Metabolism Of Foreign Compounds Research Articles

Environmentally Persistent Free Radicals stimulate CYP2E1-mediated generation of reactive oxygen species at the expense of substrate metabolism.

Environmentally persistent free radicals (EPFRs) are a recently recognized component of particulate matter that cause respiratory and cardiovascular toxicity. The mechanism of EPFR toxicity appears to be related to their ability to generate reactive oxygen species (ROS), causing oxidative damage. EPFRs were shown to affect P450 function, inducing the expression of some forms through the Ah receptor. However, another characteristic of EPFRs lies in their ability to inhibit P450 activities. CYP2E1 is one of the P450s that is inhibited by EPFR (MCP230) exposure. As CYP2E1 is also known to generate ROS, it is important to understand the ability of EPFRs to influence the function of this enzyme and to identify the mechanisms involved. CYP2E1 was shown to be inhibited by EPFRs, and to a lesser extent by non-EPFR particles. As EPFR-mediated inhibition was more robust at subsaturating NADPH-cytochrome P450 reductase (POR) concentrations, disruption of POR·CYP2E1 complex formation and electron transfer were examined. Surprisingly, neither complex formation nor electron transfer between POR and CYP2E1 were inhibited by EPFRs. Examination of ROS production showed that MCP230 generated a greater amount of ROS than the non-EPFR CuO-Si. When a POR/CYP2E1-containing reconstituted system was added to the pollutant-particle systems there was a synergistic stimulation of ROS production. The results indicate that EPFRs cause inhibition of CYP2E1-mediated substrate metabolism, yet do not alter electron transfer and actually stimulate ROS generation. Taken together, the results are consistent with EPFRs affecting CYP2E1 function by inhibiting substrate metabolism and increasing the generation of ROS. Significance Statement Environmentally persistent free radicals affect CYP2E1 function by inhibition of monooxygenase activity. This inhibition is not due to disruption of the POR·CYP2E1 complex or inhibition of electron transfer, but due to uncoupling of NADPH and oxygen consumption from substrate metabolism to the generation of ROS. These results show that EPFRs block the metabolism of foreign compounds, and also synergistically stimulate the formation of reactive oxygen species that lead to oxidative damage within the organism.

Interaction of Perfumes with Cytochrome P-450 19

Cytochrome P450 (CYP) enzymes play a key role in the metabolism of foreign compounds and in the biosynthesis and catabolism of endogenous substances, including hormones. The activity of these enzymes can be affected by various xenobiotics, such as pollutants, food constituents, pharmaceuticals, and cosmetic products, which can disrupt the endocrine system by interfering with steroidogenic CYPs. CYP19, also known as aromatase, is a crucial enzyme for testosterone conversion into 17β-estradiol, which is the final step in estrogen biosynthesis. Endocrine disruptors have the potential to inhibit CYP19 activity, leading to an imbalance in estrogen levels in the body. This imbalance can impair reproduction and cause osteoporosis, atherosclerosis, dementia, and some types of cancer. The aim of this study was to assess the effect of commercially available perfumes on testosterone aromatization to 17β-estradiol. For this purpose, we used high-performance liquid chromatography (HPLC) with UV detection and HPLC coupled with mass spectrometry (MS) to examine CYP19 activity with and without perfume. The results showed that all perfumes tested (in a 300-fold dilution) had an inhibitory effect on this enzyme-catalyzed reaction, particularly the Montale® fragrance, ‘Intense Roses Musk’, which decreased 17β-estradiol production by 88% in comparison with the control. Upon exposure to UV light, the inhibitory effect of this perfume did not decrease. But exposure to UV light significantly increased the inhibitory capacity of another perfume with a weak baseline inhibitory effect. To ascertain whether this inhibition was caused by CYP19 interactions with perfumes, we measured the catalytic activity of NADPH:cytochrome P450 oxidoreductase (CYPOR), the CYP reaction partner, with one selected perfume, ‘Intense Roses Musk’ by Montale®, and found no significant CYPOR inhibition. Accordingly, the decrease in testosterone conversion into 17β-estradiol caused by this perfume derives solely from CYP19. Combined, our findings highlight the importance of testing perfumes rather than single ingredients to determine their potential for adverse effects and to ensure consumer safety because their mixtures can interfere with a key enzyme of estrogen biosynthesis.

Analysis and Prediction of Significant Genes in Gastric Cancer Based on TCGA Database.

