Current Drug Metabolism - Volume 9, Issue 4, 2008

Although there has been much effort in recent years to unravel the complications of pharmacogenetics in drug metabolism, impacts which are ‘environmental’ in the widest sense are also important and have been relatively neglected. Typically, they underlie at least 20-30%of the variation seen in any metabolic pathway within a human population but because they often do not impact on the genome, the effects are less easy to recognise. The input is multifactorial and often comes from surprising sources, including diet [1], indoor air [2], concomitant drug therapy [3], pesticides [4] industrial accidents [5] urban dust particulate matter [6] and medical devices [7]. Some environmental contributions are more obvious than others or have clear therapeutic implications- for instance the inhibition of CYP 3A4 by fruit juices and grapefruit furanocoumarins has been investigated and shown to affect the gastrointestinal first-pass metabolism of certain statins as well as other drugs such as warfarin and felodipine [8,9]. Aromatase (CYP2C19) is an isoform of cytochrome P-450 which converts androgens such as testosterone to oestrogens such as oestradiol by catalysing the formation of the aromatic (phenolic) ring which is the distinguishing feature of the female steroid hormones. This pathway is a metabolic bottleneck since it appears to be the only means of synthesising oestrogens in mammals. Its inhibition is used in therapy for breast cancers which are usually oestrogen-dependent [10]; drugs such as anastrozole which are aromatase inhibitors are now in routine use [11]. However, like many CYP isoforms, aromatase is inhibited by dietary components at levels which may be physiologically relevant [12]. The effects of environmental modulation of CYP isoforms and glucuronyltransferases have recently been described in several papers [12,13,14]; both these pathways are central to drug metabolism but can be modulated by components, often including flavonoids, from Brassica, Cruciferae, Allium and other plant families. Dietary indoles and flavonoids can activate CYP1A expression either by direct ligand interaction with the arylhydrocarbon receptor (AhR) or by increasing the interaction of the AhR with xenobiotic response elements in CYP1A1 and other target genes. Interactions with pharmacogenetic variation can also occur [5,15]; cruciferae interact with the glucuronyltransferase polymorphism UGT1A1*28 which has decreased UGT1A1 promoter activity due to 7 thymine-adenine (TA) repeats although no interaction was seen with individuals with the wild-type 6 (TA) repeats [15]. The contributions of dietary and environmental factors to the activities of other pathways have been less well-explored and this collection of articles focuses on a range of enzymes which although they are not ‘the usual suspects’ are nevertheless main stream. Flavin monoxygenase (FMO-3) catalyses the conversion of trimethylamine, which smells of fish, to its N-oxide which does not [16]. Inhibition of this enzyme may therefore affect the social interactions of the individuals concerned and inspection of web-sites devoted to perception of body odour (eg those for trimethylaminuria/‘fish odour syndrome’) shows that this can be a major problem. The COMT (catechol-Omethyltransferase) isoforms act as methylating agents using S-adenosylmethionine as donor. Oestrogens are converted to 4-catechol oestrogens which can be metabolised to their methoxy derivatives by COMT or oxidised to catechol oestrogen-3,4-quinone metabolites. This process impacts on carcinogenesis as the oestrogen quinone metabolites are believed to react with DNA to form depurinating adducts and to induce mutations. Reduced activity of COMT might therefore be a risk factor for oestrogen-sensitive tumours and reduced expression of COMT in breast tissue has been linked with a higher susceptibility to breast cancer [17]. Inhibition of the COMT enzymes by a range of common industrial pollutants may therefore be an aetiological factor in this increasingly common disease. Recent research has shown that many persistent organic pollutants (POPS), including PCB metabolites and plasticisers, can inhibit the various isoforms of the sulphotransferase (SULT) enzymes [18]. In general, the addition of sulphate is a pathway for detoxication/inactivation and endogenous compounds such as dopamine and oestrogen give metabolites which are more water-soluble and no longer active at the receptors. Many, although not all, drug sulphate conjugates (Minoxydil is an exception) are similarly inactive pharmacologically so that inhibition of the SULT isoforms can alter both endogenous and xenobiotic metabolism. All these articles explore the effects of POPs as direct enzyme inhibitors, actions which would not be found by genomic or proteomic studies since alterations in gene/protein expression are not involved. Recent research has shown that environmental compounds have the capacity to alter the methylation status of DNA sequences and so to affect gene expression.

