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D M Grant

Publications and source records attributed to D M Grant.

At least 19 recordsLinked to original sources
PubMed1995

Genetic analysis of microsomal epoxide hydrolase in patients with carbamazepine hypersensitivity.

Carbamazepine therapy is occasionally complicated by hypersensitivity reactions, the mechanism of which is poorly understood. It has been suggested that affected individuals may have a genetically-determined defect of microsomal epoxide hydrolase. The aim of this study was to determine whether a single genetic mutation or pattern of mutations could be used to predict individual susceptibility to carbamazepine-hypersensitivity. DNA was isolated from 10 carbamazepine-hypersensitive patients and 10 healthy volunteers. The patients had developed various forms of toxicity with carbamazepine, including toxic epidermal necrolysis, Stevens-Johnson syndrome, hepatitis and pneumonitis. The technique of polymerase chain reaction single-strand conformation polymorphism analysis (PCR-SSCP) was used to screen for mutations in all nine exons of the microsomal epoxide hydrolase gene. Any new mutations detected by this method were characterised by direct sequencing of the DNA. In addition, in the most severely affected patient, we sequenced all nine exons of the gene. There was a higher frequency of mutations in the hypersensitive group when compared with the controls, but there was no consistent mutation (or pattern of mutations) in the microsomal epoxide hydrolase gene which was common to the hypersensitive group. DNA sequencing of all nine exons of the microsomal epoxide hydrolase gene from the most severely affected patient showed the sequence to be "wild-type," when compared to the previously published sequences. The results of this study suggest that a single mutation within the coding region of the microsomal epoxide hydrolase gene cannot be the sole determinant of the predisposition to carbamazepine hypersensitivity.

Adolescent↗
PubMed1995

Relationship of 13C NMR chemical shift tensors to diffraction structures.

13C chemical shift tensor measurements on single crystals provide a powerful method to study changes in the electron environment of nuclei with changes in molecular structure. Thus, diffraction structures are critical to an understanding of chemical shift tensors. This work explores the general reliability of using structural data to predict components of the symmetrical chemical shift tensor. Imprecision in the hydrogen positions introduces considerable scatter in the simulated 13C shift tensors, and optimized C-H bond distances in methyl-beta-D-glucopyranoside used with the X-ray positions of the heavier C and O atoms greatly improve the simulated chemical shifts. Acenaphthene, with two crystallographically different molecules per unit cell, offers an excellent example for comparing and contrasting structural differences in the two molecules. A recently improved X-ray structure of naphthalene obtained at low temperature provides chemical shift simulations which are comparable to those from neutron diffraction methods and appear to reflect breaks in the D2h symmetry measured in the NMR chemical shift tensors. These data illustrate the close relationship between NMR and diffraction structures.

Carbohydrate Conformation↗
PubMed1995

In vitro formation, disposition and toxicity of N-acetoxy-sulfamethoxazole, a potential mediator of sulfamethoxazole toxicity.

Variation in the formation and disposition of the hydroxylamine of (SMX-HA) is thought to play an important role in the pathogenesis of sulfamethoxazole (SMX)-induced idiosyncratic adverse drug reactions. We hypothesized that, in analogy to carcinogenic arylamines, SMX-HA might be further converted to an electrophilic N-acetoxy metabolite which could play a role in mediating SMX toxicity. Accordingly, we chemically synthesized N-acetoxy-SMX, and examined the characteristics of its formation, metabolism, cytotoxicity and mutagenicity in human and bacterial test systems. The human arylamine N-acetyl-transferases, (NAT)1 and NAT2, were capable of converting SMX-HA to N-acetoxy-SMX. NAT1 and NAT2 possessed similar affinities for SMX-HA (apparent Km values of 650 and 520 microM, respectively), but the apparent maximal velocity of the NAT1-mediated acetylation was higher than that of NAT2. (1332 vs. 37 nmol/min/U of immunoreactive NAT protein). Human peripheral blood mononuclear cells 12,000 x g supernatant fractions converted N-acetoxy-SMX mainly back to SMX-HA, and also to a lesser extent to SMX, at clinically relevant concentrations. Similar pathways were observed in human hepatic cytosolic fractions. In a cytotoxicity assay, N-acetoxy-SMX was significantly more toxic to human peripheral blood mononuclear cells than SMX-HA (16.6 vs. 11.5% dead cells at a concentration of 300 microM). N-acetoxy-SMX was weakly mutagenic to the Salmonella typhimurium TA100 strain in the Ames test. These data suggest that the N-acetoxy metabolites of sulfonamides could potentially play a role in mediating sulfonamide idiosyncratic adverse drug reactions.

