Quantification of naltrexone and 6,beta-naltrexol in plasma and milk using gas chromatography-mass spectrometry. Application to studies in the lactating sheep.

A selective gas chromatography-mass spectrometry method using solid-phase extraction has been developed for the detection and quantification of naltrexone and its metabolite, 6,beta-naltrexol in plasma and milk from humans and sheep at pharmacologically relevant concentrations. Di- or tri-acetyl derivatives were formed and quantified by selected-ion monitoring. Recoveries of naltrexone (30 microg/l) and 6,beta-naltrexol (250 microg/l) from both human plasma and milk were greater than 70%. Intra-assay and inter-day precision ranged from 3 to 21% for naltrexone and 2-18% for 6,beta-naltrexol for all matrices investigated, with an overall mean accuracy of 104% for naltrexone, and 99% for 6,beta-naltrexol. Human samples containing these analytes were stable for at least 3 weeks at -20 degrees C or 6 weeks at -80 degrees C. Analysis of the plasma and milk from the lactating sheep showed mean milk-to-plasma ratios of 55 for naltrexone and 3 for 6,beta-naltrexol.

Inactivation of human arylamine N-acetyltransferase 1 by the hydroxylamine of p-aminobenzoic acid.

Human N-acetyltransferase 1 (NAT1) is a widely distributed enzyme that catalyses the acetylation of arylamine and hydrazine drugs as well as several known carcinogens, and so its levels in the body may have toxicological importance with regard to drug toxicity and cancer risk. Recently, we showed that p-aminobenzoic acid (PABA) was able to down-regulate human NAT1 in cultured cells, but the exact mechanism by which PABA acts remains unclear. In the present study, we investigated the possibility that PABA-induced down-regulation involves its metabolism to N-OH-PABA, since N-OH-AAF functions as an irreversible inhibitor of hamster and rat NAT1. We show here that N-OH-PABA irreversibly inactivates human NAT1 both in cultured cells and cell cytosols in a time- and concentration-dependent manner. Maximal inactivation in cultured cells occurred within 4 hr of treatment, with a concentration of 30 microM reducing activity by 60 +/- 7%. Dialysis studies showed that inactivation was irreversible, and cofactor (acetyl coenzyme A) but not substrate (PABA) completely protected against inactivation, indicating involvement of the cofactor-binding site. In agreement with these data, kinetic studies revealed a 4-fold increase in cofactor K(m), but no change in substrate K(m) for N-OH-PABA-treated cytosols compared to control. We conclude that N-OH-PABA decreases NAT1 activity by a direct interaction with the enzyme and appears to be a result of covalent modification at the cofactor-binding site. This is in contrast to our findings for PABA, which appears to reduce NAT1 activity by down-regulating the enzyme, leading to a decrease in NAT1 protein content.

Characterization of an ATP-dependent pathway of activation for the heterocyclic amine carcinogen N-hydroxy-2-amino-3-methylimidazo[4, 5-f]quinoline.