Use TCGA data to screen the significantly expressed genes in gastric cancer,then analyze the biological functions of characteristic genes.To identify potential targets for the diagnosis, prognosis and therapeutic monitoring of gastric cancer. Download the high-throughput gastric cancer patient tissue mRNA expression data from the TCGA database, and used the edgeR software package to perform gene differences using the Rstudio software according to the |logFC|>1 and p<0.05 standards analysis, then performed enrichment analysis and constructed protein interaction network of the differently expressed genes to find the significant genes.Finally,analyzed the relationship between significant genes and gastric cancer patient prognosis. The RNA-seq expression data of 375 cases of gastric cancer and 32 adjacent tissues were obtained from the TCGA database. The differential analysis yielded 4320 differential genes, of which 2718 were highly expressed and 1602 were low expressed. Through enrichment analysis, it is found that the differential genes are mainly related to the neural active ligand-receptor interaction, calcium signaling pathway, Protein digestion and absorption, chemical carcinogenesis, and cytochrome P450 metabolism of foreign compounds. Further survival analysis screened out the five significant genes of ALB, AFP, IGFBP1, APOH, and TF are related to the prognosis of gastric cancer. The five significant genes screened by TCGA data may be related to the prognosis of gastric cancer and are expected to become prognostic biomarkers.

Enzyme Kinetics of Oxidative Metabolism-Cytochromes P450.

The cytochrome P450 enzymes (CYPs) are the most important enzymes in the oxidative metabolism of hydrophobic drugs and other foreign compounds (xenobiotics). The versatility of these enzymes results in some unusual kinetic properties, stemming from the simultaneous interaction of multiple substrates with the CYP active site. Often, the CYPs display kinetics that deviate from standard hyperbolic saturation or inhibition kinetics. Non-Michaelis-Menten or "atypical" saturation kinetics include sigmoidal, biphasic, and substrate inhibition kinetics (see Chapter 2 ). Interactions between substrates include competitive inhibition, noncompetitive inhibition, mixed inhibition, partial inhibition, activation, and activation followed by inhibition (see Chapters 4 and 6 ). Models and equations that can result in these kinetic profiles will be presented and discussed.

Pentose metabolism and conversion to biofuels and high-value chemicals in yeasts.

Pentose sugars are widespread in nature and two of them, D-xylose and L-arabinose belong to the most abundant sugars being the second and third by abundance sugars in dry plant biomass (lignocellulose) and in general on planet. Therefore, it is not surprising that metabolism and bioconversion of these pentoses attract much attention. Several different pathways of D-xylose and L-arabinose catabolism in bacteria and yeasts are known. There are even more common and really ubiquitous though not so abundant pentoses, D-ribose and 2-deoxy-D-ribose, the constituents of all living cells. Thus, ribose metabolism is example of endogenous metabolism whereas metabolism of other pentoses, including xylose and L-arabinose, represents examples of the metabolism of foreign exogenous compounds which normally are not constituents of yeast cells. As a rule, pentose degradation by the wild-type strains of microorganisms does not lead to accumulation of high amounts of valuable substances; however, productive strains have been obtained by random selection and metabolic engineering. There are numerous reviews on xylose and (less) L-arabinose metabolism and conversion to high value substances; however, they mostly are devoted to bacteria or the yeast Saccharomyces cerevisiae. This review is devoted to reviewing pentose metabolism and bioconversion mostly in non-conventional yeasts, which naturally metabolize xylose. Pentose metabolism in the recombinant strains of S. cerevisiae is also considered for comparison. The available data on ribose, xylose, L-arabinose transport, metabolism, regulation of these processes, interaction with glucose catabolism and construction of the productive strains of high-value chemicals or pentose (ribose) itself are described. In addition, genome studies of the natural xylose metabolizing yeasts and available tools for their molecular research are reviewed. Metabolism of other pentoses (2-deoxyribose, D-arabinose, lyxose) is briefly reviewed.

