Quantification and cellular localization of expression in human skin of genes encoding flavin-containing monooxygenases and cytochromes P450.

The expression, in adult human skin, of genes encoding flavin-containing monooxygenases (FMOs) 1, 3, 4, and 5 and cytochromes P450 (CYPs) 2A6, 2B6, and 3A4 was determined by RNase protection. Each FMO and CYP exhibits inter-individual variation in expression in this organ. Of the individuals analysed, all contained CYP2B6 mRNA in their skin, 90% contained FMO5 mRNA and about half contained mRNAs encoding FMOs 1, 3, and 4, and CYPs 2A6 and 3A4. The amount of each of the FMO and CYP mRNAs in skin is much lower than in the organ in which it is most highly expressed, namely the kidney (for FMO1) and the liver (for the others). In contrast to the latter organs, in the skin FMO mRNAs are present in amounts similar to, or greater than, CYP mRNAs. Only the mRNA encoding CYP2B6 decreased in abundance in skin with increasing age of the individual. All of the mRNAs were substantially less abundant in cultures of keratinocytes than in samples of skin from which the cells were derived. In contrast, an immortalized human keratinocyte cell line, HaCaT, expressed FMO3, FMO5, and CYP2B6 mRNAs in amounts that fall within the range detected in the whole skin samples analysed. FMO1, CYP2A6, and CYP3A4 mRNAs were not detected in HaCaT cells, whereas FMO4 expression was markedly increased in this cell line compared to whole skin. In situ hybridization showed that the expression of each of the FMOs and CYPs analysed was localized to the epidermis, sebaceous glands and hair follicles.

Ethnic differences in human flavin-containing monooxygenase 2 (FMO2) polymorphisms: detection of expressed protein in African-Americans.

The flavin-containing monooxygenases (FMOs) are a family of xenobiotic-metabolizing enzymes that are expressed in a species- and tissue-specific manner. FMO2 expression has been observed in pulmonary tissue from several species, but not human. Two human FMO2 point mutations have been reported: a cytosine to thymidine transition at position 1414 resulting in a premature stop codon and a thymidine insertion at position 1589 resulting in a frameshift. To define the frequency of these sequence variations and explore their significance, unrelated African-American, Caucasian, and Korean individuals were genotyped. In the African-American population tested (n = 180), the 1414C allele occurred at a 13% frequency; however, all of the tested Caucasians (n = 52) and Koreans (n = 100) were homozygous for the 1414T allele. The T1589 allele occurred at frequencies of 6.9 and 13.0% in African-Americans (n = 175) and Caucasians (n = 23), respectively, and appears to segregate with the 1414T allele. Thus, it would have no further impact on FMO2 activity. Western blot analysis of pulmonary microsomes failed to detect immunoreactive protein in 1414T homozygotes. A heterozygotic individual did exhibit a single band of the expected size, but no detectable FMO activity in the corresponding lung microsomes. Sequence analysis, however, was consistent with the 1414C allele encoding an active FMO2 enzyme. FMO2 mRNA expression was observed in most individuals, but failed to correlate with genotype or protein expression. In summary, functional FMO2 is expressed in only a small percentage of the overall population. However, in certain ethnic groups, active pulmonary FMO2 enzyme will be present in a significant number of individuals.

A novel mutation in the flavin-containing monooxygenase 3 gene, FM03, that causes fish-odour syndrome: activity of the mutant enzyme assessed by proton NMR spectroscopy.

We have previously shown that primary trimethylaminuria, or fish-odour syndrome, is caused by an inherited defect in the flavin-containing monooxygenase 3 (FMO3) catalysed N-oxidation of the dietary-derived malodorous amine, trimethylamine (TMA). We now report a novel causative mutation for the disorder identified in a young girl diagnosed by proton nuclear magnetic resonance (NMR) spectroscopy of her urine. Sequence analysis of genomic DNA amplified from the patient revealed that she was homozygous for a T to C missense mutation in exon 3 of the FMO3 gene. The mutation changes an ATG triplet, encoding methionine, at codon 82 to an ACG triplet, encoding threonine. A polymerase chain reaction/restriction enzyme-based assay was devised to genotype individuals for the FMO3Thr82 allele. Wild-type and mutant FMO3, heterologously expressed in a baculovirus-insect cell system, were assayed by ultraviolet spectrophotometry and NMR spectroscopy for their ability to catalyse the N-oxidation of TMA. The latter technique has the advantage of enabling the simultaneous, direct and semi-continuous measurement of both of the products, TMA N-oxide and NADP, and of one of the reactants, NADPH. Results obtained from both techniques demonstrate that the Met82Thr mutation abolishes the catalytic activity of the enzyme and thus represents the genetic basis of the disorder in this individual. The combination of NMR spectroscopy with gene sequence and expression technology provides a powerful means of determining genotype-phenotype relationships in trimethylaminuria.