2-amino-3-methylimidazo[4,5-f]quinoline (IQ) is one of several mutagenic and carcinogenic heterocyclic amines formed during the cooking process of protein-rich foods. These compounds are highly mutagenic and have been shown to produce tumours in various tissues in rodents and non-human primates. Metabolic activation of IQ is a two-step process involving N-hydroxylation by CYP1A2 followed by esterification to a more reactive species capable of forming adducts with DNA. To date, acetylation and sulphation have been proposed as important pathways in the formation of N-hydroxy esters. In this study we have demonstrated the presence of an ATP-dependent activation pathway for N-hydroxy-IQ (N-OH-IQ) leading to DNA adduct formation measured by covalent binding of [(3)H]N-OH-IQ to DNA. ATP-dependent DNA binding of N-OH-IQ was greatest in the cytosolic fraction of rat liver, although significant activity was also seen in colon, pancreas and lung. ATP was able to activate N-OH-IQ almost 10 times faster than N-hydroxy-2-amino-1-methyl-6-phenylimidazo[4, 5-b]pyridine (7.7 +/- 0.3 and 0.9 +/- 0.1 pmol/mg protein/min, respectively). Using reported intracellular concentrations of cofactor, the ability of ATP to support DNA binding was similar to that seen with 3'-phosphoadenosine 5'-phosphosulphate and approximately 50% of that seen with acetyl coenzyme A (AcCoA). In addition to DNA binding, HPLC analysis of the reaction mixtures using ATP as co-factor showed the presence of two stable, polar metabolites. With AcCoA, only one metabolite was seen. The kinase inhibitors genistein, tyrphostin A25 and rottlerin significantly inhibited both DNA binding and metabolite formation with ATP. However, inhibition was unlikely to be due to effects on enzyme activity since the broad spectrum kinase inhibitor staurosporine had no effect and the inactive analogue of genistein, daidzein, was as potent as genistein. The effects of genistein and daidzein, which are naturally occurring isoflavones from soy and other food products, on DNA adduct formation may potentially be useful in the prevention of heterocyclic amine-induced carcinogenesis.

Substrate-dependent regulation of human arylamine N-acetyltransferase-1 in cultured cells.

Arylamine N-acetyltransferase-1 (NAT1) is a polymorphically expressed enzyme that is widely distributed throughout the body. In the present study, we provide evidence for substrate-dependent regulation of this enzyme. Human peripheral blood mononuclear cells cultured in medium supplemented with p-aminobenzoic acid (PABA; 6 microM) for 24 h showed a significant decrease (50-80%) in NAT1 activity. The loss of activity was concentration-dependent (EC(50) approximately 2 microM) and selective because PABA had no effect on the activity of constitutively expressed lactate dehydrogenase or aspartate aminotransferase. PABA also induced down-regulation of NAT1 activity in several human cell lines grown at confluence. Substrate-dependent down-regulation was not restricted to PABA. Addition of other NAT1 substrates, such as p-aminosalicylic acid, ethyl-p-aminobenzoate, or p-aminophenol to peripheral blood mononuclear cells in culture also resulted in significant (P <.05) decreases in NAT1 activity. However, addition of the NAT2-selective substrates sulfamethazine, dapsone, or procainamide did not alter NAT1 activity. Western blot analysis using a NAT1-specific antibody showed that the loss of NAT1 activity was associated with a parallel reduction in the amount of NAT1 protein (r(2) = 0.95). Arylamines that did not decrease NAT1 activity did not alter NAT1 protein levels. Semiquantitative reverse transcriptase polymerase chain reaction of mRNA isolated from treated and untreated cells revealed no effect of PABA on NAT1 mRNA levels. We conclude that NAT1 can be down-regulated by arylamines that are themselves NAT1 substrates. Because NAT1 is involved in the detoxification/activation of various drugs and carcinogens, substrate-dependent regulation may have important consequences with regard to drug toxicity and cancer risk.

A comparison of high-performance liquid chromatography and fluorescence polarization immunoassay for therapeutic drug monitoring of tricyclic antidepressants.

Although the manufacturer of the polyclonal fluorescence polarization immunoassay (FPIA) for tricyclic antidepressants (TCA) only recommends its use in the diagnosis of overdose, the assay is nevertheless widely used in therapeutic drug monitoring. Using plasma samples from 337 patients taking one of eight different tricyclic antidepressants, the authors investigated the performance of the TDx assay procedure for eight different TCAs by comparison to specific high-performance liquid chromatography (HPLC) assay methods. The regression correlation between the TDx assay value and that for active tricyclic measured by HPLC was poor (r2 < 0.9) for amitriptyline, clomipramine, dothiepin, and doxepin. The regression line for amitriptyline also had a significant positive y-axis intercept. Moreover, the TDx method overestimated the concentration of active drug to an extent that varied considerably between different TCAs and within the usual therapeutic range for a single TCA. The authors conclude that the TDx assay is probably satisfactory for routine TDM of desipramine, imipramine, nortriptyline, and trimipramine. However, it significantly overestimates therapeutic concentrations of amitriptyline, clomipramine, dothiepin, and doxepin. The use of TDx and HPLC assay methods by different laboratories for sequential therapeutic drug monitoring of TCAs in the same patient may confuse physicians and confound dose adjustment and patient management. Although their study shows that the TDx assay can give satisfactory therapeutic drug monitoring results for some drugs, the authors conclude that its use should be restricted to the evaluation of overdose as recommended by the manufacturer.

