Safety assessment and risk-benefit analysis of the use of azodicarbonamide in baby food jar closure technology: putting trace levels of semicarbazide exposure into perspective--a review.

The discovery of trace levels of semicarbazide (SEM) in bottled foods (especially baby foods) led to a consideration of the safety of this hydrazine compound by regulatory agencies worldwide. Azodicarbonamide, which is used in the jar-sealing technology known as Press On-Twist Off (or Push-Twist/PT) closures for the formation of a hermetic, plastisol seal, partially degrades with the heat of processing to form trace amounts of SEM. This review has evaluated the potential toxicological risks of resulting exposure to SEM and also the benefit of the PT technology (with azodicarbonamide) in the context of possible microbial contamination. It also considers the potential impact on infant nutrition if parents come to the conclusion that commercial baby foods are unsafe. SEM shows limited genotoxicity in vitro that is largely prevented by the presence of mammalian metabolic enzymes. Negative results were found in vivo in DNA alkaline elution, unscheduled DNA synthesis and micronucleus assays. This pattern is in contrast to the genotoxic hydrazines that also have been shown to cause tumours. Carcinogenicity studies of SEM are of limited quality, show a questionable weak effect in mice at high doses, which are not relevant to human exposure at trace levels, and show no effect in the rat. The IARC has assigned SEM as Group 3, 'Not classifiable as to its carcinogenicity to humans'. Based on estimates of exposure to infants consuming baby foods (with the assumption of SEM levels at the 95th percentile of 20 ng g(-1) in all of the consumed 'ready-to-eat' foods) compared with a no observed adverse effect level (NOAEL) in developmental toxicity studies, the margin of safety is more than 21 000. Since the risk of an adverse effect is negligible, it is clear that any theoretical risk is outweighed by the benefits of continuing use of the PT closure (with azodicarbonamide blowing agent) to ensure both the microbial integrity and availability of commercial baby foods as a valuable source of infant nutrition.

Homology modelling and structural analysis of human arylamine N-acetyltransferase NAT1: evidence for the conservation of a cysteine protease catalytic domain and an active-site loop.

Arylamine N-acetyltransferases (EC 2.3.1.5) (NATs) catalyse the biotransformation of many primary arylamines, hydrazines and their N-hydroxylated metabolites, thereby playing an important role in both the detoxification and metabolic activation of numerous xenobiotics. The recently published crystal structure of the Salmonella typhimurium NAT (StNAT) revealed the existence of a cysteine protease-like (Cys-His-Asp) catalytic triad. In the present study, a three-dimensional homology model of human NAT1, based upon the crystal structure of StNAT [Sinclair, Sandy, Delgoda, Sim and Noble (2000) Nat. Struct. Biol. 7, 560-564], is demonstrated. Alignment of StNAT and NAT1, together with secondary structure predictions, have defined a consensus region (residues 29-131) in which 37% of the residues are conserved. Homology modelling provided a good quality model of the corresponding region in human NAT1. The location of the catalytic triad was found to be identical in StNAT and NAT1. Comparison of active-site structural elements revealed that a similar length loop is conserved in both species (residues 122-131 in NAT1 model and residues 122-133 in StNAT). This observation may explain the involvement of residues 125, 127 and 129 in human NAT substrate selectivity. Our model, and the fact that cysteine protease inhibitors do not affect the activity of NAT1, suggests that human NATs may have adapted a common catalytic mechanism from cysteine proteases to accommodate it for acetyl-transfer reactions.

Pharmacogenetics of the arylamine N-acetyltransferases: a symposium in honor of Wendell W. Weber.

This article is a report on a symposium sponsored by the American Society for Pharmacology and Experimental Therapeutics presented at the joint meeting of the American Society for Biochemistry and Molecular Biology and the American Society for Pharmacology and Experimental Therapeutics, June 4-8, Boston, Massachusetts. The presentations focused on the pharmacogenetics of the NAT1 and NAT2 arylamine N-acetyltransferases, including developmental regulation, structure-function relationships, and their possible role in susceptibility to breast, colon, and pancreatic cancers. The symposium honored Wendell W. Weber for over 35 years of leadership and scientific advancement in pharmacogenetics and was highlighted by his overview of the historical development of the field.

