Development of a Competition-Binding Assay to Determine Binding Affinity of Molecules to Neuromelanin via Fluorescence Spectroscopy.

Neuromelanin, the polymeric form of dopamine which accumulates in aging neuronal tissue, is increasingly recognized as a functional and critical component of a healthy and active adult human brain. Notorious in plant and insect literature for their ability to bind and retain amines for long periods of time, catecholamine polymers known colloquially as 'melanins' are nevertheless curiously absent from most textbooks regarding biochemistry, neuroscience, and evolution. Recent research has brought attention to the brain pigment due to its possible role in neurodegeneration. This linkage is best illustrated by Parkinson's disease, which is characterized by the loss of pigmented dopaminergic neurons and the 'white brain' pathological state. As such, the ability to determine the binding affinity of neurotoxic agents, as well as any potential specific endogenous ligands to neuromelanin are of interest and potential value. Neuromelanin has been shown to have saturable binding interactions with nicotine as monitored by a fluorimeter. This interaction provides a signal to allow for a competition-binding assay with target molecules which do not themselves produce signal. The current report establishes the viability of this competition assay toward three compounds with central relevance to Parkinson's disease. The Kd of binding toward neuromelanin by methyl-phenyl-pyridinium ion (MPP+), dopamine, and 6-hydroxydopamine were found to be 1 mM, 0.05 mM, and 0.1 mM, respectively in the current study. In addition, we demonstrate that 6-hydroxydopamine polymerizes to form neuromelanin granules in cultured dopaminergic neurons that treated with 2,4,5-trihydroxy-l-phenylalanine. Immunohistochemical analysis using fluor-tagged anti-dopamine antibodies suggests that the incorporation of 6-hydroxydopamine (following internalization and decarboxylation analogous to levodopa and dopamine) alters the localized distribution of bound dopamine in these cells.

Neuromelanin, one of the most overlooked molecules in modern medicine, is not a spectator.

The loss of pigmented neurons from the human brain has long been the hallmark of Parkinson's disease (PD). Neuromelanin (NM) in the pre-synaptic terminal of dopamine neurons is emerging as a primary player in the etiology of neurodegenerative disorders including PD. This mini-review discusses the interactions between neuromelanin and different molecules in the synaptic terminal and describes how these interactions might affect neurodegenerative disorders including PD. Neuromelanin can reversibly bind and interact with amine containing neurotoxins, e.g., MPTP, to augment their actions in the terminal, eventually leading to the instability and degeneration of melanin-containing neurons due to oxidative stress and mitochondrial dysfunction. In particular, neuromelanin appears to confer susceptibility to chemical toxicity by providing a large sink of iron-bound, heme-like structures in a pi-conjugated system, a system seemingly purposed to allow for stabilizing interactions including pi-stacking as well as ligand binding to iron. Given the progressive accumulation of NM with age corresponding with an apparent decrease in dopamine synthetic pathways, the immediate question of whether NM is also capable of binding dopamine, the primary functional monoamine utilized in this cell, should be raised. Despite the rather glaring implications of this finding, this idea appears not to have been adequately addressed. As such, we postulate on potential mechanisms by which dopamine might dissociate from neuromelanin and the implications of such a reversible relationship. Intriguingly, if neuromelanin is able to sequester and release dopamine in membrane bound vesicles, this intracellular pre-synaptic mechanism could be the basis for a form of chemical memory in dopamine neurons.

Saturation Binding of Nicotine to Synthetic Neuromelanin Demonstrated by Fluorescence Spectroscopy.

Neuromelanin (NM) has long been considered as an aging pigment, perhaps an unavoidable and undesirable byproduct of dopaminergic neural transmission. However, NM is carefully packaged into double membrane-bound structures within cells of the substantia nigra and other neural tissues, suggesting a beneficial function to maintaining these stores. It is well established that NM is able to concentrate toxic xenobiotics within pigmented cells due to its unique chemical environment. In doing so, such agents may confer susceptibility to Parkinson's disease (PD) as illustrated by model PD-inducing neurotoxins such as methyl-phenyl-pyridinium ion. It is possible that high-affinity binding interactions toward NM may contribute to the adverse effects of PD-inducing toxins, as well as neuroprotective agents. Here we aim to develop a generalized assay capable of elucidating the binding constants of chemical agents to synthetic and natural neuromelanins. Toward this end, a model neuromelanin synthesized from dopamine and cysteine was prepared according to published procedure. Using a UV/Visible spectroscopic assay, we show that dopamine, 6-hydroxy dopamine, and nicotine bind to the synthetic neuromelanin, while caffeine did not. More importantly, nicotine was further found to induce a fluorescence signal in the presence of NM which was used to establish a binding constant estimated at 0.65 mM. Dopamine appears to enhance this signal, also in a saturable manner, with an estimated Kd of 0.05 mM in our isolated chemical system. In summary, the micro-scale fluorescence assay described herein will allow us to overcome many of the problems inherent in the study of chemical interaction with NM through traditional spectroscopic means. Using a single standardized signal, it should now be possible to rank a number of PD-related toxins based on NM-binding affinity and shed further light on this important problem.

