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R A McKinnon

Publications and source records attributed to R A McKinnon.

At least 19 recordsLinked to original sources

Polymorphic variations in the expression of the chemical detoxifying UDP glucuronosyltransferases.

The UDP glucuronosyltransferases (UGT) are expressed predominantly in the liver and gastrointestinal tract in humans. Their expression varies widely between individuals, due in part to coding region polymorphisms that alter catalytic function and in part, to differences in the regulation of UGT genes. The latter differences are most likely the result of polymorphisms in the regulatory elements of UGT genes and in the transcription factors that bind to these elements. Several frequent polymorphisms in the promoters of UGT genes have been described; however, few of these fall within critical regulatory elements and alter UGT expression. Some rare mutations alter UGT promoter activity in in vitro systems but their effect in the clinic is still to be confirmed. Several transcription factors that regulate UGT gene expression in cells of hepatic and intestinal origin have been identified. These include positive regulators of UGT gene expression such as hepatocyte nuclear factor 1 alpha (HNF1 alpha), octamer transcription factor-1 (Oct-1) and the intestine-specific transcription factor, caudal-related homeodomain protein 2 (Cdx2). Negative regulators include the Pre B cell homeobox factor (Pbx2) and its dimerization partner, Pbx regulating protein 1 (Prep1). Polymorphisms in these transcription factors may cause differences in their interaction and binding to UGT promoters. Current work describing the effects of these transcription factor polymorphisms on UGT expression will be described. Knowledge of UGT promoter elements and the proteins that bind to these elements, as well as knowledge of polymorphisms that alter their function, may aid in the prediction of an individual's response to chemicals and in the prediction of chemical toxicities.

Gene Expression Regulation, Enzymologic↗

Towards integrated ADME prediction: past, present and future directions for modelling metabolism by UDP-glucuronosyltransferases.

Undesirable absorption, distribution, metabolism, excretion (ADME) properties are the cause of many drug development failures and this has led to the need to identify such problems earlier in the development process. This review highlights computational (in silico) approaches that have been used to identify the characteristics of ligands influencing molecular recognition and/or metabolism by the drug-metabolising enzyme UDP-gucuronosyltransferase (UGT). Current studies applying pharmacophore elucidation, 2D-quantitative structure metabolism relationships (2D-QSMR), 3D-quantitative structure metabolism relationships (3D-QSMR), and non-linear pattern recognition techniques such as artificial neural networks and support vector machines for modelling metabolism by UGT are reported. An assessment of the utility of in silico approaches for the qualitative and quantitative prediction of drug glucuronidation parameters highlights the benefit of using multiple pharmacophores and also non-linear techniques for classification. Some of the challenges facing the development of generalisable models for predicting metabolism by UGT, including the need for screening of more diverse structures, are also outlined.

Computational Biology↗

Optimizing bacterial expression of catalytically active human cytochromes P450: comparison of CYP2C8 and CYP2C9.

1. Methods for the co-expression in Escherichia coli of human cytochrome P450 (CYP) 2C8 and CYP2C9 with NADPH-cytochrome P450 reductase (OxR) to produce a catalytically active system were compared. 2. Approaches assessed were expression of a CYP:OxR fusion construct, bicistronic plasmids, simultaneous transformation with CYP and OxR plasmids, and separate expression of CYP and OxR with reconstitution of activity by mixing the bacterial membranes. Two N-terminal modifications (Delta3-20 and 17alpha-leader) of the individual P450s were additionally investigated. 3. Each approach gave efficient expression of CYP2C8 and CYP2C9, but the bicistronic constructs under the expression conditions used gave low OxR expression and low catalytic activity. CYP expression was higher with the Delta3-20 construct for CYP2C9 and with the 17alpha-presequence construct for CYP2C8. 4. Using torsemide as substrate, all methods gave catalytically active systems with K(m) values similar to human liver microsomes. Mixing bacterial membranes containing separately expressed CYP and OxR reconstituted a catalytically active system with the Delta3-20 construct for CYP2C9 but not for CYP2C8, and with neither of the 17alpha- presequence constructs. OxR co-expressed with CYP in the same membrane interacted with CYP to reconstitute activity more effectively than addition of exogenous OxR membranes. 5. Expression construct and OxR co-expression strategy should be individualized for CYP isoforms.