Compound heterozygosity for missense mutations in the flavin-containing monooxygenase 3 (FM03) gene in patients with fish-odour syndrome.

Fish-odour syndrome is a highly unpleasant disorder of hepatic trimethylamine (TMA) metabolism characterized by a body odour reminiscent of rotting fish, due to excessive excretion of the malodorous free amine. Although fish-odour syndrome may exhibit as sequelae with other conditions (e.g. liver dysfunction), many patients exhibit an inherited, more persistent form of the disease. Ordinarily, dietary-derived TMA is oxidized to the nonodorous N-oxide by hepatic flavin-containing monooxygenase 3 (FMO3). Our previous demonstration that a mutation, P153L (C to T), in the FMO3 gene segregated with the disorder and inactivated the enzyme confirmed that defects in FMO3 underlie the inherited form of fish-odour syndrome. We have investigated the genetic basis of the disorder in two further affected pedigrees and report that the three propositi are all compound heterozygotes for causative mutations of FMO3. Two of these individuals possess the P153L (C to T) mutation and a novel mutation, N61S (A to G). The third is heterozygous for novel, M4341 (G to A), and previously reported, R492W (C to T), mutations. Functional characterization of the S61, 1434 and W492 variants, via baculovirus-mediated expression in insect cells, confirmed that all three mutations either abolished, or severely attenuated, the capacity of the enzyme to catalyse TMA N-oxidation. Although 1434 and W492 were also incapable of catalysing the S-oxidation of methimazole, S61 was fully active with this sulphur-containing substrate. Since an asparagine is conserved at the equivalent position to N61 of FMO3 in mammalian, yeast and Caenorhabditis elegans FMOs, the characterization of the naturally occurring N61S (A to G) mutation may have identified this asparagine as playing a critical role specifically in FMO-catalysed N-oxidation.

The flavin-containing monooxygenase 2 gene (FMO2) of humans, but not of other primates, encodes a truncated, nonfunctional protein.

Flavin-containing monooxygenases (FMOs) are NADPH-dependent flavoenzymes that catalyze the oxidation of heteroatom centers in numerous drugs and xenobiotics. FMO2, or "pulmonary" FMO, one of five forms of the enzyme identified in mammals, is expressed predominantly in lung and differs from other FMOs in that it can catalyze the N-oxidation of certain primary alkylamines. We describe here the isolation and characterization of cDNAs for human FMO2. Analysis of the sequence of the cDNAs and of a section of the corresponding gene revealed that the major FMO2 allele of humans encodes a polypeptide that, compared with the orthologous protein of other mammals, lacks 64 amino acid residues from its C terminus. Heterologous expression of the cDNA revealed that the truncated polypeptide was catalytically inactive. The nonsense mutation that gave rise to the truncated polypeptide, a C --> T transition in codon 472, is not present in the FMO2 gene of closely related primates, including gorilla and chimpanzee, and must therefore have arisen in the human lineage after the divergence of the Homo and Pan clades. Possible mechanisms for the fixation of the mutation in the human population and the potential significance of the loss of functional FMO2 in humans are discussed.

Distribution of mRNAs encoding the peroxisome proliferator-activated receptor alpha, beta, and gamma and the retinoid X receptor alpha, beta, and gamma in rat central nervous system.