Crime victimization and the implications for individual health and wellbeing: A Sheffield case study.

Public health and criminology have developed largely independently of one another at the research and policy levels so that the links between crime victimization and health status are not well understood. Although it is not difficult to support the idea of crime as a threat to the health of individuals and the wider community, the difficulty lies in quantifying the impact of crime on public health, while controlling other variables, including gender and ethnicity. We report the results of a study, the goals of which were to: develop an understanding conceptually of the relationships between different types of crime (violent and non-violent) and health; explore the impact of victimization on quality of life and physical and psychological wellbeing; investigate the role of social and demographic factors in shaping any relationships. The study is based on 840 responses from a postal survey administered to 4,100 households in Sheffield, England, located primarily in deprived areas where overall crime rates were high. Non-violent crimes were more frequently reported than violent crimes and in general, inner city neighbourhoods were associated with higher violent crime rates. Out of 392 victims of crime, 27% of individuals detailed physical injuries resulting directly from a crime event and 31% had taken some medical steps to treat a crime-related injury. 86% experienced at least one psychological or behavioural change, including stress, sleeping difficulties, loss of confidence, and depression. Logistic regression models estimated victimization risk based on various social and demographic variables. Violent crimes were consistently linked with higher odds of seeking medical treatment and a higher likelihood of experiencing psychological ill health effects or behavioural changes. In comparison, victims of non-violent or property crimes were not significantly associated with mental health or behavioural/lifestyle effects.

Comparing the perception with the reality of walking in a hilly environment: an accessibility method applied to a University campus in Hong Kong.

The influence of hilliness on walking behavior could be a consequence of the real effect of the local topography, but individual perception of the difficulties associated with walking in a hilly environment may also be important. Previous studies have found that people's perceptions do not necessarily match well with the realities of walking in hilly environments. There are a few methods that can be used to visualize the geography of that difference for use by urban planners and public health practitioners. A walking accessibility measure that allows comparison of perception and reality is proposed and implemented in this study. We note that difficulties in calculating accessibility measures in the present context arise primarily from problems with data quality, three-dimensional pedestrian network modelling and the adequacy of accessibility methods for describing and predicting walking behavior. We present practical strategies for addressing these issues using geographic information systems. Our method is illustrated by calculating accessibility for a hilly university campus in Hong Kong. Walking behaviors on, and people's perceptions of, this hilly environment were obtained through walking diaries and a survey. The article concludes with suggested directions for the future development of walking accessibility measures along with some ideas about their applicability to the practice of planning and designing a walkable environment.

Bridging the undergraduate curriculum using an integrated course-embedded undergraduate research experience (ICURE).

The traditional undergraduate program of study incorporates a selection of classes that represent a broad spectrum of subdisciplines. Unfortunately, few curricula successfully integrate concepts in all subdisciplines, giving undergraduates the misconception that there is a lack of application or connectedness between class subjects. An integrated course-embedded research experience (ICURE) was initiated to redress this problem by bridging classes within one discipline in an effort to engage undergraduates in a long-term analysis of biodiversity. The approach was both inclusive and longitudinal: 1) the ICURE bridge brought students from different classes and levels of instruction together with faculty members in a research project with a common goal-chronicling the changing face of the local environment in biological terms; and 2) research data collected were maintained and supplemented each semester and year in an online biodiversity database. Analysis of content and attitudinal gains suggested the integrated research protocol increased student comprehension and confidence. Results are discussed in terms of future amendments to instructional design and potential research applications. Though this program was concentrated on one discipline, there is no reason to assume other disciplines could not take advantage of similar research connections.

Using knowledge fusion to analyze avian influenza H5N1 in East and Southeast Asia.