Aryl Hydrocarbon Hydroxylases↗

In silico insights: chemical and structural characteristics associated with uridine diphosphate-glucuronosyltransferase substrate selectivity.

1. Undesirable absorption, distribution, metabolism, excretion properties are the cause of many drug development failures and this has led to the need to identify such problems earlier in the development process. This work highlights computational (in silico) approaches used to identify characteristics influencing the metabolism of uridine diphosphate (UDP)-glucuronosyltransferase (UGT) substrates. Uridine diphosphate-glucuronosyltransferase facilitates conjugation between glucuronic acid and a nucleophilic site within a substrate and is one of the major drug-metabolizing enzymes. 2. An understanding of the relevant structural and chemical characteristics of the ligand and the enzyme active site will lead to greater utilization of metabolically relevant structural information in drug design. However, an X-ray crystal structure of UGT is not yet available, little has been reported about important structurally or catalytically relevant amino acids and only recently has the reported substrate profile of UGT isoforms reached an interpretable level. 3. A database of all the known substrates and non-substrates for each human UGT isoform was assembled and a range of modelling approaches assessed. Currently, pharmacophore models developed using Catalyst (Accelrys, San Diego, CA, USA) indicate that substrates of the UGT1A family share two key hydrophobic regions 3 and 6-7 A from the site of glucuronidation in a well-defined spatial geometry. Furthermore, two-dimensional quantitative structure-activity relationship models show significant reliance on substrate lipophilicity and a range of other descriptors that are known to capture information relevant to ligand-protein interactions. 4. In conclusion, substrate-based modelling of UGT appears both useful and feasible, with significant potential for determining aspects of chemical structure associated with metabolism and to quantify the nature of the relationship for UGT substrates. The development of a novel, user-defined 'glucuronidation feature' for alignment was crucial to the development of pharmacophore-based UGT models.

Glucuronosyltransferase↗

Xenobiotic metabolism in Australian marsupials.

The Australian marsupials are significant and unique Australian fauna. Xenobiotic metabolism is the process of enzymatic modification of xenobiotics, which include the chemicals, such as agricultural chemicals and natural dietary toxins, that these animals may be exposed to. Very little is known about the enzymes involved in xenobiotic metabolism in this unique group of animals. Folivore marsupials such as the koala (Phascolarctos cinereus and the brushtail possum (Trichosurus vulpecula) represent unique adaptation which has only been relatively superficially examined to date. We provide an overview of our current knowledge of marsupial xenobiotic metabolism.

Adaptation, Physiological↗

Cytochrome P450 4A, peroxisomal enzymes and nicotinamide cofactors in koala liver.

We have examined hepatic levels of microsomal lauric acid hydroxylase activity and cyanide-insensitive palmitoyl coenzyme A oxidative activity in koala (Phascolarctos cinereus) and tammar wallaby (Macropus eugenii) and compared our results to those determined in rat. Microsomal lauric acid hydroxylation was significantly higher in koala than in tammar wallaby or rat. However, cyanide-insensitive palmitoyl-CoA oxidation was absent in the koala. We have also determined the hepatic nicotinamide cofactors in these species. Hepatic nicotinamide-adenine dinucleotide (NAD) and the ratio of NAD/nicotinamide-adenine dinucleotide phosphate (NADP) were higher in koala than in tammar wallaby and rat liver. Reverse transcription of koala liver mRNA, followed by polymerase chain reaction using primers based on highly conserved areas in the CYP4A family led to the cloning of a partial, near full length, cDNA clone with approximately 70% nucleotide and deduced amino acid sequence identity to human CYP4A11. The CYP has been named CYP4A15.

Amino Acid Sequence↗

Characterisation of tolbutamide hydroxylase activity in the common brushtail possum, (Trichosurus vulpecula) and koala (Phascolarctos cinereus): inhibition by the eucalyptus terpene 1,8-cineole.