The cytosolic sulfotransferase enzymes (SULT isoforms) utilise PAPS (3'-phosphoadenosine-5'-phosphosulfate) as co-factor to transfer sulfonate groups onto a wide range of substrates. SULT1A3 has catecholamines such as dopamine as substrates while SULT 1E1 sulfonates oestrogens. SULT 1A1 sulfonates phenols and also oestrogens at a higher Km than SULT 1E1. SULT 2A1 mainly sulfonates DHEA and some steroids, with hydroxy derivatives of polycyclic aromatic hydrocarbons. Studies on these isoforms with a range of environmental chemicals and dietary components have shown that SULT 1A1 is significantly inhibited by flavonoids; all flavones and flavonols with a 3',4'-dihydroxy motif had an IC50 of ⋚ 100nm against 3μM 4-nitrophenol as the standard substrate. SULTs 1A3 and 2A1 were less strongly inhibited by flavonoids or isoflavonoids although tricin (3',5'-dimethoxy-4',5,7-trihydroxyflavone is a competitive inhibitor of SULT 1E1 with an inhibition constant of ∼1nM. Fruit and vegetable cytosols also inhibit SULT isoforms, as do long-chain alkylphenols and chlorinated phenols. Phthalates (used as plasticisers) inhibited SULTs 1E1 and 2A1. As these environmental contaminants and dietary components all act at the same site, their effects would be expected to be additive and could potentially therefore reduce sulfonation of drugs and lead to altered pharmacological responses.

Previously we have shown that E2 down regulates S-COMT expression. Here the effects of four phthalate esters and 4-(tertoctyl) phenol on the intra-cellular levels of S-COMT and COMT activity were studied in MCF-7 cells as a measure of estrogenic activity of these compounds. The four phthalate esters caused significant reductions in both S-COMT protein and COMT activity levels. These effects were inhibited by the ERα receptor antagonist ICI182780. 4-(tert-octyl)phenol also caused reductions in these parameters, but the effects were not abolished by ICI182780. Assay of S-COMT protein levels represents a simple and convenient method of assessing the estrogenic potential of a compound.

Whilst the scientific community was celebrating the truly momentous discovery of a ‘mixed function oxidase’ another oxidase was quietly working behind the scenes, mopping up soft nucleophiles and, as it had undoubtedly being doing for aeons, aiding then unknown in the metabolism of xenobiotics and the protection of life forms. This enzyme, flavin mono-oxygenase, has subsequently been shown to be a major player, if not yet an equal partner with cytochrome(s) P450, in the metabolism of both endogenous biochemicals and foreign compounds that enter the human organism. This article outlines the importance of the flavin mono-oxygenases and examines their susceptibility to activity modulation by exogenous factors.

The effects of four compounds, bis(2-ethylhexyl)phthalate (BEHP); diisodecylphthalate (DIP); 4-n-octylphenol (OP); 4- chloro-3-methylphenol (CMP), on gene expression (steady-state mRNA levels) across the whole human genome were studied in human TE671 cells. Effects were studied using the Affymetrics GeneChip® Human Genome U133 Plus 2.0, HG-U133 Plus 2.0 arrays, The array analyses the expression of 47,000 transcripts and variants, including ∼38,500 well characterised. All four compounds exerted statistically significant actions, affecting between 4 and 6.5% of all genes. Each compound had its own expression signature. In most instances where there was an effect steady-state mRNA levels were decreased, although not always. CMP reatment caused most increases in mRNA levels. .A mixture of DIP and CMP caused fewer changes in mRNA levels than either of the individual compounds. Conclusions: These plasticisers affected the steady-state mRNA levels of many human genes. Exposure to these compounds over many years has the potential to influence human health.