Highly pathogenic avian influenza (HPAI) H5N1, a disease associated with high rates of mortality in infected human populations, poses a serious threat to public health in many parts of the world. This article reports findings from a study aimed at improving our understanding of the spatial pattern of the highly pathogenic avian influenza, H5N1, risk in East-Southeast Asia where the disease is both persistent and devastating. Though many disciplines have made important contributions to our understanding of H5N1, it remains a challenge to integrate knowledge from different disciplines. This study applies genetic analysis that identifies the evolution of the H5N1 virus in space and time, epidemiological analysis that determines socio-ecological factors associated with H5N1 occurrence, and statistical analysis that identifies outbreak clusters, and then applies a methodology to formally integrate the findings of the three sets of methodologies. The present study is novel in two respects. First it makes the initiative attempt to use genetic sequences and space-time data to create a space-time phylogenetic tree to estimate and map the virus' ability to spread. Second, by integrating the results we are able to generate insights into the space-time occurrence and spread of H5N1 that we believe have a higher level of corroboration than is possible when analysis is based on only one methodology. Our research identifies links between the occurrence of H5N1 by area and a set of socio-ecological factors including altitude, population density, poultry density, and the shortest path distances to inland water, coastlines, migrating routes, railways, and roads. This study seeks to lay a solid foundation for the interdisciplinary study of this and other influenza outbreaks. It will provide substantive information for containing H5N1 outbreaks.

Applying Geostatistical Analysis to Crime Data: Car-Related Thefts in the Baltic States.

Geostatistical methods have rarely been applied to area-level offense data. This article demonstrates their potential for improving the interpretation and understanding of crime patterns using previously analyzed data about car-related thefts for Estonia, Latvia, and Lithuania in 2000. The variogram is used to inform about the scales of variation in offense, social, and economic data. Area-to-area and area-to-point Poisson kriging are used to filter the noise caused by the small number problem. The latter is also used to produce continuous maps of the estimated crime risk (expected number of crimes per 10,000 habitants), thereby reducing the visual bias of large spatial units. In seeking to detect the most likely crime clusters, the uncertainty attached to crime risk estimates is handled through a local cluster analysis using stochastic simulation. Factorial kriging analysis is used to estimate the local- and regional-scale spatial components of the crime risk and explanatory variables. Then regression modeling is used to determine which factors are associated with the risk of car-related theft at different scales.

Inference from ecological models: estimating the relative risk of stroke from air pollution exposure using small area data.

Maheswaran et al. (2006) analysed the effect of outdoor modelled NO(x) levels, classified into quintiles, on stroke mortality using a Poisson Bayesian hierarchical model with spatial random effects. An association was observed between higher levels of NO(x) and stroke mortality at the small area (enumeration district) level. As this model is framed in an ecological perspective, the relative risk estimates suffer from ecological bias. In this paper we use a different model specification based on Jackson et al. (2008), modelling the number of cases of mortality due to stroke as a binomial random variable where p(i) is the probability of dying from stroke in area i. The within-area variation in outdoor modelled NO(x) levels is used to determine the proportion of the population in area i falling into each of the five exposure categories in order to estimate the probability of an individual dying from stroke given the kth level of NO(x) exposure assuming a homogeneous effect across the study region. The inclusion of within-area variability in an ecological regression model has been demonstrated to help reduce the ecological bias (Jackson et al., 2006, 2008). Revised estimates of relative risk are obtained and compared with previous estimates.

Expression and functional analysis of CYP2D6.24, CYP2D6.26, CYP2D6.27, and CYP2D7 isozymes.