Acetyltransferases↗
PubMed1992

Improvements in the computerized analysis of 2D INADEQUATE spectra.

The carbon skeleton of a molecule can be determined by using the powerful 2D INADEQUATE experiment, but the method suffers from very poor sensitivity at natural carbon-13 abundance. A computer program, described previously, has been significantly improved in its ability to recognize AB spectral patterns corresponding to carbon-carbon bonds which makes it possible to evaluate reliably spectra with rms S/N ratio as low as 2.5, i.e., nearly 1 order of magnitude below the level required for routine manual interpretation. Application of the INADEQUATE experiment to samples containing as little as 20 mumol of a compound of interest is now possible. The method is described in detail and critically evaluated by means of examples and simulations.

Carbon Isotopes↗
PubMed1992

Applications of the improved computerized analysis of 2D INADEQUATE spectra.

This paper illustrates the use of the program CCBond to determine the carbon skeletons of bioorganic molecules in low concentration samples. Discussed is the structure elucidation of bistramide A, a compound extracted from a Fijian Lissoclinum sp. and cholesterol in 71- and 20-mumol samples, respectively. The detection limit of the automated bond extraction is shown to be dramatically improved compared to the manual interpretation of 2D INADEQUATE spectra.

Acetamides↗
PubMed1992

Salmonella typhimurium strains expressing human arylamine N-acetyltransferases: metabolism and mutagenic activation of aromatic amines.

Epidemiological studies have established the carcinogenic risk of occupational exposure to aromatic amines such as benzidine, beta-naphthylamine, and 4-aminobiphenyl. Metabolic activation of these chemicals to reactive, genotoxic electrophiles, via enzymatic N-oxidation and subsequent conjugation reactions, is necessary for their carcinogenic potential to be realized. Many aromatic amines are mutagenic in prokaryotic test systems, in the presence of exogenous mammalian activating enzymes such as those contained in hepatic 9000 x g supernatant. However, in the Ames (Salmonella typhimurium) assay, induction of mutations by aromatic amines and nitroarenes is also almost completely dependent upon the activity of the endogenous bacterial enzyme, N-acetyltransferase/O-acetyltransferase. The relevance of this assay to the prediction of the carcinogenic potential of aromatic amines in humans is thus restricted by the likelihood that the bacterial and human enzymes possess different substrate specificities. In this paper we report the construction and use of new tester strains of S. typhimurium that express high levels of functional human arylamine N-acetyltransferases, NAT1 and NAT2, retaining characteristic arylamine substrate specificities that are distinct from those of the bacterial enzyme. These new strains support the mutagenic activation of benzidine, 2-aminofluorene and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline in the Ames test and may provide a new tool for evaluating the carcinogenic potential of aromatic amines.

Arylamine N-Acetyltransferase↗
PubMed1992

Site-directed mutagenesis of recombinant human arylamine N-acetyltransferase expressed in Escherichia coli. Evidence for direct involvement of Cys68 in the catalytic mechanism of polymorphic human NAT2.

The single coding exons of the cloned genes encoding two human arylamine N-acetyltransferases (NAT1 and NAT2) were amplified by expression-cassette polymerase chain reaction and subcloned into the tac promoter-based phagemid vector pKEN2 for production of the recombinant proteins in Escherichia coli strain XA90. Induction of cultures grown from selected bacterial transformants resulted in the production of substantial quantities of soluble recombinant human NAT1 and NAT2 with identical electrophoretic, immunologic and catalytic properties to those expressed in mammalian cell culture or in human liver. Oligonucleotide-directed mutagenesis of recombinant human NAT2 was then employed to determine the relative importance of 3 highly conserved cysteine residues in the enzyme's catalytic mechanism. Substitution of cysteine with glycine at position 68 of the 290 amino acid protein molecule (Cys68----Gly) resulted in the production of normal quantities of immunoreactive NAT2 which was completely devoid of enzyme activity, suggesting that the sulfhydryl group of Cys68 is directly involved in the transfer of acetate from the essential cofactor CoASAc to acceptor amine substrates. On the other hand, the mutations producing Cys44----Gly and Cys223----Gly led to the production of enzymatically active NAT2 proteins with markedly reduced in vitro stability, suggesting that substitution of either of these amino acids may cause alterations in the tertiary structure of the native enzyme.