Functional polymorphism of the human arylamine N-acetyltransferase type 1 gene caused by C190T and G560A mutations.

Human N-acetyltransferase type 1 (NAT1) catalyses the N- or O-acetylation of various arylamine and heterocyclic amine substrates and is able to bioactivate several known carcinogens. Despite wide inter-individual variability in activity, historically, NAT1 was considered to be monomorphic in nature. However, recent reports of allelic variation at the NAT1 locus suggest that it may be a polymorphically expressed enzyme. In the present study, peripheral blood mononuclear cell NAT1 activity in 85 individuals was found to be bimodally distributed with approximately 8% of the population being slow acetylators. Subsequent sequencing of the individuals having slow acetylator status showed all to have either a C190T or G560A base substitution located in the protein encoding region of the NAT1 gene. The C190T base substitution changed a highly conserved Arg64, which others have shown to be essential for fully functional NAT1 protein. The C190T mutation has not been reported previously and we have named it NAT1 x 17. The G560A mutation is associated with the base substitutions previously observed in the NAT1 x 10 allele and this variant (NAT1 x 14) encodes for a protein with reduced acetylation capacity. A novel method using linear PCR and dideoxy terminators was developed for the detection of NAT1 x 14 and NAT1 x 17. Neither of these variants was found in the rapid acetylator population. We conclude that both the C190T (NAT1 x 17) and G560A (NAT1 x 14) NAT1 structural variants are involved in a distinct NAT1 polymorphism. Because NAT1 can bioactivate several carcinogens, this polymorphism may have implications for cancer risk in individual subjects.

Metabolic activation of aromatic amines by human pancreas.

Epidemiologic studies have suggested that aromatic amines (and nitroaromatic hydrocarbons) may be carcinogenic for human pancreas. Pancreatic tissues from 29 organ donors (13 smokers, 16 non-smokers) were examined for their ability to metabolize aromatic amines and other carcinogens. Microsomes showed no activity for cytochrome P450 (P450) 1A2-dependent N-oxidation of 4-aminobiphenyl (ABP) or for the following activities (and associated P450s): aminopyrine N-demethylation and ethylmorphine N-demethylation (P450 3A4); ethoxyresorufin O-deethylation (P450 1A1) and pentoxyresorufin O-dealkylation (P450 2B6); p-nitrophenol hydroxylation and N-nitrosodimethyl-amine N-demethylation (P450 2E1); lauric acid omega-hydroxylation (P450 4A1); and 4-(methylnitrosamino)-1-(3-pyridyl-1-butanol) (NNAL) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) alpha-oxidation (P450 1A2, 2A6, 2D6). Antibodies were used to examine microsomal levels of P450 1A2, 2A6, 2C8/9/18/19, 2E1, 2D6, and 3A3/4/5/7 and epoxide hydrolase. Immunoblots detected only epoxide hydrolase at low levels; P450 levels were <1% of liver. Microsomal benzidine/prostaglandin hydroperoxidation activity was low. In pancreatic cytosols and microsomes, 4-nitrobiphenyl reductase activities were present at levels comparable to human liver. The O-acetyltransferase activity (AcCoA-dependent DNA-binding of [3H]N-hydroxy-ABP) of pancreatic cytosols was high, about twothirds the levels measured in human colon. Cytosols showed high activity for N-acetylation of p-aminobenzoic acid, but not of sulfamethazine, indicating that acetyltransferase-1 (NAT1) is predominantly expressed in this tissue. Cytosolic sulfotransferase was detected at low levels. Using 32P-post-labeling enhanced by butanol extraction, putative arylamine-DNA adducts were detected in most samples. Moreover, in eight of 29 DNA samples, a major adduct was observed that was chromatographically identical to the predominant ABP-DNA adduct, N-(deoxyguanosin-8-yl)-ABP. These results are consistent with a hypothesis that aromatic amines and nitroaromatic hydrocarbons may be involved in the etiology of human pancreatic cancer.