Diversity and evolution of the P450 family in arthropods

The P450 family (CYP genes) of arthropods encodes diverse enzymes involved in the metabolism of foreign compounds and in essential endocrine or ecophysiological functions. The P450 sequences (CYPome) from 40 arthropod species were manually curated, including 31 complete CYPomes, and a maximum likelihood phylogeny of nearly 3000 sequences is presented. Arthropod CYPomes are assembled from members of six CYP clans of variable size, the CYP2, CYP3, CYP4 and mitochondrial clans, as well as the CYP20 and CYP16 clans that are not found in Neoptera. CYPome sizes vary from two dozen genes in some parasitic species to over 200 in species as diverse as collembolans or ticks. CYPomes are comprised of few CYP families with many genes and many CYP families with few genes, and this distribution is the result of dynamic birth and death processes. Lineage-specific expansions or blooms are found throughout the phylogeny and often result in genomic clusters that appear to form a reservoir of catalytic diversity maintained as heritable units. Among the many P450s with physiological functions, six CYP families are involved in ecdysteroid metabolism. However, five so-called Halloween genes are not universally represented and do not constitute the unique pathway of ecdysteroid biosynthesis. The diversity of arthropod CYPomes has only partially been uncovered to date and many P450s with physiological functions regulating the synthesis and degradation of endogenous signal molecules (including ecdysteroids) and semiochemicals (including pheromones and defense chemicals) remain to be discovered. Sequence diversity of arthropod P450s is extreme, and P450 sequences lacking the universally conserved Cys ligand to the heme have evolved several times. A better understanding of P450 evolution is needed to discern the relative contributions of stochastic processes and adaptive processes in shaping the size and diversity of CYPomes.

Drug–protein adducts: past, present, and future

Research over the past half-century has demonstrated that the metabolism of drugs and other foreign compounds to chemically reactive intermediates that bind covalently to endogenous proteins can, in certain cases, lead to organ damage or to immune-mediated adverse reactions. While the chemistry of metabolic activation is now relatively well understood, the molecular events that link exposure to reactive metabolites to toxic sequalae remain ill-defined. In particular, the role of covalent protein binding in drug-induced toxicities is unclear, and has been a controversial issue in drug discovery efforts. In this article, the covalent binding of drugs and other xenobiotics to proteins is reviewed from a historical perspective, and the evolution of the field is traced from an early dependence on radiolabeled tracers that provided limited information on adduct structure to contemporary label-free approaches based on powerful chemical proteomics and mass spectrometry methodology that provide a global perspective on proteome reactivity. Currently evolving databases of the proteins targeted by safe and toxic xenobiotics likely will lead in the future to predictive algorithms that can be exploited by medicinal chemists to assist in the design of safe therapeutic agents.

Survey of Machine Learning Techniques for Prediction of the Isoform Specificity of Cytochrome P450 Substrates.

Determination or prediction of the Absorption, Distribution, Metabolism, and Excretion (ADME) properties of drug candidates and drug-induced toxicity plays crucial roles in drug discovery and development. Metabolism is one of the most complicated pharmacokinetic properties to be understood and predicted. However, experimental determination of the substrate binding, selectivity, sites and rates of metabolism is time- and recourse- consuming. In the phase I metabolism of foreign compounds (i.e., most of drugs), cytochrome P450 enzymes play a key role. To help develop drugs with proper ADME properties, computational models are highly desired to predict the ADME properties of drug candidates, particularly for drugs binding to cytochrome P450. This narrative review aims to briefly summarize machine learning techniques used in the prediction of the cytochrome P450 isoform specificity of drug candidates. Both single-label and multi-label classification methods have demonstrated good performance on modelling and prediction of the isoform specificity of substrates based on their quantitative descriptors. This review provides a guide for researchers to develop machine learning-based methods to predict the cytochrome P450 isoform specificity of drug candidates.

Molecular and functional characterization of cytosolic sulfotransferases in cynomolgus macaque