The objectives of this study were to compare the drug-metabolizing activity of human CYP2D6.24 (I297L), CYP2D6.26 (I369T), and CYP2D6.27 (E410K) allelic isoforms with wild-type CYP2D6.1 and to express the CYP2D7 protein derived from an indel polymorphism (CYP2D7 138delT) and investigate its possible codeine O-demethylase activity. Successful creation of individual cDNAs corresponding to CYP2D6*24 (2853 A>C), CYP2D6*26 (3277 T>C), and CYP2D6*27 (3853 G>A) allelic variants and CYP2D7 was achieved via molecular cloning. The corresponding proteins, CYP2D6.24, CYP2D6.26, CYP2D6.27, and CYP2D7, were expressed in insect cells by using a baculovirus-mediated expression system. All CYP2D proteins showed the empirical carbon monoxide difference spectra. We were surprised to find that the CYP2D7 protein was detected mainly in mitochondrial fractions, whereas all CYP2D6 allelic isoforms were present in the microsomal fraction. Furthermore, CYP2D7 did not produce any morphine from codeine. In contrast, CYP2D6.24, CYP2D6.26, and CYP2D6.27 allelic isoforms all showed active drug-metabolizing activities toward both codeine and dextromethorphan O-demethylation. Whereas CYP2D6.24 exhibited the highest intrinsic clearance in dextromethorphan O-demethylation (approximately 6-fold higher than that by CYP2D6.1), it had the lowest enzyme efficiency in codeine O-demethylation (approximately 50% lower than that by CYP2D6.1). Overall, the enzymatic consequences of CYP2D6 allelic isozymes are substrate dependent. These data would help preclinical and clinical assessments of the metabolic elimination of drugs that are mediated by human CYP2D enzyme.

Cytochrome P450-catalyzed pathways in human brain: metabolism meets pharmacology or old drugs with new mechanism of action?

The true importance of cytochrome P450 enzymes, not just in drug metabolism but also in pharmacology, is only beginning to be appreciated. Though originally discovered through their role in the biotransformation of xenobiotics, the P450 enzyme super family is ubiquitous in nature and necessarily evolved around endogenous pathways. The extent of tissue- and cell-specific expression of individual P450 isoforms has led many investigators to hypothesize localized roles in endogenous biochemical pathways for isoforms traditionally thought of as drug-metabolizing. In some cases, direct evidence from humanized transgenic animal models can confirm the degree to which such enzymes modulate endogenous pathways. However, overlapping P450 substrate specificities may mask genetic or biochemical deficiencies, such that many of these reactions appear nonessential. Nonetheless, the drug-induced alteration of local biochemical concentrations in extrahepatic tissues due to metabolism by and inhibition of P450 isoforms has tremendous potential for introducing unexpected pharmacological effects. Nowhere is this truer than in the CNS. On the other hand, if we can harness the power of in silico modeling to create highly specific inhibitors of identified brain isoforms, a novel avenue for drug design using P450 as drug targets may be at hand. This article highlights some notable examples in which the catalytic state of specific P450 isoforms involved in endogenous biochemical reaction pathways are influenced by pharmacological agents. The implications of inhibition of P450-catalzyed oxidation steps that are known or speculated to influence arachadonic acid, cholesterol, and catecholamine neurotransmitters pathways in human brain will be considered.

Outdoor NOx and stroke mortality: adjusting for small area level smoking prevalence using a Bayesian approach.

There is increasing evidence, mainly from daily time series studies, linking air pollution and stroke. Small area level geographical correlation studies offer another means of examining the air pollution-stroke association. Populations within small areas may be more homogeneous than those within larger areal units, and census-based socioeconomic information may be available to adjust for confounding effects. Data on smoking from health surveys may be incorporated in spatial analyses to adjust for potential confounding effects but may be sparse at the small area level. Smoothing, using data from neighbouring areas, may be used to increase the precision of smoking prevalence estimates for small areas. We examined the effect of modelled outdoor NOx levels on stroke mortality using a Bayesian hierarchical spatial model to incorporate random effects, in order to allow for unmeasured confounders and to acknowledge sampling error in the estimation of smoking prevalence. We observed an association between NOx and stroke mortality after taking into account random effects at the small area level. We found no association between smoking prevalence and stroke mortality at the small area level after modelling took into account imprecision in estimating smoking prevalence. The approach we used to incorporate smoking as a covariate in a single large model is conceptually sound, though it made little difference to the substantive results.

Expression, purification, and characterization of mouse CYP2d22.