Plant constituents such as terpenes are major constituents of the essential oil in Eucalyptus sp. 1,8-Cineole and p-cymene (Terpenes present in high amounts in Eucalyptus leaves) are potential substrates for the CYP family of enzymes. We have investigated tolbutamide hydroxylase as a probe substrate reaction in both koala and terpene pretreated and control brushtail possum liver microsomes and examined inhibition of this reaction by Eucalyptus terpenes. The specific activity determined for tolbutamide hydroxylase in the terpene treated brushtails was significantly higher than that for the control animals (1865+/-334 nmol/mg microsomal protein per min versus 895+/-27 nmol/mg microsomal protein per min). The activity determined in koala microsomes was 8159+/-370 nmol/mg microsomal protein per min. Vmax values and Km values for the terpene treated possum, control, possum and koala were 1932-2225 nmol/mg microsomal protein per min and 0.80 0.81 mM; 1406-1484 nmol/mg microsomal protein per min and 0.87-0.92 mM and 5895-6403 nmol/mg microsomal protein per min and 0.067-0.071 mM, respectively. Terpenes were examined as potential inhibitors of tolbutamide hydroxylase activity. 1,8-Cineole was found to be a competitive inhibitor for the enzyme responsible for tolbutamide hydroxylation (Ki 15 microM) in the possum. In koala liver microsomes stimulation of tolbutamide hydroxylase activity was observed when concentrations of cineole were increased. Therefore, although inhibition was observed, the type of inhibition could not be determined.

Animals↗

Testosterone dehydrogenase activity in koala liver: characterisation of cofactor and steroid substrate differences.

We have studied the hepatic microsomal 17beta-hydroxysteroid dehydrogenase (17beta-HSD) capacity of koala (Phascolarctos cinereus) and tammar wallaby (Macropus eugenii). A detailed comparison of the activity in hepatic fractions from koala and rat was made. Hepatic microsomal NADP-supported 17beta-HSD activity was significantly higher in koala (11.64+/-3.35 nmoles/mg protein/min), (mean+/-S.D.) than in tammar wallaby liver (1.52+/-0.79 nmoles/mg protein/min). However, when NAD was utilised as cofactor the activity was similar in both marsupial species (2.83+/-2.03 nmoles/mg protein/min, koala; 0.70+/-0.71 nmoles/mg protein/min, tammar wallaby). Data for rat indicated a cofactor preference for NAD rather than NADP (17.94+/-6.40 nmoles/mg protein/min, NAD; 2.18+/-1.04 nmoles/mg protein/min, NADP). Michaelis-Menten parameters for the kinetics of 17beta-HSD testosterone oxidation by NADP and NAD were determined in the koala. The Km for testosterone was of the order of 10.0-24.0 microM (n = 6) irrespective of the cofactor used, whilst the Km for NADP was 0.28-0.43 microM (n = 2) and for NAD was 13.9-18.5 microM (n = 2). 17beta-estradiol was found to be an inhibitor of both NAD- and NADP- supported 17beta-HSD activity. These findings indicate that NADP-mediated, but not NAD-mediated testosterone dehydrogenation is a major pathway of steroid biotransformation in koala liver; the reaction is less extensive in fractions from wallaby, human and rat. Such species-related differences in cofactor preference may contribute along with species differences in gene expression to observed rates of 17beta-HSD activity in mammals.

17-Hydroxysteroid Dehydrogenases↗

Refining the mouse chromosomal location of Cdm, the major gene associated with susceptibility to cadmium-induced testicular necrosis.

Cadmium (Cd++) is a widespread environmental pollutant and classifed as an IARC 'Category I' human carcinogen. Cd++ can also cause severe renal toxicity and may be involved clinically in cardiovascular disease and osteoporosis. Genetic differences in sensitivity to cadmium toxicity have been noted in humans, whereas, among inbred mouse strains, unequivocal genetic data exist. Resistance to cadmium-induced testicular damage was reported in 1973 to be associated with a single major recessive gene, named Cdm, which has now been localized to mouse chromosome (Chr) 3. Using polymorphic microsatellite markers and semiquantitative histological parameters, we have corroborated the original 1973 data concerning mendelian inheritance and have further refined the region containing the Cdm gene from more than 24 cM to 0.64 cM (estimated 40-80 genes). We phenotyped 26 recombinant inbred lines generated from C57BL/6J (B6, resistant) and DBA/2J (D2, sensitive) inbred mice, and determined that the Cdm gene maps between microsatellite markers D3Mit110 and D3Mit255. Although toxicity to numerous heavy metals is well known, virtually no molecular mechanisms have yet been uncovered either in humans or laboratory animals. Identification and characterization of the mouse Cdm gene should enhance our understanding of heavy metal toxicity by identifying and characterizing, for the first time, a major mammalian gene responsible for susceptibility to diseases caused by heavy metal toxicity.