Arylamine N-Acetyltransferase↗
PubMed1992

Selective saturation and inversion of multiple resonances in high-resolution solid-state 13C experiments using slow spinning CP/MAS and tailored DANTE pulse sequences.

Taking advantage of the long 13C T1 values generally encountered in solids, selective saturation and inversion of more than one resonance in 13C CP/MAS experiments can be achieved by sequentially applying several DANTE pulse sequences centered at different transmitter frequency offsets. A new selective saturation pulse sequence is introduced composed of a series of 90 degrees DANTE sequences separated by interrupted decoupling periods during which the selected resonance is destroyed. Applications of this method, including the simplification of the measurement of the principal values of the 13C chemical shift tensor under slow MAS conditions, are described. The determination of the aromaticity of coal using a relatively slow MAS rate is also described.

Anisoles↗
PubMed1991

Molecular mechanism of slow acetylation of drugs and carcinogens in humans.

The acetylation polymorphism is one of the most common genetic variations in the transformation of drugs and chemicals. More than 50% of individuals in Caucasian populations are homozygous for a recessive trait and are of the "slow acetylator" phenotype. They are less efficient than "rapid acetylators" in the metabolism of numerous drugs and environmental and industrial chemicals. The acetylation polymorphism is associated with an increased risk of drug toxicity and with an increased frequency of certain cancers. We report the identification of the primary mutations in two alleles of the gene for the N-acetyltransferase (NAT; acetyl-CoA:arylamine N-acetyltransferase, EC 2.3.1.5) isozyme NAT2 associated with slow acetylation. These alleles, M1 and M2, account for more than 90% of slow acetylator alleles in the European population we have studied. M1 and M2 were identified by restriction fragment length polymorphisms with Kpn I and Msp I and subsequently cloned and sequenced. M1 and M2 each are characterized by a combination of two different point mutations, one causing an amino acid substitution (Ile-113----Thr in M1, Arg-197----Gln in M2), the other being silent (C 481----T in M1, C 282----T in M2). Functional expression of M1 and M2 and of chimeric gene constructs between mutant and wild-type NAT2 in COS-1 cells suggests that M1 causes a decrease of NAT2 protein in the liver by defective translation, whereas M2 produces an unstable enzyme. On the basis of the mutations described here and a rare mutant allele (M3) reported recently, we have developed a simple DNA amplification assay that allows the predictive genotyping of more than 95% of slow and rapid acetylator alleles and the identification of individuals at risk.

Acetylation↗
PubMed1991

Detoxification pathways in the liver.

The liver plays an important rôle in protecting the organism from potentially toxic chemical insults through its capacity to convert lipophiles into more water-soluble metabolites which can be efficiently eliminated from the body via the urine. This protective ability of the liver stems from the expression of a wide variety of xenobiotic biotransforming enzymes whose common underlying feature is their ability to catalyse the oxidation, reduction and hydrolysis (Phase I) and/or conjugation (Phase II) of functional groups on drug and chemical molecules. The broad substrate specificity, isoenzyme multiplicity and inducibility of many of these enzyme systems make them particularly well adapted to handling the vast array of different chemical structures in the environment to which we are exposed daily. However, some chemicals may also be converted to more toxic metabolites by certain of these enzymes, implying that variations in the latter may be important predisposing factors for toxicity. Pharmacogenetic defects of xenobiotic biotransformation enzymes, a subclass of inborn errors of metabolism which are manifested only upon drug challenge, introduce marked variation into human populations for the pharmacokinetics and pharmacodynamics of therapeutic and toxic agents, and thus may have important clinical consequences for drug efficacy and toxicity.