Metabolism of the prototype human CYP2D6 substrates debrisoquine and bufuralol proceeds at a much slower rate in mice; therefore, the mouse has been proposed as an animal model for the human CYP2D6 genetic deficiency. To interpret the molecular mechanism of this deficiency, a cDNA belonging to the CYP2D gene subfamily (Cyp2d22) has been cloned and sequenced from a mouse mammary tumor-derived cell line. In the current study, Cyp2d22 enzyme was overexpressed and purified from insect cells using a baculovirus-mediated system. The activity of this purified enzyme was directly compared with purified human CYP2D6 toward codeine, dextromethorphan, and methadone as substrates. Purified Cyp2d22 was found to catalyze the O-demethylation of dextromethorphan with significantly higher K(m) values (250 microM) than that (4.2 microM) exhibited by purified human CYP2D6. The K(m) for dextromethorphan N-demethylation by Cyp2d22 was found to be 418 microM, much lower than that observed with human CYP2D6 and near the K(m) for dextromethorphan N-demethylation catalyzed by CYP3A4. CYP2D6 catalyzed codeine O-demethylation, whereas Cyp2d22 and CYP3A4 mediated codeine N-demethylation. Furthermore, methadone, a known CYP3A4 substrate and CYP2D6 inhibitor, was N-demethylated by Cyp2d22 with a K(m) of 517 microM and V(max) of 4.9 pmol/pmol/min. Quinidine and ketoconazole, potent inhibitors to CYP2D6 and CYP3A4, respectively, did not show strong inhibition toward Cyp2d22-mediated dextromethorphan O- or N-demethylation. These results suggest that mouse Cyp2d22 has its own substrate specificity beyond CYP2D6-like-deficient activity.

A protocol for investigation of the effects of outdoor air pollution on stroke incidence, phenotypes and survival using the South London Stroke Register.

Stroke is a major cause of death and disability. About 5.3 million people die every year from stroke worldwide with over 9 million people surviving at any one time after suffering a stroke. About 1 in 4 men and 1 in 5 women aged 45 years will suffer a stroke if they live to their 85th year. It is estimated that by 2023 there will be an absolute increase in the number of people experiencing a first ever stroke of about 30% compared with 1983. In the UK, stroke is the third commonest cause of death and the most common cause of adult physical disability and consumes 5% of the health and social services budget. Stroke is assuming strategic public health importance because of increased awareness in society, an ageing population and emerging new treatments. It is an NHS health service and research priority, being identified as a target in Our Healthier Nation and the NSF for Older People for prevention and risk factor control and in the NHS Plan as a disease requiring intermediate care planning and reduction in inequalities of care. Whilst a number of risk factors for stroke are well known (e.g. increasing age, ethnicity, socioeconomic deprivation, hypertension), the potential importance of outdoor air pollution as a modifiable risk factor is much less well recognised. This is because studies to date are inconclusive or have methodological limitations. In Sheffield, we estimated that 11% of stroke deaths may be linked to current levels of outdoor air pollution and this high figure is explained by the fact that so many people are exposed to air pollution.We plan to study the effects of outdoor air pollution on stroke using a series of epidemiological (i.e. population based) studies. The purpose of this project is: to examine if short term increases in pollution can trigger a stroke in susceptible individuals, to investigate if the occurrence of stroke is higher amongst people living in more polluted areas (which would be explained by a combination of exposure to short term increases and longer term exposure to higher pollution levels), and to see if people living in more polluted areas have reduced survival following their stroke. We will use geographical information systems, robust statistical methods and powerful grid computing facilities to link and analyse the data. The datasets we will use are the South London Stroke Register database, daily monitored pollution data from national monitoring networks and modelled pollution data for London from the Greater London Authority. The South London Stroke Register records information on all patients who suffer a stroke ("incident" cases) living within a defined area. This stroke incidence dataset offers major advantages over previous studies examining the effects of pollution on hospital admissions and mortality, as not all patients with stroke are admitted or die and there may be a delay between the onset of stroke and admission or death. In addition, it contains other useful information, particularly the type of stroke people have suffered. Air pollution is a potentially modifiable risk factor for stroke. This study will provide robust population level evidence regarding the effects of outdoor air pollution on stroke. If it confirms the link, it will suggest to policy-makers at national and international levels that targeting policy interventions at high pollution areas may be a feasible option for stroke prevention.

The human intestinal cytochrome P450 "pie".