Cytosolic sulfotransferases (SULTs), drug-metabolizing enzymes essential for the metabolism of endogenous biochemicals and foreign compounds, have been characterized in humans, but remain to be investigated in cynomolgus macaques, important species in drug metabolism studies. In this study, based on the genome data, cynomolgus SULT1A1, SULT1A3, SULT1B1, SULT1C2v1, SULT1C2v2, SULT1C4, SULT1E1, and SULT2A1 cDNAs were isolated and characterized. Among these, cynomolgus SULT1C2v2 was highly homologous to human SULT1C2P1 (pseudogene). These cynomolgus SULT cDNAs had high sequence identities (95–97%) to, and closely clustered with their human orthologs in a phylogenetic tree. Gene structure and genomic organization of each cynomolgus SULT were similar to those of the human ortholog. Among the 10 tissue types analyzed, cynomolgus SULTs showed distinct expression patterns similar to human SULTs; more specifically, mRNA was most abundantly expressed in livers (SULT1A1, SULT1C2v2, SULT1C4, and SULT2A1), jejunum (SULT1A3, SULT1B1, and SULT1E1), or kidneys (SULT1C2v1). The most abundant SULT mRNA was SULT2A1, SULT1E1, and SULT1C4 found in livers, jejunum, and kidneys, respectively. Recombinant cynomolgus SULT1A1, SULT1A3, SULT1B1, SULT1C2v1, SULT1C2v2, SULT1C4, SULT1E1, and SULT2A1 in bacterial cytosolic fractions mediated 3′-phosphoadenosine-5′-phosphosulfate-dependent sulfate conjugations of typical human SULT substrates, 1-naphthol, p-nitrophenol, dopamine, dehydroepiandrosterone, and estradiol. Taken together, these results suggest molecular and functional similarities of SULTs between cynomolgus macaques and humans.

Brain Control of Sexually Dimorphic Liver Function and Disease: The Endocrine Connection.

A multistep signaling cascade originates in brain centers that regulate hypothalamic growth hormone-releasing hormone (Ghrh) and somatostatin expression levels and release to control the pattern of GH secretion. This process is sexually fine-tuned, and relays important information to the liver where GH receptors can be found. The temporal pattern of pituitary GH secretion, which is sex-specific in many species (episodic in males and more stable in females), represents a major component in establishing and maintaining the sexual dimorphism of hepatic gene transcription. The liver is sexually dimorphic exhibiting major differences in the profile of more than 1000 liver genes related to steroid, lipid, and foreign compound metabolism. Approximately, 90% of these sex-specific liver genes were shown to be primarily dependent on sexually dimorphic GH secretory patterns. This proposes an interesting scenario in which the central nervous system, indirectly setting GH profiles through GHRH and somatostatin control, regulates sexual dimorphism of liver activity in accordance with the need for sex-specific steroid metabolism and performance. We describe the influence of the loss of sexual dimorphism in liver gene expression due to altered brain function. Among other many factors, abnormal brain sexual differentiation, xenoestrogen exposure and D2R ablation from neurons dysregulate the GHRH-GH axis, and ultimately modify the liver capacity for adaptive mechanisms. We, therefore, propose that an inefficient brain control of the endocrine growth axis may underlie alterations in several metabolic processes through an indirect influence of sexual dimorphism of liver genes.

Cloning, heterologous expression, and activity analysis of NADPH-cytochrome P450 reductase from the Chinese white rabbit

ABSTRACTCytochrome P450 reductase (CPR) is an integral component of the P450 oxidoreductase system (P450s). It serves as the electron donor for most cytochromes in P450s, which are involved in the metabolism of foreign compounds and the synthesis of endocrine hormones and have tremendous biotechnological potential for the synthesis of pharmaceuticals and fine chemicals. However, commercially available CPR is very expensive, and heterologous expression in Escherichia coli is a more affordable way to obtain enough CPR. In the present study, a full-length cDNA encoding a CPR was isolated from the liver of the Chinese white rabbit using reverse transcription-polymerase chain reaction (RT-PCR). The cDNA contains a 2,040-bp open reading frame, which is predicted to encode an enzyme of 679 amino acids. The deduced peptide shares 99.5% amino acid similarity with CPR of Oryctolagus cuniculus, showing that the Chinese white rabbit is a close genetic relative of the European rabbit. The cloned CPR has the typical hallmarks, including an N-terminal membrane anchor and flavin adenine dinucleotide (FAD)-, flavin mononucleotide (FMN)- and nicotinamide adenine dinucleotide phosphate (NADPH)-binding domains. An N-terminally truncated protein was heterologously expressed in E. coli BL21 (DE3) cells and purified, and the specific activity of the recombinant enzyme was determined. The enzyme activity analysis indicated that electrons were passed from NADPH to Cyt C at a rate of 2.3174 μmol/(min/mg protein). The present study provides an efficient procedure for preparing large amounts of recombinant CPR, which would facilitate the synthesis of pharmaceuticals and fine chemicals with P450s.

Cytochrome P450-derived eicosanoids and heart function.