Animals↗

Polymorphisms in UDP glucuronosyltransferase genes: functional consequences and clinical relevance.

As glucuronidation is a major process for the metabolism and removal of lipophilic chemicals, polymorphic variations in genes encoding the enzymes involved in this process, the UDP glucuronosyltransferases (UGT), may have a significant impact on our capacity to detoxify and eliminate drugs and toxins. Although 24 human UGT genes have been identified to date, only polymorphisms in five UGTs, viz. UGT1A1, UGT1A6, UGT2B4, UGT2B7 and UGT2B15 have been described. Polymorphisms in UGT1A1, the major bilirubin-glucuronidating form, often result in a decreased capacity to glucuronidate bilirubin, such as observed in Gilbert Syndrome and some forms of perinatal jaundice. The frequencies of individual UGT1A1 polymorphisms show extensive variability across ethnic groups. Two variants of UGT1A6 and UGT2B4 and one variant of UGT2B7 and UGT2B15 have been identified. However, the clinical significance of these variants is unclear. More UGT polymorphisms will undoubtedly be discovered when the human genome is sequenced. However, unless the UGT in question is responsible for the exclusive metabolism of a particular drug or chemical (e.g. UGT1A1 and bilirubin) or is the predominant or only UGT present in the cell, it is unlikely that these polymorphisms will be of major clinical significance.

Animals↗

Hepatic microsomal enzyme activity in the koala and tammar wallaby: high 17beta-hydroxysteroid oxidoreductase activity in koala liver microsomes.

We have studied the hepatic microsomal xenobiotic metabolising capacity of koala (Phascolarctos cinereus) and tammar wallaby (Macropus eugenii). Total cytochrome P450 content in hepatic microsomes from koala (0.87 +/- 0.18 nmol/mg protein, n = 4, mean (S.D.) and rat were comparable while tammar wallaby displayed reduced P450 content (0.24 +/- 0.04 nmol/mg protein). Associated microsomal activities (NADPH cytochrome P450 reductase, aminopyrine N-demethylation, aniline hydroxylation, and androstenedione 6beta- and 16alpha-hydroxylation) in koala liver were similar to or reduced relative to rat. Hepatic microsomal NADPH-supported 17beta-hydroxysteroid oxidoreductase (17beta-HSOR) activity was significantly higher in koala (9.99+/-3.08 nmol/mg protein/min) than in tammar wallaby liver (0.86 +/- 0.16 nmol/mg protein/min). However, when NADH was utilised as cofactor the activity was similar in both marsupial species (koala, 1.44 +/- 0.84 nmol/mg protein/min; tammar wallaby, 1.52 +/- 0.44 nmol/mg protein/min). Michaelis-Menten parameters for the kinetics of 17beta-HSOR androstenedione reduction by NADPH and NADH were determined in the koala. The Km for androstenedione was of the order of 1.9-4 microM (n = 4) irrespective of the cofactor used, whilst the Km for NADPH was 0.04-0.05 microM (n = 2) and for NADH was 134-430 microM (n = 2). Potential inhibitors were evaluated for their effects on NADPH-mediated 17beta-HSOR activity with menadione and, to lesser extents, menthone, benzaldehyde and metyrapone eliciting significant inhibition. From detailed kinetic studies menthone was found to be an uncompetitive inhibitor of the activity in koala liver (Ki 220 microM).

Animals↗

Role of CYP2A5 and 2G1 in acetaminophen metabolism and toxicity in the olfactory mucosa of the Cyp1a2(-/-) mouse.