Commercial PCB mixtures have been shown to induce liver tumors in female rats and this effect has been attributed to the effects of PCBs on estrogen metabolism. Catechol metabolites of PCBs are potent inhibitors of COMT activity and are likely to contribute significantly to reduced clearance of genotoxic catechol metabolites of estrogen. The effect of PCB metabolites on COMT expression in cultured cells was investigated to explore potential mechanisms by which PCB exposure alters catechol estrogen clearance. We hypothesize that estrogenic PCB metabolites may contribute to reduction of COMT expression via interaction with the estrogen receptor. To test this hypothesis, human MCF-7 cells were exposed to PCB analogues and the expression of COMT determined. Western blot analysis demonstrated that COMT protein levels were statistically significantly reduced by both the phenolic and the catechol compounds, an effect which was abolished by the anti-estrogen, ICI182780. The above suggests that COMT levels may be reduced by estrogenic PCB metabolites, via interactions between PCB metabolites and the ER. It supports the hypothesis that both phenolic and catechol metabolites of PCBs may contribute to PCB-mediated carcinogenesis through reduction of COMT levels and activities and subsequent reduction in clearance of endogenous and xenobiotic catechols.

Human cytochrome P450 (CYP) 3A4 is the most abundant hepatic and intestinal phase I enzyme that metabolizes approximately 50% marketed drugs. The crystal structure of bound and unbound CYP3A4 has been recently constructed, and a small active site and a peripheral binding site are identified. A recent study indicates that CYP3A4 undergoes dramatic conformational changes upon binding to ketoconazole or erythromycin with a differential but substantial (>80%) increase in the active site volume, providing a structural basis for ligand promiscuity of CYP3A4. A number of important drugs have been identified as substrates, inducers and/or inhibitors of CYP3A4. The ability of drugs to act as inducers, inhibitors, or substrates for CYP3A is predictive of whether concurrent administration of these compounds with a known CYP3A substrate might lead to altered drug disposition, efficacy or toxicity. The substrates of CYP3A4 considerably overlap with those of P-glycoprotein (P-gp). To date, the identified clinically important CYP3A4 inhibitors mainly include macrolide antibiotics (e.g., clarithromycin, and erythromycin), anti-HIV agents (e.g., ritonavir and delavirdine), antidepressants (e.g. fluoxetine and fluvoxamine), calcium channel blockers (e.g. verapamil and diltiazem), steroids and their modulators (e.g., gestodene and mifepristone), and several herbal and dietary components. Many of these drugs are also mechanism-based inhibitors of CYP3A4, which involves formation of reactive metabolites, binding to CYP3A4 and irreversible enzyme inactivation. A small number of drugs such as rifampin, phenytoin and ritonavir are identified as inducers of CYP3A4. The orphan nuclear receptor, pregnane X receptor (PXR), have been found to play a critical role in the induction of CYP3A4. The inhibition or induction of CYP3A4 by drugs often causes unfavorable and long-lasting drug-drug interactions and probably fatal toxicity, depending on many factors associated with the enzyme, drugs and the patients. The study of interactions of newly synthesized compounds with CYP3A4 has been incorporated into drug development and detection of possible CYP3A4 inhibitors and inducers during the early stages of drug development is critical in preventing potential drug-drug interactions and side effects. Clinicians are encouraged to have a sound knowledge on drugs that behave as substrates, inhibitors or inducers of CYP3A4, and take proper cautions and close monitoring for potential drug interactions when using drugs that are CYP3A4 inhibitors or inducers.

The present review aims to give an overview of the cytochrome P450 8B (CYP8B) and cytochrome P450 4A (CYP4A) subfamilies in relation to biosynthesis of bile acids, in particular trihydroxy bile acids. Trihydroxy bile acids are basically required in most species and have an impact on cholesterol and lipid metabolism. The primary trihydroxy bile acid in most mammals is cholic acid. Some species produce other important trihydroxy bile acids, for example the adult pig which produce hyocholic acid instead of cholic acid. The position of the third hydroxyl group in cholic acid and hyocholic acid, 12α or 6α position, respectively, has a profound effect on the hydrophilic- hydrophobic property of the trihydroxy bile acids. The CYP8B subfamily is required for introduction of the 12α-hydroxyl group in cholic acid biosynthesis. The enzyme responsible for 6α-hydroxylation in hyocholic acid biosynthesis, however, varies among species. This review will discuss, in particular, porcine members of the CYP8B and CYP4A subfamilies because interesting findings regarding members of these subfamilies have recently been recognized in this species. CYP8B1 was for a long time believed to be absent in the pig but was recently found to be expressed in fetal pig liver. The enzyme catalyzing the 6α-hydroxylation in hyocholic acid biosynthesis in pig was found to be an atypical member of the CYP4A subfamily, denoted CYP4A21. The review presents bile acid biosynthesis in view of these findings and discusses physiochemical properties and developmental-dependent aspects related cholic acid and hyocholic acid biosynthesis.