The cytochrome P450 monooxygenase system (CYP) is a multigene superfamily of enzymes, which are important in the metabolism of foreign and endogenous compounds. CYP isoforms metabolize a number of n-3 and n-6 polyunsaturated fatty acids (PUFA), including linoleic acid (18:2n6, LA), arachidonic acid (20:4n6, AA), ecosapentaenoic acid (20:5n3, EPA) and docosahexaenoic acid (22:6n3, DHA) into bioactive lipid mediators, termed eicosanoids. CYP-derived eicosanoids have numerous effects toward physiological and pathophysiological events within the body, which depends on the type, quantity and timing of metabolites produced. Alterations in fatty acid composition and concentrations have been shown to have a role in cardiovascular disease (CVD). The functional role of CYP isozymes and CYP-derived eicosanoids toward physiological and pathophysiological processes in the heart is a rapidly expanding field of research. Numerous studies have investigated the beneficial and detrimental effects of CYP epoxygenase derived metabolites of AA, epoxyeicosatrienoic acids (EET) and CYP ω-hydroxylase products, hydroxyeicosatetraenoic acids (HETE), toward both cardiac and vascular function and disease. Emerging research is revealing the importance of other lipid mediators generated from CYP isozymes, such as epoxyeicosatetraenoic acids (EEQ) and epoxydocosapentaenoic acids (EDP), formed from the metabolism of EPA and DHA and metabolites of LA. Important determinants such as genetics, gender and age have a role in regulating the CYP-derived eicosanoids produced from the metabolism n-3 and n-6 PUFA. Obtaining a better understanding of the complex role CYP-derived eicosanoids have within the heart will provide valuable insight for both basic and clinical researchers investigation CVD.

Physical Studies of P450-P450 Interactions: Predicting Quaternary Structures of P450 Complexes in Membranes from Their X-ray Crystal Structures.

Cytochrome P450 enzymes, which catalyze oxygenation reactions of both exogenous and endogenous chemicals, are membrane bound proteins that require interaction with their redox partners in order to function. Those responsible for drug and foreign compound metabolism are localized primarily in the endoplasmic reticulum of liver, lung, intestine, and other tissues. More recently, the potential for P450 enzymes to exist as supramolecular complexes has been shown by the demonstration of both homomeric and heteromeric complexes. The P450 units in these complexes are heterogeneous with respect to their distribution and function, and the interaction of different P450s can influence P450-specific metabolism. The goal of this review is to examine the evidence supporting the existence of physical complexes among P450 enzymes. Additionally, the review examines the crystal lattices of different P450 enzymes derived from X-ray diffraction data to make assumptions regarding possible quaternary structures in membranes and in turn, to predict how the quaternary structures could influence metabolism and explain the functional effects of specific P450–P450 interactions.

Inhibition of cytochrome P450 2B4 by environmentally persistent free radical-containing particulate matter

Combustion processes generate particulate matter (PM) that can affect human health. The presence of redox-active metals and aromatic hydrocarbons in the post-combustion regions results in the formation of air-stable, environmentally persistent free radicals (EPFRs) on entrained particles. Exposure to EPFRs has been shown to negatively influence pulmonary and cardiovascular functions. Cytochromes P450 (P450/CYP) are endoplasmic reticulum resident proteins that are responsible for the metabolism of foreign compounds. Previously, it was shown that model EPFRs, generated by exposure of silica containing 5% copper oxide (CuO–Si) to either dicholorobenzene (DCB230) or 2-monochlorophenol (MCP230) at ≥ 230°C, inhibited six forms of P450 in rat liver microsomes (Toxicol. Appl. Pharmacol. (2014) 277:200–209). In this study, the inhibition of P450 by MCP230 was examined in more detail by measuring its effect on the rate of metabolism of 7-ethoxy-4-trifluoromethylcoumarin (7EFC) and 7-benzyloxyresorufin (7BRF) by the purified, reconstituted CYP2B4 system. MCP230 inhibited the CYP2B4-mediated metabolism of 7EFC at least 10-fold more potently than non-EPFR controls (CuO-Si, silica, and silica generated from heating silica and MCP at 50°C, so that EPFRs were not formed (MCP50)). The inhibition by EPFRs was specific for the P450 and did not affect the ability of the redox partner, P450 reductase (CPR) from reducing cytochrome c. All of the PM inhibited CYP2B4-mediated metabolism noncompetitively with respect to substrate. When CYP2B4-mediated metabolism of 7EFC was measured as a function of the CPR concentration, the mechanism of inhibition was competitive. EPFRs likely inhibit CYP2B4-mediated substrate metabolism by physically disrupting the CPR·P450 complex.