Acetaminophen (AP) is a widely-used analgesic agent that has been linked to human liver and kidney disease with prolonged or high-dose usage. In rodents, the target organs that are affected include liver, kidney, and the olfactory mucosa. AP toxicity requires cytochrome P450(CYP)-mediated metabolic activation, and the isozymes CYP1A2, 2E1, and 3A are known to activate AP in the human. In the present study, we determined that olfactory mucosal toxicity of AP was not different between the Cyp1a2(+/+) wild-type and the Cyp1a2(-/-) knockout mouse, whereas the hepatic toxicity of AP was significantly diminished in Cyp1a2(-/-) mice. Western blots of olfactory mucosa revealed that CYP2E1 and CYP3A levels are similar between untreated Cyp1a2(+/+) and Cyp1a2(-/-) mice. Diallyl sulfide (DAS), a known inhibitor of CYP2E1 and of CYP2A10/2A11 (the rabbit orthologue of mouse CYP2A5), completely eliminated olfactory toxicity of AP in both the Cyp1a2(-/-) and wild-type mouse olfactory mucosa. We found that heterologously expressed mouse CYP2A5 and CYP2G1 enzymes (known to be present in olfactory mucosa) form 3-hydroxyacetaminophen (3-OH-AP) and 3-(glutathion-S-yl)acetaminophen (GS-AP); CYP2A5 is considerably more active than 2G1. Addition of GSH caused increases in GS-AP proportional to decreases in 3-OH-AP, suggesting that these two metabolites arise from a common precursor or are formed by way of competing pathways. We also found that both CYP2A5 and CYP2G1 are inhibitable by DAS in vitro. These studies provide strong evidence that, in addition to CYP2E1, CYP2A5 and 2G1 are important in AP bioactivation in the mouse olfactory mucosa and that CYP1A2 appears to be of minor importance for AP olfactory toxicity.

Acetaminophen↗

Human thiopurine methyltransferase: no evidence of activation by its substrates.

A HPLC assay was developed to assay baculovirus expressed human thiopurine methyltransferase activity. Using 6-mercaptopurine as substrate, the expressed thiopurine methyltransferase was found to have an apparent Km of 0.99 mM and a Vmax of 19 nmoles/mg/min. These values are in agreement with those determined using the standard radiometric assay for thiopurine methyltransferase activity. The effects of 6-thioguanine on 6-mercaptopurine metabolism were determined. 6-Thioguanine was found to be a mixed inhibitor of 6-mercaptopurine methylation.

Animals↗

Cytochrome P450 knockout mice: new toxicological models.

1. Despite the frenetic pace of cytochrome P450 (CYP) research, important questions remain about the possible role of xenobiotic-metabolizing CYP in development and cellular homeostasis. 2. The recent experimental development of CYP null mouse lines has provided valuable new models for addressing these issues, as well as providing novel in vivo models to study the precise role of CYP in chemical-mediated toxicity. 3. The present review summarizes results from the three Cyp gene knockout mouse lines generated so far and looks at likely future directions of this research field. 4. Also discussed are null mouse lines with targeted mutations in genes encoding receptors having primary roles in Cyp gene regulation.

Animals↗

Sensitivity of CYP1A1 mRNA inducibility by dioxin is the same in Cyp1a2(+/+) wild-type and Cyp1a2(-/-) null mutant mice.

In mammals, the induction of experimental porphyria by halogenated aromatic hydrocarbons (HAHs) seems to be influenced by the levels of hepatic CYP1A2. The pharmacokinetics and relative rates of uptake and storage of HAHs in the liver are correlated with hepatic CYP1A2 concentrations. It is possible that these rates of HAH uptake and storage might affect the expression of other HAH-inducible genes. The differential inducibility of liver CYP1A1 mRNA by dioxin was therefore compared in Cyp1a2(+/+) wild-type mice, Cyp1a2(+/-) heterozygotes, and Cyp1a2(-/-) homozygous null mutants. Using doses of dioxin over eight orders of magnitude (from 10[-12] to 10[-4] g/kg), we could detect no differences in the sensitivity of CYP1A1 mRNA inducibility. These data indicate that the complete absence of the microsomal CYP1A2 enzyme has no measurable effect on hepatic expression of the Cyp1a1, gene, the only other known member of the mammalian CYP1A cytochrome P450 subfamily.

Animals↗

The role of xenobiotic metabolizing enzymes in arylamine toxicity and carcinogenesis: functional and localization studies.