Uptake of the food-derived heterocyclic amine carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and its N-hydroxy metabolite into rat pancreatic acini and hepatocytes in vitro.

Sinc DNA adducts of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) are formed at relatively high levels in the rat pancreas but not liver, we examined the uptake of PhIP and its N-hydroxy metabolite (N-OH-PhIP) into pancreatic acini and hepatocytes to determine if differential tissue uptake was a factor modulating the formation of PhIP-DNA adducts. In addition, since the precursors of PhIP formation are two amino acids and since various amino acid transporters have been identified in the pancreas, the possible involvement of these transporters in the uptake of PhIP and N-OH-PhIP was investigated. The uptake both heterocyclic compounds into both tissue preparations was rapid, with maximal uptake occurring with 1-2 min. However, PhIP uptake into pancreatic acini was significantly (2-way ANOVA, P < 0.05) greater than uptake of N-OH-PhIP into pancreatic acini and the uptake of both PhIP and N-OH-PhIP into hepatocytes. Although uptake was rapid, efflux of both compounds from both tissue preparations was also rapid. However, the efflux rate constant (1.86 +/- 0.6/min, mean +/- SEM) for PhIP) was significantly lower (Student's t-test, P < 0.05) than that for N-OH-PhIP (4.14 +/- 0.04/min) from pancreatic acini. This, combined with the increased uptake of PhIP into pancreatic acini , suggests that there is substantial but reversible binding of PhIP in the pancreas. The uptake of both PhIP and N-OH-PhIP into pancreatic acini and hepatocytes was not affected by the presence of various amino acids in the incubation buffer, indicating that amino acid transporters are not involved in uptake of these compounds. Furthermore, uptake of both compounds did not appear to be dependent on metabolic energy supply. The above data, together with the high octanol:buffer partition coefficients (logP = 1.322 and 1.301 for PhiP and N-OH-PhIP respectively) suggest that both uptake and efflux of PhIP and N-OH-PhIP are consistent with a process of passive diffusion. The tissue binding characteristics for PhIP in the pancreas may create conditions whereby pancreatic cytochrome P450 1A1 can catalyse the formation of N-OH-PhIP. While N-OH-PhIP is not the ultimate reactive DNA binding species, it has been shown to directly bind to and form DNA adducts. Therefore, it is possible that the apparent selective accumulation of PhIP may contribute to the high level of PhIP-DNA adducts formed in the rat pancreas.

Polymorphism in the N-acetyltransferase 1 (NAT1) polyadenylation signal: association of NAT1*10 allele with higher N-acetylation activity in bladder and colon tissue.

Exposures to carcinogens present in the diet, in cigarette smoke, or in the environment have been associated with increased risk of bladder and colorectal cancer. The aromatic amines and their metabolites, a class of carcinogen implicated in these exposures, can be N- or O-acetylated by the NAT1 and NAT2 enzymes. Acetylation may result in activation to DNA-reactive metabolites or, in some cases, detoxification. Many studies have focused on genetic variation in NAT2 and its potential as a risk factor in bladder and colorectal cancer; however, NAT1 activity is higher in bladder and colonic mucosa than NAT2, and the NAT1 enzyme also exhibits phenotypic variation among human tissue samples. We hypothesized that specific genetic variants in the polyadenylation signal of the NAT1 gene would alter tissue levels of NAT1 enzyme activity and used a PCR-based method to distinguish polymorphic NAT1 alleles in samples obtained from 45 individuals. When the NAT1 genotype was compared with the NAT1 phenotype in bladder and colon tissue samples (p-aminobenzoic acid activity), we observed a approximately 2-fold higher NAT1 enzyme activity in samples from individuals who inherited a variant polyadenylation signal (NAT1*10 allele). This is the first observation relating a genetic polymorphism in NAT1 to a rapid/slow NAT1 phenotype in humans.