Role of Cytochrome P450 Monooxygenase in Carcinogen and Chemotherapeutic Drug Metabolism.

The purpose of this chapter is to provide insight into which human cytochromes P450 (CYPs) may be involved in metabolism of chemical carcinogens and anticancer drugs. A historical overview of this field and the development of literature using relevant animal models and expressed human CYPs have provided information about which specific CYPs may be involved in carcinogen metabolism. Definition of the biochemical properties of CYP activity came from several groups who studied the reaction stoichiometry of butter yellow and benzo[α]pyrene, including their role in induction of these enzyme systems. This chapter will list as much as is known about the human CYPs involved in carcinogen and anticancer drug metabolism, as well as summarize studies with rodent CYPs. A review of three major classes of anticancer drugs and their metabolism in humans is covered for cyclophosphamide, procarbazine, and anthracycline antibiotics, cancer chemotherapeutic compounds extensively metabolized by CYPs. The emerging information about human CYP gene polymorphisms as well as other enzymes involved in foreign compound metabolism provides considerable information about how these genetic variants affect carcinogen and anticancer drug metabolism. With information available from individual's genomic sequences, consideration of populations who may be at risk due to environmental exposure to carcinogens or how to optimize their cancer therapy regimens to enhance efficacy of the anticancer drugs appears to be an important field of study to benefit individuals in the future.

The Intestinal Metabolome: An Intersection Between Microbiota and Host

Recent advances that allow us to collect more data on DNA sequences and metabolites have increased our understanding of connections between the intestinal microbiota and metabolites at a whole-systems level. We can also now better study the effects of specific microbes on specific metabolites. Here, we review how the microbiota determines levels of specific metabolites, how the metabolite profile develops in infants, and prospects for assessing a person's physiological state based on their microbes and/or metabolites. Although data acquisition technologies have improved, the computational challenges in integrating data from multiple levels remain formidable; developments in this area will significantly improve our ability to interpret current and future data sets.

Molecular and functional characterization of flavin-containing monooxygenases in cynomolgus macaque

Flavin-containing monooxygenases (FMOs), drug-metabolizing enzymes essential for the metabolism of endogenous biochemicals and foreign compounds, have been characterized in human (including FMO1-5 and FMO6P), but remain to be investigated in cynomolgus macaque. In this study, cDNAs of cynomolgus FMO1-5 and FMO6 were isolated and characterized. Amino acid sequences of cynomolgus FMO1-5, respectively, shared high sequence identities (94–98%) and were closely clustered in a phylogenetic tree, with human FMO1-5. Eight different transcripts, due to alternative splicing, were isolated for cynomolgus FMO6, which is highly identical (~96%) to human FMO6P. Among the 10 tissue types analyzed, cynomolgus FMO1, FMO2, FMO4, and FMO6 were most abundantly expressed in kidney, while cynomolgus FMO3 and FMO5 were most abundantly expressed in liver. In kidney and liver, the most abundantly expressed cynomolgus FMO genes were FMO1 and FMO3, respectively. Cynomolgus FMO1, FMO2, FMO3, and FMO5 metabolized benzydamine, and FMO1/FMO3 and FMO3 also metabolized methimazole and trimethylamine, respectively. Rates of benzydamine N-oxygenation (catalyzed by FMO3) varied (approximately 20-fold) among the 28 cynomolgus livers and were significantly correlated with FMO3 protein expression, indicating that the inter-animal variations in benzydamine N-oxygenation might be partly accounted for by the variable FMO3 expression. Cynomolgus FMO6 metabolized benzydamine only slightly, but minimal expression of FMO6 in all tissue precludes the importance of FMO6 in drug metabolism, unlike cynomolgus FMO1, FMO2, FMO3, and FMO5 which were all functional. Abundant expression of FMO1 and FMO3 in kidney and liver, respectively, suggest their importance in drug metabolism in cynomolgus macaque, similar to human.