Pharmacogenetics of the human arylamine N-acetyltransferases.

This review briefly describes current understanding of one of the earliest discovered pharmacogenetic polymorphisms of drug biotransformation affecting acetylation of certain homo- and heterocyclic aromatic amines and hydrazines. This so-called acetylation polymorphism arises from allelic variation in one of the two known human arylamine N-acetyltransferase genes, namely NAT2, which results in production of NAT2 proteins with variable enzyme activity or stability. The NAT1 gene locus encodes a structurally related enzyme, NAT1, with catalytic specificity for arylamine acceptor substrates distinct from that exhibited by NAT2. NAT1 function is also genetically variable in human populations. Clinical and toxicological consequences of genetic variation in NAT1 and NAT2 activity are discussed.

Identification of amino acids imparting acceptor substrate selectivity to human arylamine acetyltransferases NAT1 and NAT2.

The human arylamine N-acetyltransferases NAT1 and NAT2 catalyse the acetyl-CoA-dependent N- and O-acetylation of primary arylamine and hydrazine xenobiotics and their N-hydroxylated metabolites. We previously used a panel of recombinant NAT1/NAT2 chimaeric proteins to identify linear amino acid segments that have roles in imparting the distinct catalytic specificities to these proteins [Dupret, Goodfellow, Janezic and Grant (1994) J. Biol. Chem. 269, 26830-26835]. These studies indicated that a conserved central region (residues 112-210) distinct from that containing the active-site cysteine residue Cys(68) was important in determining NAT substrate selectivity. In the present study we have refined our analysis through further chimaera generation of this conserved region and by subsequent site-directed mutagenesis of individual amino acids. Enzyme-kinetic analysis of these mutant proteins with the NAT1-selective and NAT2-selective substrates p-aminosalicylic acid (PAS) and sulphamethazine (SMZ) respectively suggests that residues 125, 127 and 129 are important determinants of NAT1-type and NAT2-type substrate selectivity. Modification of Arg(127) had the greatest effect on specificity for PAS, whereas changing Phe(125) had the greatest effect on specificity for SMZ. Selected NAT mutants exhibited K(m) values for acetyl-CoA that were comparable with those of the wild-type NATs, implying that the mutations affected acceptor substrate specificity rather than cofactor binding affinity. Taken together with previous observations, these results suggest that residues 125, 127 and 129 might contribute to the formation of the active-site pocket surrounding Cys(68) and function as important determinants of NAT substrate selectivity.

Human acetyltransferase polymorphisms.

Conjugation of primary amino and hydroxylamino groups with acetate, catalyzed by acetyl CoA-dependent arylamine acetyltransferase (NAT) enzymes, may play an important role in the intricate series of metabolic pathways that produce or prevent toxicity following exposure to homo- and heterocyclic arylamine and hydrazine xenobiotics. Two independently regulated and kinetically distinct human acetyltransferases are now known to exist, namely NAT1 and NAT2. Interindividual variation in NAT2 function is associated with the classical isoniazid acetylation polymorphism which was discovered over forty years ago. At last count, fifteen variant alleles at the NAT2 gene locus have been linked to the isoniazid 'acetylator phenotype', and each of these can be identified in population studies using specific PCR-based genotyping tests. On the other hand, NAT1 shows kinetic selectivity for compounds whose disposition is unrelated to the classical isoniazid acetylation polymorphism. NAT1 expression is also phenotypically variable in human populations, at least in part due to allelic differences at the NAT1 gene locus. Nine NAT1 variant alleles have been described to date, of which NAT1* 14 and NAT1* 15 clearly produce defective NAT1 proteins and lead to functional impairment in the metabolism of NAT1-selective substrates both in vivo and in vitro. On the other hand, it has been reported that the NAT1* 10 variant associates with elevated NAT1 activity and increased risk for cancers of the bladder and colon. Because of the important toxicologic consequences of allelic variation in NAT1 and NAT2 function for the metabolic activation of arylamine and heterocyclic amine procarcinogens, further studies are needed to improve our understanding of the extent of NAT allelic variation, to determine the functional capacity of each variant gene product, and to develop accurate methods of detecting them in population and epidemiological studies.