Metabolic activation and DNA adduct detection of PhIP in dogs, rats, and humans in relation to urinary bladder and colon carcinogenesis.

The metabolic activation of the heterocyclic amine carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), was examined in dogs and rats as models for urinary bladder and colon carcinogenesis, respectively. The results indicate that unconjugated N-OH-PhIP is not excreted in the urine after oral dosing with PhIP and that the two isomeric N-glucuronides of N-OH-PhIP, which are formed as major metabolites, are stable under acidic conditions. These data suggest that PhIP is unlikely to serve as a urinary bladder carcinogen in either species. Using metabolic inhibitors, bile duct ligation, and intravenous dosing studies, a new hypothesis for colorectal carcinogenesis is proposed involving N-oxidation of PhIP by hepatic cytochrome P-4501A2 (CYP1A2) and O-acetylation by the polymorphic acetyltransferase (NAT2). The resulting N-hydroxy and N-acetoxy metabolites both appear to be transported through the circulation to the colon mucosa, forming covalent DNA adducts. Glucuronidation and reaction with glutathione appear to serve as detoxification pathways. In humans, individuals who are phenotypically rapid metabolizers for both CYP1A2 and NAT2 are significantly higher (p = 0.0015) in colorectal cancer/poly cases vs. controls; and PhIP-DNA adducts can be detected in human colon samples. These studies provide strong evidence that PhIP and other heterocyclic amines play an important role in the etiology of human colorectal cancer.

Metabolic activation and carcinogen-DNA adduct detection in human larynx.

Putative carcinogen-DNA adducts in human larynx tissues (n = 25) from smoker and non/ex-smoker patients were examined by 32P-postlabeling and compared with the metabolic activation capacity of larynx microsomes and cytosols from the same tissues. Hydrophobic DNA adducts were evident only in smokers, and chromatographic profiles of the adducts were similar using either the butanol extraction or nuclease P1 enhancement method, which suggested that the adducts may be derived from polycyclic aromatic hydrocarbons but not aromatic amines. Immunoblots of larynx microsomes using anti-cytochrome P450 1A1/1A2, 2C, 3A4, 2E1, and 2A6 antibodies showed intensities ranging from 1-10% of that typically observed with human liver microsomes. Enzymatic assays of larynx microsomes showed appreciable activity for benzo(a)pyrene hydroxylation (P450 1A1 and 2C) but not for 4-aminobiphenyl N-oxidation (P450 1A2), which indicated that the observed immunoreactivity was for P450 1A1; this represents the highest level of this P450 yet detected in human extrahepatic tissues. Accordingly, total DNA adduct levels in the larynx correlated strongly with levels of P450 2C, 1A1, and 3A4 but not with P450 2E1 or 2A6. Larynx cytosols also showed appreciable aromatic amine N-acetyl-transferase activity for p-aminobenzoic acid (NAT-1) but not for sulfamethazine (NAT-2); however, NAT-1 activity was not correlated with total DNA adducts, which is again consistent with the lack of aromatic amine-DNA adducts detected by 32P-postlabeling. Thus, these results suggest that the DNA adducts detected in human larynx are largely derived from metabolic activation of polycyclic aromatic hydrocarbons in cigarette smoke by P450 2C, 3A4, and/or 1A1.

Metabolic activation pathway for the formation of DNA adducts of the carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in rat extrahepatic tissues.