In both animal models and humans, the first and obligatory step in the activation of arylamines is N-hydroxylation. This pathway is primarily mediated by the phase-I enzymes CYP1A1, CYP1A2 and CYP4B1. In the presence of flavonoids such as alpha-naphthoflavone and flavone, both CYP3A4 and CYP3A5 have also been shown to play a minor role in the activation of food-derived heterocyclic amines. The further activation of N-hydroxyarylamines by phase-II metabolism can involve both N, O-acetylation and N, O-sulfonation catalyzed by N-acetyltransferases (NAT1 and NAT2) and sulfotransferases, respectively. Using an array of techniques, we have been unable to detect constitutive CYP1A expression in any segments of the human gastrointestinal tract. This is in contrast to the rabbit where CYP1A1 protein was readily detectable on immunoblots in microsomes prepared from the small intestine. In humans, CYP3A3/3A4 expression was detectable in the esophagus and all segments of the small intestine. Northern blot analysis of eleven human colons showed considerable heterogeneity in CYP3A mRNA between individuals, with the presence of two mRNA species in some subjects. Employing the technique of hybridization histochemistry (also known as in situ hybridization), CYP4B1 expression was observed in some human colons but not in the liver or the small intestine. Hybridization histochemistry studies have also demonstrated variable NAT1 and NAT2 expression in the human gastrointestinal tract. NAT1 and NAT2 mRNA expression was detected in the human liver, small intestine, colon, esophagus, bladder, ureter, stomach and lung. Using a general aryl sulfotransferase riboprobe (HAST1), we have demonstrated marked sulfotransferase expression in the human colon, small intestine, lung, stomach and liver. These studies demonstrate that considerable variability exists in the expression of enzymes involved in the activation of aromatic amines in human tissues. The significance of these results in relation to a role for heterocyclic amines in colon cancer is discussed.

Amines↗

Genetic polymorphisms in human drug-metabolizing enzymes: potential uses of reverse genetics to identify genes of toxicological relevance.

The human mind was engaged with fundamental questions on the nature of heredity long before the study of genetics became a scientific discipline. Many traits, such as height, eye color, blood pressure, or cancer susceptibility, have been known to run in families, although the genes or combination of genes that underlie these observable characteristics remain unknown in most cases. Differences in susceptibility to environmental agents in humans are likewise determined by variations in genetic background--genetic polymorphisms. In this article, we review the current status of studies on human polymorphisms in drug-metabolizing enzymes and discuss various approaches to the analysis of genetic polymorphisms. We expect that in the near future, novel methods in genetic analysis of human populations will be likely to play a key role in the identification of genes of toxicological relevance.

Cytochrome P-450 Enzyme System↗

Cyp1a2(-/-) null mutant mice develop normally but show deficient drug metabolism.

Cytochrome P450 1A2 (CYP1A2) is a predominantly hepatic enzyme known to be important in the metabolism of numerous foreign chemicals of pharmacologic, toxicologic, and carcinogenic significance. CYP1A2 substrates include aflatoxin B1, acetaminophen, and a variety of environmental arylamines. To define better the developmental and metabolic functions of this enzyme, we developed a CYP1A2-deficient mouse line by homologous recombination in embryonic stem cells. Mice homozygous for the targeted Cyp1a2 gene, designated Cyp1a2(-/-), are completely viable and fertile; histologic examination of 15-day embryos, newborn pups, and 3-week-old mice revealed no abnormalities. No CYP1A2 mRNA was detected by Northern blot analysis. Moreover, mRNA levels of Cyp1a1, the other gene in the same subfamily, appear unaffected by loss of the Cyp1a2 gene. Because the muscle relaxant zoxazolamine is a known substrate for CYP1A2, we studied the Cyp1a2(-/-) genotype by using the zoxazolamine paralysis test: the Cyp1a2(-/-) mice exhibited dramatically lengthened paralysis times relative to the Cyp1a2(+/+) wild-type animals, and the Cyp1a2(+/-) heterozygotes showed an intermediate effect. Availability of a viable and fertile CYP1A2-deficient mouse line will provide a valuable tool for researchers wishing to define the precise role of CYP1A2 in numerous metabolic and pharmacokinetic processes.

Animals↗