Tacrine belongs to the group of acetylcholinesterse (AChE) inhibitors used as drugs for treatment of Alzheimer's disease (AD). The formation of hydroxyderivatives of tacrine is well-established step in the metabolization of this drug in liver by microsomal cytochrome P450 enzymes family. Genetic polymorphism of cytochrome P450 enzymes is probably responsible for balance between a number of stable and non-toxic metabolites and highly protein-reactive and toxic ones. By this manner may be explained why the hepatotoxicity of tacrine was observed only in the part of persons and why not every patient with AD responds to the treatment by this drug.

Capecitabine is a drug that requires the consecutive action of three enzymes: carboxylesterase 2 (CES 2), cytidine deaminase (CDD), and thymidine phosphorylase (TP) for transformation into 5-fluorouracil (5FU). The metabolism of 5FU requires the activity of thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) among other enzymes. The present study prospectively examined the possible relationship between the toxicity and efficacy of capecitabine and 14 different polymorphisms in CES 2, CDD, TS and DPD. Between 2003 and 2005, a total of 136 patients with advanced breast or colorectal cancer treated with capecitabine were prospectively enrolled. The presence of two polymorphisms (CDD 943insC and CES 2 Exon3 6046 G/A) were associated with a non-statistically significant higher incidence of grade 3 hand-foot syndrome (HFS) (p=0.07) and grade 3-4 diarrhoea (p=0.09), respectively. Patients heterozygous or homozygous for the polymorphism CES 2 5'UTR 823 C/G exhibited a significantly greater response rate to capecitabine, and time to progression of disease (59%, 8.7 months) than patients with the wild type gene sequence (32%, p=0.015; 5.3 months, p=0.014). For the first time, an association between a polymorphism in the CES2 gene and the efficacy of capecitabine has been described, providing preliminary evidence of its predictive and prognostic value.

The metabolism of drugs and other xenobiotics is exemplified by cytochrome P450-mediated oxidation to more hydrophilic compounds. Enzymatic oxidation of some functional groups, however, can result in transient reactive intermediates -- a transformation that is common in nature. Some drugs and many phytochemicals that contain, for example, a thiophene ring are oxidized by cytochrome P450 to reactive intermediates, such as sulfoxides, that can covalently bond to thiol nucleophiles in macromolecules, such as proteins. Many other functional groups can be oxidized to reactive intermediates -- for instance, olefins, alkynes, alkylamines, furans, and paminophenols. Because any consequence of a biological reactive intermediate (BRI) is dependent on many factors a number of BRIs are benign. Toxicity is determined by complex and specific arrays of parallel and/or serial pathways and cellular states, not by entities or individual reactions. Because the formation of reactive intermediates can correlate with unacceptable toxicity, it is important to understand if or how a compound may undergo this type of transformation. Not all substances that form reactive intermediates are toxic; many are not. Therefore, it is critical to understand the mechanism of metabolism in considering any toxicity relevance and in evaluating animal models. Indeed, the complex nature of these many interactions and entities underscores the fact that the presence of a BRI is not an accurate indicator of human toxicity. A BRI is neither good nor a priori bad. It is a potential detoxication product and an incidental fate of the xenobiotic.

The sensory contact model can induce various different psychopathological states in male mice (anxious depression, catalepsy, social withdrawal, pathological aggression, cognition disturbances, anhedonia, alcoholism etc.). Additionally, this model facilitates the screening of drugs for therapeutic properties, preventive properties and efficiency under simulated clinical conditions. This approach can reveal the action of drugs at different stages of disease development. It is proposed that this pharmacological approach can be applied for the screening of various novel psychotropic drugs.