We report the isolation, by RT-PCR, of partial cDNAs encoding the rat peroxisome proliferator-activated receptor (PPAR) isoforms PPAR alpha, PPAR beta, and PPAR gamma and the rat retinoid X receptor (RXR) isoforms RXR alpha, RXR beta, and RXR gamma. These cDNAs were used to generate antisense RNA probes to permit analysis, by the highly sensitive and discriminatory RNase protection assay, of the corresponding mRNAs in rat brain regions during development. PPAR alpha, PPAR beta, RXR alpha, and RXR beta mRNAs are ubiquitously present in different brain regions during development, PPAR gamma mRNA is essentially undetectable, and RXR gamma mRNA is principally localised to cortex. We demonstrate, for the first time, the presence of PPAR and RXR mRNAs in primary cultures of neonatal meningeal fibroblasts, cerebellar granule neurons (CGNs), and cortical and cerebellar astrocytes and in primary cultures of adult cortical astrocytes. PPAR alpha, PPAR beta, RXR alpha, and RXR beta mRNAs are present in all cell types, albeit that PPAR alpha and RXR alpha mRNAs are at levels near the limit of detection in CGNs. PPAR gamma mRNA is expressed at low levels in most cell types but is present at levels similar to those of PPAR alpha mRNA in adult astrocytes. RXR gamma mRNA is present either at low levels, or below the level of detection of the assay, for all cell types studied.

Molecular cloning of rat mitochondrial 3-hydroxy-3-methylglutaryl-CoA lyase and detection of the corresponding mRNA and of those encoding the remaining enzymes comprising the ketogenic 3-hydroxy-3-methylglutaryl-CoA cycle in central nervous system of suckling rat.

We have investigated, by RNase protection assays in rat brain regions and primary cortical astrocyte cultures, the presence of the mRNA species encoding the three mitochondrially located enzymes acetoacetyl-CoA thiolase, mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (mt. HMG-CoA synthase) and HMG-CoA lyase (HMG-CoA lyase) that together constitute the ketogenic HMG-CoA cycle. As a prerequisite we obtained a full-length cDNA encoding rat HMG-CoA lyase by degenerate oligonucleotide-primed PCR coupled to a modification of PCR-rapid amplification of cDNA ends (PCR-RACE). We report here: (1) the nucleotide sequence of rat mt. HMG-CoA lyase, (2) detection of the mRNA species encoding all three HMG-CoA cycle enzymes in all regions of rat brain during suckling, (3) approximately twice the abundance of mt. HMG-CoA synthase mRNA in cerebellum than in cortex in 11-day-old suckling rat pups, (4) significantly lower abundances of mt. HMG-CoA synthase mRNA in brain regions derived from rats weaned to a high-carbohydrate/low-fat diet compared with the corresponding regions derived from the suckling rat, and (5) the presence of mt. HMG-CoA synthase mRNA in primary cultures of neonatal cortical astrocytes at an abundance similar to that found in liver of weaned animals. These results provide preliminary evidence that certain neural cell types possess ketogenic potential and might thus have a direct role in the provision of fatty acid-derived ketone bodies during the suckling period.

Missense mutation in flavin-containing mono-oxygenase 3 gene, FMO3, underlies fish-odour syndrome.

Individuals with primary trimethylaminuria exhibit a body odour reminiscent of rotting fish, due to excessive excretion of trimethylamine (TMA; refs 1-3). The disorder, colloquially known as fish-odour syndrome, is inherited recessively as a defect in hepatic N-oxidation of dietary-derived TMA and cannot be considered benign, as sufferers may display a variety of psychosocial reactions, ranging from social isolation of clinical depression and attempted suicide. TMA oxidation is catalyzed by flavin-containing mono-oxygenase (FMO; refs 7,8), and tissue localization and functional studies have established FMO3 as the form most likely to be defective in fish-odour syndrome. Direct sequencing of the coding exons of FMO3 amplified from a patient with fish-odour syndrome identified two missense mutations. Although one of these represented a common polymorphism, the other, a C-->T transition in exon 4, was found only in an affected pedigree, in which it segregated with the disorder. The latter mutation predicts a proline-->leucine substitution at residue 153 and abolishes FMO3 catalytic activity. Our results indicate that defects in FMO3 underlie fish-odour syndrome and that the Pro 153-->Leu 153 mutation described here is a cause of this distressing condition.

Structural organization of the human flavin-containing monooxygenase 3 gene (FMO3), the favored candidate for fish-odor syndrome, determined directly from genomic DNA.