Animals↗
PubMed1991

Rickettsiae in gill epithelial cells of the hard clam, Mercenaria mercenaria.

Rickettsiae are found in the gill epithelium of the hard clam, Mercenaria mercenaria. The procaryotes occur free in the cytoplasm of the epithelial cells at the tip of the filament and in the more proximal cells that support the lateral J cilia. The fine structure of the organisms, showing rippled cell walls, is typical of the rickettsiae. The increasing size of the inclusion representing late phase growth often culminates in lysis of the host cell. Masses (Gram-negative, Feulgen-positive) in ova, similar to those observed in the gill epithelium, suggest that transovarian transmission may occur.

Animals↗
PubMed1991

Expression of monomorphic arylamine N-acetyltransferase (NAT1) in human leukocytes.

The expression of arylamine N-acetyltransferase (NAT) in leukocytes was investigated using p-aminobenzoic acid (PABA) and sulfamethazine (SMZ), substrates which are preferentially acetylated by the monomorphic NAT1 and polymorphic NAT2 enzymes, respectively. Activity towards both substrates was detected in mononuclear leukocytes (MNL; preparation containing approximately 80% lymphocytes), monocytes and neutrophils. PABA and SMZ acetylation rates were highly correlated in each of the isolated cell types. The NAT in leukocytes displayed a much higher affinity and turnover rate for the acetylation of PABA than for SMZ. These kinetic characteristics suggest that the acetylating activity in human leukocytes is predominantly attributable to the monomorphic enzyme NAT1. Neutrophils showed evidence of biphasic kinetics for SMZ which would indicate the coexpression of NAT1 and low levels of the polymorphic enzyme, NAT2. NAT activity in MNL was not influenced by the acetylator phenotype of the individual. There was, however, a significant correlation between NAT activity in MNL and the in vivo acetylation (urinary metabolite ratio) of p-aminosalicylic acid, which is monomorphically acetylated in humans. The expression of NAT1 in leukocytes and the virtuall absence of NAT2 may have important toxicological implications. The in vitro/in vivo correlation suggests that leukocytes may be a useful marker of systemic NAT1 activity.

4-Aminobenzoic Acid↗
PubMed1991

Monomorphic and polymorphic human arylamine N-acetyltransferases: a comparison of liver isozymes and expressed products of two cloned genes.

A genetic polymorphism of human liver arylamine N-acetyltransferase (NAT; EC 2.3.1.5) enzyme activity divides populations into distinguishable "slow acetylator" and "rapid acetylator" phenotypes. Two human genes, NAT1 and NAT2, encoding NAT proteins [DNA Cell Biol. 9:193-203 (1990)] were transiently expressed in cultured monkey kidney COS-1 cells, and the resulting recombinant NAT1 and NAT2 proteins were compared with N-acetyltransferase activities in human liver cytosol with respect to their stability, chromatographic behavior on anion exchange columns, electrophoretic mobility, and arylamine acceptor substrate specificity. NAT1 was far less stable in vitro than NAT2. Under conditions designed to optimize enzyme stability, anion exchange chromatography experiments revealed that enzymes corresponding to both recombinant NAT1 and NAT2 were expressed in human liver. Recombinant and human liver NAT1 enzymes showed the same characteristic selectivity (low apparent Km, high Vmax) for the "monomorphic" substrates p-aminosalicylic acid and p-aminobenzoic acid. Such substrates fail to discriminate between the acetylator phenotypes in vivo. The same criteria established that recombinant NAT2 was indistinguishable from one of two previously observed N-acetyltransferases (NAT2A and NAT2B) whose liver contents correlate with acetylator phenotype in human populations. Recombinant NAT2 and the liver NAT2 isoforms NAT2A and NAT2B selectivity N-acetylated the "polymorphic" substrates sulfamethazine and procainamide, whose disposition in vivo is affected by the acetylation polymorphism. Interestingly, the carcinogen 2-aminofluorene was very efficiently metabolized by both NAT1 and NAT2. Independent regulation of NAT1 and NAT2 genes was suggested by a lack of correlation of NAT1 and NAT2 enzyme activities in cytosols from 39 human livers. The results provide strong evidence that the NAT2 locus is the site of the human acetylation polymorphism. In addition, the use of recombinant NAT1 and NAT2 will allow us to predict whether any given arylamine will be polymorphically acetylated in humans.

