Current Drug Metabolism - Volume 8, Issue 1, 2007

Tert-butylhydroquinone (tBHQ) has been commonly used as a synthetic food antioxidant to prevent oils and fats from oxidative deterioration and rancidity due to its potent anti-lipid peroxidation activity. In North America, the maximum level of tBHQ allowed in fat products is 0.02% with an acceptable daily intake of 0 - 0.7 mg/kg body weight. Extensive studies have demonstrated that tBHQ exhibit anti-carcinogenic effect. The ability of tBHQ to induce phase II xenobiotic metabolizing enzymes through an Nrf2-dependent pathway is thought to be responsible for the observed protective effect of tBHQ. It has been proposed that tBHQ enhances Nrf2-mediated transcription by promoting reactive oxygen species-mediated dissociation of Nrf2-Keap1, Nrf2 stabilization, phosphatidylinositol 3-kinase (PI3K)/Akt activity, and MAPK pathway activation. In contrast to the beneficial effects of tBHQ, a number of studies have shown that chronic exposure to tBHQ may induce carcinogenicity. However, the precise mechanisms of tBHQ carcinogenicity are not well understood. The toxicity or carcinogenicity of tBHQ has been attributed to the formation of reactive GSH-conjugates, generation of reactive species, CYP1A1 induction, caspase activation and reduced GSH/ATP levels. This review provides an account of recent mechanisms proposed for both chemoprotective and carcinogenic effect of tBHQ.

Objectives: Induction or inhibition of cytochrome P450 (CYP) enzyme activities, enzymes that activate or detoxify xenobiotics, is one mechanism by which vegetables may alter cancer risk. As the effect of food on CYP enzyme activities have not been studied in the Jordanian population, we examined the effect of supplementing the diet with broccoli on CYP1A2 and CYP2A6 activities. Methods: Five men and five women, non-smokers, consumed a standard diet of broccoli (500 g) for 6 days. Enzyme activities were determined by measuring urinary metabolite ratios after a 100 mg caffeine tablet on the seventh day. Results: The mean CYP1A2 activity for men (21.1 ± 3.2) was significantly lower than that for women (27.6 ± 1.6) before the consumption of broccoli (P <0.05). These activities were significantly induced in both men (52.5 ± 6.6) and women (36.6 ± 8.4) after a standard diet of broccoli (P <0.005). Similarly, the mean value of CYP2A6 activity for men was 0.061 ± 0.040 and for women, 0.144 ± 0.039 before consumption of broccoli, which were significantly different (P <0.05). The activity of CYP2A6 was induced in both groups significantly after broccoli consumption (P <0.05). The mean value for men was 0.193 ± 0.02 and for women, 0.214 ± 0.064. Conclusion: Our study on a group of Jordanians confirmed the well-established observation that broccoli induces CYP1A2 activity. This study also demonstrates the effect of gender and broccoli consumption on CYP2A6 activity in Jordanians.

Hyperhomocysteinemia (HHCY) is a risk factor for cardiovascular (CVD) and neurodegenerative diseases, osteoporotic fractures and pregnancy complications. HHCY is common and is mostly related to B-vitamin deficiency. Retrospective and prospective studies emphasise the causal relationship between HHCY and CVD risk. Some reported vitamin intervention trials, however, did not demonstrate lower risk of CVD after treatment. Confounding factors on the one hand and low subject numbers on the other hand reduced the statistical power of the results. Re-analysis of the VISP study (after excluding renal failure and vitamin B12 status tampering factors), detected a 21% decrease in the risk of stroke. This number has been confirmed by results from the HOPE 2 vitamin intervention trial. A significant decline of strokemortality (8 to 16 %) has been observed in the USA and Canada after fortification of grain products with folate. Despite negative results from secondary prevention trials regarding the CVD risk reduction there is convincing evidence about the effectiveness of B-vitamin supplementation in lowering the risk of stroke (approximately 20%). Additionally, HHCY was recently linked to the occurrence and severity of chronic heart insufficiency. HHCY is also a risk factor for osteoporotic fractures and vitamin treatment can lower the fracture risk. HHCY predicts the decline in cognitive function with age. Hypomethylation is among the central mechanisms through which HHCY may damage the brain. HHCY and low folate are causal factors for pregnancy complications. In addition to the recommended folate supplementation, vitamin B12 supplementation may also decrease pregnancy complications.

Reported predictions of human in vivo hepatic clearance from in vitro data have used a variety of values for the scaling factors human microsomal protein (MPPGL) and hepatocellularity (HPGL) per gram of liver, generally with no consideration of the extent of their inter-individual variability. We have collated and analysed data from a number of sources, to provide weighted meangeo values of human MPPGL and HPGL of 32 mg g-1 (95% Confidence Interval (CI); 29 - 34 mg.g-1) and 99 x 106 cells.g-1 (95% CI; 74 131 mg.g-1), respectively. Although inter-individual variability in values of MPPGL and HPGL was statistically significant, gender, smoking or alcohol consumption could not be detected as significant covariates by multiple linear regression. However, there was a weak but statistically significant inverse relationship between age and both MPPGL and HPGL. These findings indicate the importance of considering differences between study populations when forecasting in vivo pharmacokinetic behaviour. Typical clinical pharmacology studies, particularly in early drug development, use young, fit, healthy male subjects of around 30 years of age. In contrast, the average age of patients for many diseases is about 60 years of age. The relationship between age and MPPGL observed in this study estimates values of 40 mg.g-1 for a 30 year old individual and 31 mg.g-1 for a 60 year old individual. Investigators may wish to consider the reported covariates in the selection of scaling factors appropriate for the population in which estimates of clearance are being predicted. Further studies are required to clarify the influence of age (especially in paediatric subjects), donor source and ethnicity on values of MPPGL and HPGL. In the meantime, we recommend that the estimates (and their variances) from the current meta-analysis be used when predicting in vivo kinetic parameters from in vitro data.