The food-borne mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) induces tumors in colon of male rats and has been implicated in the etiology of human cancers, particularly colorectal cancer. This study was conducted to examine: (1) the biliary and/or circulatory transport of N-hydroxy-PhIP and its N-glucuronides, N-sulfonyloxy-PhIP and N-acetoxy-PhIP; (2) their role as proximate and ultimate carcinogenic metabolites of PhIP; (3) the potential role of glutathione in modulating PhIP-DNA adduct formation. PhIP-DNA adducts, measured by the 32P-postlabeling method, were highest in the pancreas (361 adducts/10(8) nucleotides or 100%), followed by colon (56%), lung (28%), heart (27%) and liver (2%), at 24 h after a single oral dose of PhIP (220 mumol/kg) to male rats. In each tissue examined, we observed two major adducts, each of which accounted for 35-45% of the total, and one minor adduct, which represented about 10-20% of the total. One of the major adducts was identified as N-(deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine by chromatographic comparisons with an authentic standard. The major urinary metabolites of PhIP in these rats were 4'-hydroxy-PhIP and its glucuronide and sulfate conjugates, followed by N-hydroxy-PhIP N3-glucuronide, N-hydroxy-PhIP N2-glucuronide and unchanged PhIP. In bile duct-ligated rats, the urinary excretion of the N-OH-PhIP N3-glucuronide was increased two-fold, but there was no effect on PhIP-DNA adduct formation in the colon, heart, lung, pancreas or liver. 2,6-Dichloro-4-nitrophenol, which strongly inhibits arylsulfo-transferase-mediated DNA binding in vivo, had no effect on PhIP-DNA adduct levels in liver or in extrahepatic tissues. Pretreatment of rats with buthionine sulfoximine, which results in hepatic glutathione depletion, caused a five-fold increase in adduct formation in the liver. Intravenous administration (10 mumol/kg) of N-hydroxy-PhIP and N-acetoxy-PhIP each led to high levels of PhIP-DNA adducts in each of the extrahepatic tissues examined. Adduct levels ranged from two- to six-fold higher (for N-hydroxy-PhIP) and four- to 28-fold higher (for N-acetoxy-PhIP) as compared to that after an i.v. dose of the parent compound, indicating that these two bioactivated derivatives of PhIP are sufficiently stable to be transported through the circulation to extrahepatic tissues.(ABSTRACT TRUNCATED AT 400 WORDS)

Expression of monomorphic and polymorphic N-acetyltransferases in human colon.

The metabolism of sulfamethazine (SMZ) and p-aminobenzoic acid (PABA) by N-acetyltransferase (NAT) was measured in human colorectal cytosols from 12 slow and 11 rapid acetylators whose genotype was determined independently by a specific polymerase chain reaction. SMZ metabolism was significantly greater in the rapid than in the slow phenotype (192 +/- 22 versus 94 +/- 11 pmol N-acetylsulfamethazine/min/mg protein), while PABA metabolism was similar in both phenotypes (23.7 +/- 4.4 versus 23.0 +/- 3.9 nmol N-acetyl-p-aminobenzoic acid/min/mg protein). Both monomorphic and polymorphic NAT mRNAs were detected by the polymerase chain reaction in the colorectal mucosa of most samples. The finding that polymorphic NAT is expressed in a phenotype-dependent manner in colorectal mucosa indicates that this tissue has the capacity to participate in local bioactivation of dietary and environmental aryl- or heterocyclic amine carcinogens and may explain, in part, the phenotype-dependent occurrence of colorectal cancer.

Role of acetylation in colorectal cancer.

Acetylator phenotype is a common genetic trait in humans as well as other mammals. It results from the presence of several mutations in one of the genes encoding for arylamine N-acetyltransferase. The polymorphism has been associated with several disease states including colorectal cancer. Several epidemiological studies suggest that rapid acetylators are more susceptible to colorectal cancer than slow acetylators. Moreover, individuals that are both rapid acetylators and exhibit a high cytochrome P450 1A2 activity appear to have an even higher risk of colorectal cancer. These observations not only suggest an interesting genetic link to non-familial colon cancer but also suggest that carcinogens that are activated by N-acetyltransferase and cytochrome P450 1A2 may contribute to the etiology of this disease. Heterocyclic amines present in cooked food such as "well done" red meat are carcinogenic in experimental animals forming tumours in several target tissues including the small intestines. We have shown that human polymorphic N-acetyltransferase is present in human colon tissue and that it is capable of activating several heterocyclic amine carcinogens present in cooked food. These studies provide good circumstantial evidence that rapid acetylators may be predisposed to colorectal cancer.