Cytochromes P450 (P450s) 3A, 2C, and 1A2 constitute the major "pieces" of the human liver P450 "pie" and account, on average, for 40, 25, and 18%, respectively, of total immunoquantified P450s (J Pharmacol Exp Ther 270:414-423, 1994). The P450 profile in the human small intestine has not been fully characterized. Therefore, microsomes prepared from mucosal scrapings from the duodenal/jejunal portion of 31 human donor small intestines were analyzed by Western blot using selective P450 antibodies. P450s 3A4, 2C9, 2C19, and 2J2 were detected in all individuals and ranged from 8.8 to 150, 2.9 to 27, <0.6 to 3.9, and <0.2 to 3.1 pmol/mg, respectively. CYP2D6 was detected in 29 individuals and ranged from <0.2 to 1.4 pmol/mg. CYP3A5 was detected readily in 11 individuals, with a range (average) of 4.9 to 25 (16) pmol/mg that represented from 3 to 50% of total CYP3A (CYP3A4 + CYP3A5) content. CYP1A1 was detected readily in three individuals, with a range (average) of 3.6 to 7.7 (5.6) pmol/mg. P450s 1A2, 2A6, 2B6, 2C8, and 2E1 were not or only faintly detected. As anticipated, average CYP3A content (50 pmol/mg) was the highest. Excluding CYP1A1, the remaining enzymes had the following rank order: 2C9 > 2C19 > 2J2 > 2D6 (8.4, 1.1, 0.9, and 0.5 pmol/mg, respectively). Analysis of a pooled preparation of the 31 donor specimens substantiated these results. In summary, as in the liver, large interindividual variation exists in the expression levels of individual P450s. On average, CYP3A and CYP2C9 represents the major pieces of the intestinal P450 pie, accounting for 80 and 15%, respectively, of total immunoquantified P450s.

Outdoor air pollution, mortality, and hospital admissions from coronary heart disease in Sheffield, UK: a small-area level ecological study.

AIMS: To examine the hypothesis that coronary heart disease mortality and emergency hospital admission rates are higher in areas with higher outdoor air pollution levels. METHODS AND RESULTS: Modelled nitrogen oxides (NO(x)), particulate matter (PM(10)), and carbon monoxide (CO) levels were interpolated to 1030 census enumeration districts using an ecological study design. Results, based on 6857 deaths and 11,407 admissions from 1994-98 and a population of 199,682 aged >or=45 years, were adjusted for age, sex, deprivation, and smoking prevalence. Mortality rate ratios were 1.17 (95% CI 1.06-1.29), 1.08 (95% CI 0.96-1.20), and 1.05 (95% CI 0.95-1.16) in the highest relative to the lowest NO(x), PM(10), and CO quintile categories, respectively. Corresponding admission rate ratios were 1.00 (95% CI 0.90-1.10), 1.01 (95% CI 0.90-1.14), and 0.88 (95% CI 0.79-0.98). CONCLUSION: The results are consistent with an excess risk of coronary heart disease mortality in areas with high outdoor NO(x), a proxy for traffic-related pollution, but residual confounding cannot be ruled out. If causality were assumed, 6% of coronary heart disease deaths would have been attributable to outdoor NO(x,) and targeting pollution reduction measures at high pollution areas would be an option for coronary mortality prevention.

CYP2C-catalyzed delta9-tetrahydrocannabinol metabolism: kinetics, pharmacogenetics and interaction with phenytoin.

Delta9-tetrahydrocannabinol (delta9-THC), the primary psychoactive constituent of marijuana, is subject to first pass hepatic metabolism primarily by hydroxylation to yield active and inactive oxygenated products. The primary metabolite is formed via oxidation of the allylic methyl group to yield 11-hydroxy-delta9-THC, which is oxidized further to 11-nor-9-carboxy-delta9-THC. The hydroxylation is thought to be mediated by CYP2C9. The present study was designed to address the kinetics and pharmacogenetics of CYP2C-mediated metabolism of (delta9)-THC by studying its metabolism in human liver microsomes and expressed enzymes. Expressed CYP2C9.1 exhibited high affinity for the hydroxylation of delta9-THC (apparent Km, 2 microM), similar to that observed in human liver microsomes (apparent Km 0.8 microM). In contrast, the calculated intrinsic clearance (apparent Vm/Km) for CYP2C9.2 and CYP2C9.3 was approximately 30% that of the wild type, CYP2C9.1. Given the high affinity of CYP2C9 for the hydroxylation of delta9-THC, we evaluated the potential for an interaction between delta9-THC, 11-hydroxy-delta9-THC, or 11-nor-9-carboxy-delta9-THC and the CYP2C9 substrate, phenytoin. Surprisingly, delta9-THC increased the rate of phenytoin hydroxylation in human liver microsomes and expressed CYP2C9 enzyme. Similar increases in rate were observed with co-incubation of 11-hydroxy-delta9-THC and 11-nor-9-carboxy-delta9-THC with phenytoin. These in vitro data suggest the potential for an interaction from the concomitant administration of delta9-THC and phenytoin that could result in decreased phenytoin concentrations in vivo.