The inherited metabolic disorder trimethylaminuria (fish-odor syndrome) is associated with defective hepatic N-oxidation of dietary-derived trimethylamine catalyzed by flavin-containing monooxygenase (FMO). As FMO3 encodes the major form of FMO expressed in adult human liver, it represents the best candidate gene for the disorder. The structural organization of FMO3 was determined by sequencing the products of exon-to-exon and vectorette PCR, the latter through the use of vectorette libraries constructed directly from genomic DNA. The gene contains one noncoding and eight coding exons. Knowledge of the exon/intron organization of the human FMO3 gene enabled each of the coding exons of the gene, together with their associated flanking intron sequences, to be amplified from genomic DNA and will thus facilitate the identification of mutations in FMO3 in families affected with fish-odor syndrome.

Localization of human flavin-containing monooxygenase genes FMO2 and FMO5 to chromosome 1q.

The human flavin-containing monooxygenase (FMO) gene family comprises at least five distinct members (FMO1 to FMO5) that code for enzymes responsible for the oxidation of a wide variety of soft nucleophilic substrates, including drugs and environmental pollutants. Three of these genes (FMO1, FMO3, and FMO4) have previously been localized to human chromosome 1q, raising the possibility that the entire gene family is clustered in this chromosomal region. Analysis by polymerase chain reaction of DNA isolated from a panel of human-rodent somatic cell hybrids demonstrates that the two remaining identified members of the FMO gene family, FMO2 and FMO5, also are located on chromosome 1q.

Differential developmental and tissue-specific regulation of expression of the genes encoding three members of the flavin-containing monooxygenase family of man, FMO1, FMO3 and FM04.

We have previously described the isolation and sequencing of cDNA clones encoding flavin-containing monooxygenases (FMOs) 1 and 4 of man [Dolphin, C., Shephard, E. A., Povey, S., Palmer, C. N. A., Ziegler, D. M., Ayesh, R., Smith, R. L. & Phillips, I. R. (1991) J. Biol. Chem. 266, 12379-12385; Dolphin, C., Shephard E. A., Povey, S., Smith, R. L. & Phillips, I. R. (1992) Biochem. J. 287, 261-267]. We present here the isolation of a cDNA for FM03 of man. The sequence of this CDNA and the amino acid sequence deduced from it differ substantially from those previously reported for this member of the FMO family of man. In addition, we have investigated, by quantitative RNase protection assays, the expression in several foetal and adult human tissues of genes encoding FMO1, FMO3 and FMO4, Our results demonstrate that, in the adult, FMO1 is expressed in kidney but not in liver, whereas in the foetus it is expressed in both organs. The lack of expression of FMO1 in adult human liver is in marked contrast to the situation in other mammals, such as pig and rabbit, in which FMO1 constitutes a major form of the enzyme in the liver of the adult animal. The mRNA encoding FMO3 is abundant in adult liver and is also present, in low abundance, in some foetal tissues. Thus, FMO1 and FMO3 are both subject to developmental and tissue-specific regulation, with a developmental switch in the expression of the genes taking place in the liver. FMO4 mRNA is present in low abundance in several foetal and adult tissues and thus the corresponding gene appears to be expressed constitutively.

The molecular biology of the flavin-containing monooxygenases of man.

cDNA clones encoding five distinct members of the FMO family of man (FMOs 1, 2, 3, 4 and 5) were isolated by a combination of library screening and reverse transcription-polymerase chain reaction techniques. The deduced amino acid sequences of the human FMOs have 82-87% identity with their known orthologues in other mammal but only 51-57% similarity to each other. The hydropathy profiles of the proteins are very similar. From the calculated rate of evolution of FMOs (a 1% change in sequence per 6 million years) it would appear that individual members of the FMO gene family arose by duplication of a common ancestral gene some 250-300 million years ago. Each of the FMO genes was mapped by the polymerase chain reaction to the long arm of human chromosome 1. The localization of the FMO1 gene was further refined to 1q23-q25 by in situ hybridization of human metaphase chromosomes. RNase protection assays demonstrated that in man each FMO gene displays a distinct developmental and tissue-specific pattern of expression. In the adult, FMO1 is expressed in kidney but not in liver, whereas in the foetus its mRNA is abundant in both organs. FMO3 expression is essentially restricted to the liver in the adult and the mRNA is either absent, or present in low amounts, in foetal tissues. FMO4 is expressed more constitutively. Human FMO1 and FMO3 cDNAs were functionally expressed in prokaryotic and eukaryotic cells. FMO1 and FMO3, expressed in either system, displayed product stereoselectivity in their catalysis of the N-oxidation of the pro-chiral tertiary amines, N-ethyl-N-methylaniline (EMA) and pargyline. Both enzymes were stereoselective with respect to the production of the (-)-S-enantiomer of EMA N-oxide. But in the case of pargyline, the enzymes displayed opposite stereoselectivity, FMO1 producing solely the (+)-enantiomer and FMO3 predominantly the (-)-enantiomer of the N-oxide.