Acetylation phenotype and genotype in aboriginal leprosy patients from the north-west region of Western Australia.

N-Acetyltransferases (NAT1, NAT2) play an important role in biotransformation of a number of drugs and carcinogens. A polymorphism in the metabolism of such compounds by NAT2 has been known for many years but it is only recently that the underlying molecular genetics has been elucidated. In the present study, we have correlated acetylation phenotype and genotype in a group of 49 Australian Aborigines (26 males and 23 females; mean age = 50.5 yr) from the Derby region of Western Australia. Phenotype was determined using caffeine and genotype by an allele-specific polymerase chain reaction. The percentages of slow and rapid phenotypes were 36.7 and 63.3%, respectively, while the distribution of alleles for the NAT2 gene was 41% for the wildtype and 2, 17 and 40% for the M1, M2 and M3 mutations, respectively. This is the highest proportion of M3 mutations reported for any ethnic population. The observed genotype proportions were not significantly different from those predicted by the Hardy-Weinberg Law (chi 2 = 1.07, p > 0.05). Phenotype was predictable from genotype in 100% of patients. At the time of study, 29 of the Aborigines were receiving acedapsone intramuscularly for control of leprosy. Plasma dapsone concentrations in these patients were similar for both slow (n = 11) and rapid (n = 18) acetylators, suggesting that phenotype is unlikely to influence treatment outcome. The data show that Aborigines have a similar phenotype distribution to that of some Asian populations, but that there are differences in the frequencies of the M1, M2 and M3 mutant alleles.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylation phenotype and cytochrome P450IA2 phenotype are unlikely to be associated with peripheral arterial disease.

The hypothesis that cytochrome P450IA2 (CYPIA2) and/or N-acetyltransferase 2 (NAT2) may be involved in the pathogenesis of peripheral arterial disease was investigated in 90 Australian patients with significant disease and 81 matched control subjects. CYPIA2 and NAT2 phenotypes were determined from urinary metabolite patterns after an oral dose of caffeine. NAT2 phenotype was similar (chi 2 = 0.01; p = 0.98) in both atherosclerotic patients (43.3% rapid) and control subjects (42.0% rapid). CYPIA2 metabolism as measured by the median ratio of (1,7-dimethylxanthine + 1,7-dimethyluric acid)/caffeine was significantly induced by smoking in both patients with atherosclerosis (ratio of 6.5 in nonsmokers and 12.4 in smokers; p < 0.05) and control subjects (ratio of 8.2 in nonsmokers and 14.8 in smokers; p < 0.05), but values in atherosclerotic and control nonsmokers and smokers were similar. Probit transformation of the data revealed a trimodal distribution of ratios in control subjects who were nonsmokers, with 5% classified as poor metabolizers (homozygous rapid) and 95% as extensive metabolizers. The distribution of ratios in control subjects who were smokers was unimodal, whereas among the patients with arterial disease, both smokers and nonsmokers exhibited a bimodal pattern with 8.2% to 16% poor metabolizer and 84% to 91.8% extensive metabolizer phenotypes. When data from both nonsmokers and smokers were combined, the overall proportion of subjects who were poor metabolizers was not significantly different (chi 2 = 1.82; p = 0.18) between control subjects (3.8%) and patients with atherosclerosis (10.6%). Thus biotransformation of environmental or dietary aromatic or heterocyclic amines by NAT2 or CYPIA2 is unlikely to have a significant role in the cause or pathogenesis of peripheral arterial disease.

Metabolic activation, DNA adducts, and H-ras mutations in human neoplastic and non-neoplastic laryngeal tissue.