ATP-binding cassette (ABC) transporters comprise a family of critical membrane bound proteins functioning in the translocation of molecules across cellular membranes. Substrates for transport include lipids, cholesterol and pharmacological agents. Mutations in ABC transporter genes cause a variety of human pathologies and elicit drug resistance phenotypes in cancer cells. ABCA2, the second member the A subfamily to be identified, was highly expressed in ovarian carcinoma cells resistant to the anti-cancer agent, estramustine, and more recently, in human vestibular schwannomas. Cells expressing elevated levels of ABCA2 show resistance to variety of compounds, including estradiol, mitoxantrone and a free radical initiator, 2,2'-azobis-(2-amidinopropane). ABCA2 is expressed in a variety of tissues, with greatest abundance in the central nervous system and macrophages. This transporter, along with other proteins that have a high degree of homology to ABCA2, including ABCA1 and ABCA7, are up-regulated in human macrophages during cholesterol import. Recent studies have shown ABCA2 also plays a role in the trafficking of low-density lipoprotein (LDL)- derived free cholesterol and to be coordinately expressed with sterol-responsive genes. A single nucleotide polymorphism in exon 14 of the ABCA2 gene was shown to be linked to early onset Alzheimer disease (AD) in humans, supporting an earlier study showing ABCA2 expression influences levels of APP and β-amyloid peptide, the primary component of senile plaques. Studies thus far implicate ABCA2 as a sterol transporter, the deregulation of which may affect a cellular phenotype conducive to the pathogenesis of a variety of human diseases including AD, atherosclerosis and cancer.

Cytochrome P450 (CYP) 2D6 is one of the most important drug metabolizing enzymes and the rationalization and prediction of potential CYP2D6 substrates is therefore advantageous in the discovery and development of new drugs. Experimentally, the active site of CYP2D6 can be probed by site directed mutagenesis studies. Such studies can be designed from structural models of enzyme-substrate complexes. Modeling approaches can subsequently be used to rationalize the observed effect of mutations on metabolism and inhibition. The current paper will present the construction, refinement and validation of the CYP2D6 homology model used in our laboratory for the prediction and rationalisation of CYP2D6 substrate metabolism and CYP2D6-ligand interactions. The model could explain reported site-directed mutagenesis data (for example, mutation of E216 and D301). Furthermore, based on the model, new CYP2D6 mutants were constructed and studied in our lab, and also for these mutants a rationalization of experimentally observed characteristics could be achieved (I106E, F120A, T309V, F483A). CYP2D6-substrate interaction fingerprint analysis of docked substrates in our homology model suggests that several other active site residues are probably interacting with ligands as well, opening the way for further mutagenesis studies. Our homology model was found to agree with most of the details of the recently solved substrate-free CYP2D6 crystal structure [Rowland et al. J. Biol. Chem. 2006, 281, 7614-7622]. Structural differences between the homology model and crystal structure were the same differences observed between substrate-free and substrate-bound structures of other CYPs, suggesting that these conformational changes are required upon substrate binding. The CYP2D6 crystal structure further validates our homology modeling approach and shows that computational chemistry is a useful and valuable tool to provide models for substrate-bound complexes of CYPs which give insight into CYP-ligand interactions. This information is essential for successful pre-experimental virtual screening, as well as accurate hypothesis generation for in vitro studies in drug discovery and development.

A simple LC-MS/MS method has been developed and validated for the simultaneous determination of morphine (M) and morphine-3-glucuronide (M3G) in rat serum, bile, and urine. Deuterated D3-M and D3-M3G were used as internal standards (IS) for M and M3G, respectively. Serum samples were processed by acetonitrile precipitation. Bile samples were prepared by solid-phase extraction (SPE) using Oasis MCX cartridges. Urine samples were directly analyzed after dilution with mobile phase. Chromatography was performed using a Luna C18 column (5 μm, 150 x 2.1 mm I.D.). The mobile phase consisted of acetonitrile (ACN) and 7.5 mM ammonium formate (pH 9.3) delivered from separate pumps with a simple gradient. The method was validated to quantify M in the range of 1-1000 ng/ml in bile and serum, and 0.025-25 μg/ml in urine. M3G was quantified in the range of 1-1000 ng/ml in serum, 0.1-100 μg/ml in bile, and 0.05- 25 μg/ml in urine. The method was applied to study the pharmacokinetics and disposition of M and M3G in Wistar-Kyoto (WKY) rats, and the effect of phenobarbital (PB) on M and M3G disposition. M is metabolized to M3G at a lower rate in male than female rats leading to higher M levels and lower M3G levels in serum, urine, and bile of male than female rats. PB administration induces M glucuronidation to M3G in male, but not female WKY rats, and abolishes the gender differences in M and M3G pharmacokinetics.