Acetylation↗
PubMed1990

Human arylamine N-acetyltransferase genes: isolation, chromosomal localization, and functional expression.

N-Acetylation by hepatic arylamine N-acetyltransferase (NAT, EC 2.3.1.5) is a major route in the metabolism and detoxification of numerous drugs and foreign chemicals. NAT is the target of a common genetic polymorphism of clinical relevance in human populations. We have used our recently isolated rabbit cDNA rnat to clone three human NAT genes from human leukocyte DNA. None of the three genomic coding sequences was interrupted by introns. Two genes, designated NAT1 and NAT2, each possessed open reading frames of 870 bp. Both genes have been assigned to human chromosome 8, pter-q11. Following transfection they were transiently expressed in monkey kidney COS-1 cells. NAT1 and NAT2 gave rise to functional NAT proteins, as judged by their NAT enzyme activity with the arylamine substrate sulfamethazine. Western blots with NAT-specific antisera detected proteins of apparent molecular weight of 33 and 31 kD in NAT1- and NAT2-transfected cultures, respectively. The product of NAT2 had an identical apparent molecular weight as that of NAT detected in human liver cytosol. The deduced amino acid sequence of NAT2 also contained 6 peptide sequences which had previously been determined from tryptic peptides of the polymorphic NAT purified from human liver. These data suggest that NAT2 encodes the polymorphic NAT protein. The third gene, NATP, had multiple deleterious mutations and did not encode a functional NAT protein; it most likely represents a pseudogene.

Acetyltransferases↗
PubMed1990

Acetylation pharmacogenetics. The slow acetylator phenotype is caused by decreased or absent arylamine N-acetyltransferase in human liver.

The biochemical basis underlying the genetic polymorphism of drug N-acetylation was investigated using a combination of in vivo and in vitro assays for arylamine N-acetyltransferase (NAT) activity and content in human liver. The acetylator phenotype of 26 surgical patients was determined using caffeine as an innocuous probe drug by measurement of the 5-acetyl-amino-6-formylamino-3-methyluracil to 1-methylxanthine molar ratio in urine. Liver wedge biopsies from these patients and livers from 24 organ donors were then used for measurement of N-acetyltransferase activity with the substrate sulfamethazine and for quantitation of immunoreactive N-acetyl-transferase protein. In vivo (caffeine metabolites in urine) and in vitro (sulfamethazine acetylation) measures of N-acetyl-transferase activity correlated very highly (r = 0.98). Moreover, in all subjects tested, slow acetylation both in vivo and in vitro was associated with a decrease in the quantity of immunodetectable N-acetyltransferase protein in liver cytosol relative to that seen in cytosols from rapid acetylator livers. Two kinetically distinct enzyme activities, designated NAT-1 and NAT-2, were partially purified from low- and high-activity livers and their relationship to acetylator status was determined. Low acetylation capacity was related to decreases in the liver content of both of these immunologically related proteins. The results demonstrate that genetically defective arylamine N-acetylation is due to a parallel decrease in the quantity of two structurally and functionally similar acetylating enzymes.

Acetylation↗
PubMed1989

Evidence for two closely related isozymes of arylamine N-acetyltransferase in human liver.

Acetyl CoA-dependent arylamine N-acetyltransferase (EC 2.3.1.5) is the target of a genetic polymorphism in the metabolism of drugs and carcinogens. N-Acetyltransferase was purified 1000-fold from cytosol of human liver and its identity was verified by amino acid sequence homology of two of its tryptic peptides with published rabbit and chicken N-acetyltransferase sequences. Enzyme activity correlated with the presence of two proteins, NAT-1 and NAT-2, with indistinguishable molecular masses (31 kDa). NAT-1 and NAT-2 could be separated by anion-exchange chromatography and were functionally distinguished by their different apparent affinities for the acceptor amine sulfamethazine (SMZ). Antibodies raised against NAT-1 were able to recognize both isozymes on Western blots.

Acetyltransferases↗