Metabolic activation, DNA adducts, and H-ras mutations were examined in human laryngeal tissue (n = 16) from both smoker and non/ex-smoker patients with laryngeal cancer. DNA adducts detected by 32P-postlabelling were evident only in smokers (n = 13); in fact, smoking cessation for as little as 10 months resulted in no DNA adducts detected (n = 3). Total DNA adduct levels in these samples were significantly correlated with levels of cytochromes P-4502C and 1A1 in laryngeal microsomes. Moreover, the P-4501A1 levels represent the highest yet found in human tissues. In contrast, laryngeal microsomes did not have detectable P-4501A2 activity, while laryngeal cytosols showed appreciable N-acetyltransferase activity for p-aminobenzoic acid (NAT1) but not sulfamethazine (NAT2). DNA was extracted from laryngeal specimens and amplified by PCR. Nylon filter dot or slot blots were hybridized with 32P-labelled probes for codons 12, 13, and 61 of the H-ras gene. Sixty percent of specimens demonstrated mutations in either codon 12, 13, or 61; a single common and specific mutation was a Gln-->Glu transversion in codon 61. This mutation appeared in 5 laryngeal specimens, all from smokers. These results implicate cigarette smoke components, bioactivated by CYP1A1 and/or CYP2C, in DNA adduct formation. These results also demonstrate a probable smoking-related H-ras Gln-->Glu transversion in codon 61.

Comparison of cyclosporine measurement in whole blood by high-performance liquid chromatography, monoclonal fluorescence polarization immunoassay, and monoclonal enzyme-multiplied immunoassay.

Monitoring of cyclosporine concentrations in whole blood is used routinely as a guide to adjusting dose so as to achieve optimal therapeutic benefit with minimal adverse effects. In the present study, we have compared a specific high-performance liquid chromatography (HPLC) assay with a fluorescence polarization immunoassay (TDx) and an enzyme-multiplied immunoassay (Emit). Both Emit and TDx assays employ a monoclonal antibody to cyclosporin A and therefore have the potential for a high degree of specificity. Blood specimens (EDTA as anticoagulant) were obtained from 113 patients (71 renal transplants, 17 liver transplants, and 25 other categories) taking cyclosporine and analysed by all three methods. There were significant correlations between results for HPLC and Emit (Emit = 10.54 + 1.07 x HPLC; r2 = 0.82, p less than 0.001) and between results for HPLC and TDx (TDx = 9.16 + 1.42 x HPLC; r2 = 0.82, p less than 0.001). Compared to HPLC analysis, 74% and 96%, respectively, of Emit and TDx results were to the left of the line of identity. The TDx monoclonal antibody appears to have a lesser degree of specificity than that used in the Emit assay. Mean concentrations of cyclosporine measured by Emit and TDx were 17% and 51% higher, respectively, than those measured by HPLC. Because of this overestimation, we suggest that both Emit and TDx methods may find their most appropriate use in routine therapeutic monitoring of renal transplant patients in whom metabolite concentrations are less variable over time.

N-and O-acetylation of aromatic and heterocyclic amine carcinogens by human monomorphic and polymorphic acetyltransferases expressed in COS-1 cells.

Human monomorphic and polymorphic arylamine acetyltransferases (EC 2.3.1.5) were expressed in monkey kidney COS-1 cells and used to study the N- and O-acetylation of a number of carcinogenic amines and their N-hydroxy metabolites. The monomorphic enzyme N-acetylated the aromatic amines, 2-aminofluorene and 4-aminobiphenyl, and also O-acetylated their N-hydroxy derivatives. None of the food-derived heterocyclic amines (Glu-P-1, PhIP, IQ, MeIQx) were substrates and their N-hydroxy metabolites were poorly O-acetylated by this isozyme. By contrast, the polymorphic acetyltransferase catalyzed the N-acetylation of both aromatic amines, and to a lesser extent, Glu-P-1 and PhIP. However, all six N-hydroxy amine substrates were readily O-acetylated to form DNA-bound adducts by the polymorphic isozyme. These data suggest that, for the heterocyclic amine carcinogens, rapid acetylator individuals will be predisposed to their genotoxicity.