A nomenclature for the mammalian flavin-containing monooxygenase gene family based on amino acid sequence identities.

A nomenclature based on comparisons of amino acid sequences is proposed for the members of the mammalian flavin-containing monooxygenase (FMO) gene family. This nomenclature is based on evidence of a single gene family composed of five genes. The percentage identities of the amino acid sequences of the five known forms of mammalian FMO are between 52 and 57% in rabbit and between 50 and 58% across species lines. The identities of all orthologs are greater than 82%. There is no evidence for multiple, highly related forms of the enzyme or for more than one mammalian FMO gene family. In the proposed system, the mammalian flavin-containing monooxygenase gene family is designated as "FMO" and the individual genes are distinguished by an Arabic numeral. The FMOs known as the "liver" and "lung" enzymes become FMO1 and FMO2, and the more recently described forms of the enzymes become FMO3, FMO4, and FMO5. Human FMO gene designations, FMO1 and FMO3, remain unchanged, but the gene designated FMO2 becomes FMO4. Following convention, the genes and cDNA designations will be italicized and the mRNA and protein designations will be nonitalicized. The purpose of the proposed nomenclature is to provide for the unambiguous identification of orthologous forms of mammalian FMOs, regardless of the species or tissue in question. The proposed classification considers only members of the mammalian flavin-containing monooxygenase gene family and has no bearing on the generally accepted definition of a multisubstrate flavin-containing monooxygenase.

Localization of genes encoding three distinct flavin-containing monooxygenases to human chromosome 1q.

We have used the polymerase chain reaction to map the gene encoding human flavin-containing monooxygenase (FMO) form II (N. Lomri, Q. Gu, and J. R. Cashman, 1992, Proc. Natl. Acad. Sci. USA 89: 1685-1689) to chromosome 1. We propose the designation FMO3 for this gene as it is the third FMO gene to be mapped. The two other human FMO genes identified to date, FMO1 and FMO2, are also located on chromosome 1 (C. Dolphin, E. A. Shephard, S. Povey, C. N. A. Palmer, D. M. Ziegler, R. Ayesh, R. L. Smith, and I. R. Phillips, 1991, J. Biol. Chem. 266: 12379-12385; C. Dolphin, E. A. Shephard, S. F. Povey, R. L. Smith, and I. R. Phillips, 1992, Biochem. J. 286: 261-267). The localization of FMO1, FMO2, and FMO3 has been refined to the long arm of chromosome 1. Analysis of human metaphase chromosomes by in situ hybridization confirmed the mapping of FMO1 and localized this gene more precisely to 1q23-q25.

Cloning, primary sequence and chromosomal localization of human FMO2, a new member of the flavin-containing mono-oxygenase family.

We have previously reported the cloning of cDNAs for a flavin-containing mono-oxygenase (FMO) of man, designated FMO1 [Dolphin, Shephard, Povey, Palmer, Ziegler, Ayesh, Smith & Phillips (1991) J. Biol. Chem. 266, 12379-12385], that is the orthologue of pig and rabbit hepatic FMOs. We now describe the isolation and characterization of cDNA clones for a second human FMO, which we have designated FMO2. The polypeptide encoded by the cDNAs is 558 amino acid residues long, has a calculated M(r) of 63337, and contains putative FAD- and NADP-binding sites that align exactly with those described in other mammalian FMOs. Human FMO2 has 51-53% primary sequence identity with human FMO1, rabbit pulmonary FMO and rabbit liver FMO form 2, and thus represents a fourth, distinct, member of the mammalian FMO family. The corresponding mRNA is present in low abundance in adult human liver. Southern blot hybridization with single-exon probes demonstrated that human FMO2 and FMO1 are the products of single genes. The gene encoding FMO2 (designated FMO2) was mapped, by the polymerase chain reaction, to human chromosome 1, the same chromosome on which FMO1 is located.

The gene CYP3 encoding P450pcn1 (nifedipine oxidase) is tightly linked to the gene COL1A2 encoding collagen type 1 alpha on 7q21-q22.1.