Evaluation of Level of Precursors of N-Nitrosamine in Vitro in Wistar Rats Fed Different Levels of Dietary Protein

This study compares the level of Nitrite in urine, protein concentration and nitrite concentration in post mitochondrial fraction of rats fed different levels of dietary protein with concurrent administration of precursors of N-nitrosamine; dimethylamine hydrochloride (DMA-HCl) and sodium nitrite (NaNO2). Thirty Male Wistar rats were divided into three groups and were kept for four weeks. Group one was given high protein diet (64%), group two was given a normal protein diet (27%) and group three was given low protein diet (3.5%). All the groups were administered with 3mg NaNO2 and 20mg DMA-HCL/kg, using the application of spectrophotometric analysis, centrifugation, as well as colorimetric methods. Following administrations of the chemicals to the test animal groups, the concentration of 24 hours urinary excretion of nitrite was 7.417µg/ml in high protein fed rats, 2.063µg/ml in normal protein fed rats and 0.569 µg/ml in low protein fed rats. There was a significant difference (p<0.05) in the nitrite concentration in the group fed high protein diet with concurrent administration of NaNO2 and DMA-HCl. The wistar rats fed with high protein diet, excreted 5.8 to 7 times more nitrite in urine than the severely protein deprived animals. The protein and nitrite concentration of the post mitochondrial fraction of liver was highest in rats that were fed high protein diet. This study has revealed that nutrition status affects metabolism of foreign compounds including nitrites and dimethylamine hydrochloride.

Dexamethasone regulates differential expression of carboxylesterase 1 and carboxylesterase 2 through activation of nuclear receptors

Carboxylesterases (CESs) play important roles in the metabolism of endogenous and foreign compounds in physiological and pharmacological responses. The aim of this study was to investigate the effect of dexamethasone at different doses on the expression of CES1 and CES2. Imidapril and irinotecan hydrochloride (CPT-11) were used as special substrates for CES1 and CES2, respectively. Rat hepatocytes were cultured and treated with different concentrations of dexamethasone. The hydrolytic activity of CES1 and CES2 was tested by incubation experiment and their expression was quantitated by real-time PCR. A pharmacokinetic study was conducted in SD rats to further evaluate the effect of dexamethasone on CESs activity in vivo. Western blotting was performed to investigate the regulatory mechanism related to pregnane X receptor (PXR) and glucocorticoid receptor (GR). The results showed that exposure of cultured rat hepatocytes to nanomolar dexamethasone inhibited the imidapril hydrolase activity, which was slightly elevated by micromolar dexamethasone. For CES2, CPT-11 hydrolase activity was induced only when dexamethasone reached micromolar levels. The real-time PCR demonstrated that CES1 mRNA was markedly decreased by nanomolar dexamethasone and increased by micromolar dexamethasone, whereas CES2 mRNA was significantly increased by micromolar dexamethasone. The results of a complementary animal study showed that the concurrent administration of dexamethasone significantly increased the plasma concentration of the metabolite of imidapril while the ratio of CPT-11 to its metabolite SN-38 was significantly decreased. PXR protein was gradually increased by serial concentrations of dexamethasone. However, only nanomolar dexamethasone elevated the level of GR protein. The different concentrations of dexamethasone required suggested that suppression of CES1 may be mediated by GR whereas the induction of CES2 may result from the role of PXR. It was concluded that dexamethasone at different concentrations can differentially regulate CES1 and CES2.

A Method for Efficient Calculation of Diffusion and Reactions of Lipophilic Compounds in Complex Cell Geometry

A general description of effects of toxic compounds in mammalian cells is facing several problems. Firstly, most toxic compounds are hydrophobic and partition phenomena strongly influence their behaviour. Secondly, cells display considerable heterogeneity regarding the presence, activity and distribution of enzymes participating in the metabolism of foreign compounds i.e. bioactivation/biotransformation. Thirdly, cellular architecture varies greatly. Taken together, complexity at several levels has to be addressed to arrive at efficient in silico modelling based on physicochemical properties, metabolic preferences and cell characteristics. In order to understand the cellular behaviour of toxic foreign compounds we have developed a mathematical model that addresses these issues. In order to make the system numerically treatable, methods motivated by homogenization techniques have been applied. These tools reduce the complexity of mathematical models of cell dynamics considerably thus allowing to solve efficiently the partial differential equations in the model numerically on a personal computer. Compared to a compartment model with well-stirred compartments, our model affords a more realistic representation. Numerical results concerning metabolism and chemical solvolysis of a polycyclic aromatic hydrocarbon carcinogen show good agreement with results from measurements in V79 cell culture. The model can easily be extended and refined to include more reactants, and/or more complex reaction chains, enzyme distribution etc, and is therefore suitable for modelling cellular metabolism involving membrane partitioning also at higher levels of complexity.