DOX. Furthermore, DOX was observed to be a better substrate than DAUN for the wild-type enzyme and its variants. HapMap analysis indicated that a haplotype carrying the C4Y and V244M mutations may occur in some individuals in the 11 ethnic populations studied in the HapMap project. Our preparation of the double mutant indicated a significant reduction in activity compared with the wild-type enzyme and single-mutant preparations. These findings suggest that commonly occurring ns-SNPs in human CBR3 significantly alter the in vitro metabolism of DOX and DAUN.

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