CYP3, the gene which encodes the hepatic cytochrome P450pcn1, the isozyme responsible for the metabolic oxidation of the calcium channel-blocking drug nifedipine, has recently been mapped to human chromosome 7 using somatic cell hybrids. Using multilocus linkage analysis in CEPH families, we examined the linkage of a cDNA probe (hPCN1) for CYP3 to the oncogene MET, the pro-alpha 2(1) collagen gene COL1A2, and the T-cell receptor beta-chain gene TCRB, together with three arbitrary loci D7S8, D7S13, and D7S16, defined by the anonymous DNA probes pJ3.11, pB79a, and p7C22, respectively. From 70 CEPH parents screened with a StyI RFLP for hPCN1, four informative families were found each with both parental and maternal grandparents and 6-11 children per family. Tight linkage emerged between CYP3 and COL1A2, with a maximum combined lod score of 5.72 at theta = 0, suggesting the most likely subchromosomal localization of CYP3 is 7q21.3-q22.1.

The role of intestinal microflora in the formation of the methylthio adduct metabolites of paracetamol. Studies in neomycin-pretreated and germ-free mice.

The contribution of the gastrointestinal microflora to the formation of methylthio adducts from paracetamol has been studied by comparing the fate of this drug in conventional mice with that in germ-free and neomycin-treated animals. In both germ-free and neomycin-treated mice there was a highly significant reduction in the urinary excretion of 3-methylthioparacetamol, its glucuronic acid and sulfate conjugates and its sulfoxide, with no other systemic alteration to the overall fate of the drug. These data are consistent with the gut flora playing a major role in the C-S cleavage of paracetamol-3-cysteine, thereby reducing the excretion of the array of methylthio adducts subsequently formed by tissue enzymes from 3-thioparacetamol, the putative product of the C-S cleavage.

Effect of poly rI:rC treatment upon the metabolism of [14C]-paracetamol in the BALB/cJ mouse.

The effect of the immunomodulator, poly rI:rC, upon the in vivo metabolism of [14C]-paracetamol has been investigated in male BALB/cJ mice. In both poly rI:rC treated and control groups of mice the major part of the dose was excreted in the 0-24 hr urine and the major urinary metabolites were the glucuronic acid and sulphate conjugates. The urinary excretion of these two conjugates and of free paracetamol was not significantly altered following poly rI:rC treatment. Following enzymic hydrolysis of glucuronides and sulphates, the 3-cysteine, 3-mercapturate, 3-thiomethyl and 3-methylsulphoxide metabolites of paracetamol were all identified in the 0-24 hr urine together with very small amounts of 3-methoxy paracetamol. Although poly rI:rC treatment reduced the proportional urinary excretion of each of the thio adducts the individual differences were not significant. However, total thio adduct excretion, an indirect estimate of the metabolic activation of paracetamol, was significantly lower following poly rI:rC treatment. This depression in the urinary excretion of thio adducts following poly rI:rC treatment is discussed in relation to possible implications for paracetamol toxicity.

Effect of interferon synthesis upon the metabolism of [carboxyl-14C]-aspirin in the mouse.

The effect of stimulation of interferon (IFN) synthesis with the immunomodulators, polyriboinosinic polyribocytidylic acid (poly rI:rC) and Newcastle Disease Virus (NDV) upon the in vivo metabolism of [carboxyl-14C]-aspirin was investigated in male mice of three different strains. Following poly rI:rC administration to DBA/2 mice, the metabolic conjugation of salicylic acid with glycine was significantly increased, glucuronidation was little changed and there was an approximately 10-fold reduction in the excretion of the oxidation product, gentisic acid. Prolongation of hexobarbitone-induced sleeping time confirmed the ability of this agent to depress oxidative metabolism in vivo. Poly rI:rC administration to C57BL/6By and BALB/cBy mice resulted in similar changes in aspirin metabolism in vivo. Treatment of C57BL/6By mice with NDV produced high levels of circulating IFN and produced alterations in aspirin metabolism similar to those seen after poly rI:rC treatment. Comparable studies in BALB/cBy mice, which did not produce detectable levels of IFN in response to NDV challenge, revealed no change in the fate of aspirin metabolism following treatment with this agent. These data indicate that the in vivo oxidation of salicylate, but not its conjugation, can be depressed, at least indirectly, by an interferon-associated mechanism.