Study of the role of the highly conserved residues Arg9 and Arg64 in the catalytic function of human N-acetyltransferases NAT1 and NAT2 by site-directed mutagenesis.

The arylamine N-acetyltransferases (NATs) NAT1 and NAT2 are responsible for the biotransformation of many arylamine and hydroxylamine xenobiotics. It has been proposed that NATs may act through a cysteine-linked acetyl-enzyme intermediate in a general base catalysis involving a highly conserved arginine residue such as Arg64. To investigate this possibility, we used site-directed mutagenesis and expression of recombinant human NAT1 and NAT2 in Escherichia coli. Sequence comparison with NATs from other species indicated that Arg9 and Arg64 are the only invariant basic residues. Either mutation of the presumed catalytic Cys68 residue or the simultaneous mutation of Arg9 and Arg64 to Ala produced proteins with undetectable enzyme activity. NAT1 or NAT2 singly substituted at Arg9 or Arg64 with Ala, Met, Gln or Lys exhibited unaltered Km values for arylamine acceptor substrates, but a marked loss of activity and stability. Finally, double replacement of Arg9/Arg64 with lysine in NAT1 altered the Km for arylamine substrates (decreased by 8-14-fold) and for acetyl-CoA (elevated 5-fold), and modified the pH-dependence of activity. Thus, through their positively charged side chains, Arg9 and Arg64 seem to contribute to the conformational stability of NAT1 and NAT2 rather than acting as general base catalysts. Our results also support a mechanism in which Arg9 and Arg64 are involved in substrate binding and transition-state stabilization of NAT1.

Structure-function studies of human arylamine N-acetyltransferases NAT1 and NAT2. Functional analysis of recombinant NAT1/NAT2 chimeras expressed in Escherichia coli.

The human arylamine N-acetyltransferases NAT1 and NAT2 catalyze the biotransformation of primary aromatic amine or hydrazine drugs and xenobiotics. These enzymes share 81% amino acid sequence identity, yet differ markedly with respect to their acceptor substrate selectivities and intrinsic in vitro stabilities. To define the contribution of large regions of NAT1 and NAT2 polypeptide structure to enzyme integrity and catalytic specificity, we used selected restriction endonuclease digestions and fragment religation into the tac promoter-based phagemid pKEN2 to construct a panel of 18 NAT1/NAT2 hybrid gene vectors for heterologous expression in Escherichia coli. Induction of hybrid gene expression in recombinant transformants of E. coli strain XA90 led to the production of soluble, catalytically active acetylating enzymes in all cases. Chimeric proteins produced in this fashion were then compared to wild-type NAT1 and NAT2 with respect to their enzyme kinetic constants (apparent Km, Vmax, and Vmax/Km) for the NAT1-selective and NAT2-selective substrates p-aminosalicylic acid and sulfamethazine, respectively, and for their in vitro stabilities at 37 degrees C. The ratio of the Vmax/Km for sulfamethazine to that for p-aminosalicylic acid allowed for the unambiguous classification of each enzyme as either NAT1 or NAT2 type, except for one novel chimera possessing a low Michaelis constant and a high maximal velocity for the acetylation of both substrates. A central region (amino acids 112-210) within the 290-residue polypeptide appeared to play a role in determining NAT1- or NAT2-type behavior. On the other hand, the region (residues 47-111) encompassing the putative active site cysteine (Cys68) was important in contributing to a low apparent Km for p-aminosalicylic acid but not for sulfamethazine, while amino acids 211-250 affected Km for sulfamethazine and 251-290 influenced Km for both substrates. Maximal velocities were highest for both substrates when the central 112-210 amino acid region was derived from NAT1. Finally, the region from amino acids 211-250 in NAT2 was important in determining its greater intrinsic enzyme stability than that exhibited by NAT1.