Anticonvulsant hypersensitivity syndrome. In vitro assessment of risk.

Arene oxide metabolites of aromatic anticonvulsants (phenytoin, phenobarbital, and carbamazepine) may be involved in the pathogenesis of hypersensitivity reactions. We investigated 53 patients with clinical sensitivity to anticonvulsants by exposing their lymphocytes in vitro to drug metabolites generated by a murine hepatic microsomal system. The diagnosis of a hypersensitivity reaction was corroborated by in vitro rechallenge for each drug (phenytoin, n = 34; phenobarbital, n = 22; carbamazepine, n = 25) when cytotoxicity (% dead cells) exceeded 3 SD above the mean result for controls. Cross-reactivity among the drugs was noted. 7 out of 10 patients who had received all three anticonvulsants had adverse reactions to each. 40 out of 50 patients tested to all three drugs in vitro were positive to each. Adverse reactions were indistinguishable among anti-convulsants. Skin rash (87%), fever (94%), hepatitis (51%), and hematologic abnormalities (51%) were common clinical features of each drug. 62% of reactions involved more than two organs. Cells from patients' parents exhibited in vitro toxicity that was intermediate between values for controls and patients. In vitro testing can help diagnose hypersensitivity to anticonvulsants. Cells from patients may also be used for prospective individualization of therapy to decrease risk of adverse reaction. Cross-reactivity among the major anticonvulsants is common and should be considered before deciding on alternative therapy.

Nitrofurantoin cytotoxicity. In vitro assessment of risk based on glutathione metabolism.

Nitrofurantoin, a commonly used urinary tract antiseptic, has been associated with idiosyncratic pulmonary and hepatic damage. We have used human lymphocytes in vitro to explore the biochemical basis of susceptibility to nitrofurantoin toxicity. The drug itself did not damage the cells as assessed by trypan blue dye exclusion. In the presence of a mouse hepatic microsomal drug-activating system, however, nitrofurantoin metabolites produced dose dependent toxicity to the lymphocytes. Inhibition of the enzyme epoxide hydrolase did not enhance toxicity; the metabolite thus does not appear to be a furan epoxide. Binding of reactive metabolites to cell macromolecules may lead directly to cell death, or in vivo, by acting as haptens to secondary immunologic responses. The metabolite caused a dose-dependent depletion of lymphocyte glutathione content. Cells from a patient with glutathione synthetase deficiency showed markedly enhanced nitrofurantoin toxicity. The findings suggest that glutathione plays a major role in protecting cells from nitrofurantoin-induced damage, and that studies of lymphocyte toxicity and glutathione metabolism in patients experiencing idiosyncratic reactions to nitrofurantoin may lead to elucidation of the biochemical and genetic basis of drug susceptibility.

Biochemical heterogeneity in glutathione synthetase deficiency.

Two different clinical syndromes are associated with glutathione synthetase deficiency, one presenting with hemolytic anemia and 5-oxoprolinuria, the other with isolated hemolysis. We have differentiated these disorders on an enzymatic basis. In 5-oxoprolinuria, all cell types examined have grossly deficient enzyme activity and glutathione content. In contrast, in the nonoxoprolinuric variant, erythrocytes have decreased enzyme activity and glutathione content, whereas nucleated cells maintain substantial levels of both. The enzyme in this disorder is unstable in vitro and has shortened survival in intact erythrocytes. Nucleated cells appear able to maintain sufficient enzyme activity and concentrations of glutathione to suppress overproduction of 5-oxoproline.

Metabolism of leukotriene A4 into C4 by human platelets.

Tritium-labelled leukotriene A4 is converted by a suspension of human platelets into leukotriene C4. The conversion is stimulated by reduced glutathione and is dependent on the platelet concentration. Formation of leukotriene C4 is temperature and time dependent and is destroyed by heating the platelets at 100 degrees C for 5 min. Verification of leukotriene C4 formation was obtained by conversion into leukotriene D4 during reaction of the HPLC-purified platelet-derived leukotriene C4 with commercial gamma-glutamyl transpeptidase. In separate experiments we incubated authentic tritiated leukotriene C4 with human platelets and we showed the formation of tritiated leukotriene D4, demonstrating the presence of gamma-glutamyl transpeptidase activity in these cells. This activity could be blocked by the presence of reduced glutathione in the incubation mixture. In contrast, erythrocytes converted tritiated leukotriene A4 almost exclusively into leukotriene B4. Although platelets have been reported to lack 5-lipoxygenase activity, our study demonstrates that platelets possess the necessary machinery to transform leukotriene A4 into leukotrienes C4 and D4. Our results suggest that an intracellular interaction between platelets and leukotriene A4-forming cells, e.g., polymorphonuclear leukocytes, could lead to the formation of these potent peptidolipids in the circulation.

Characterization of the microsomal epoxide hydrolase gene in patients with anticonvulsant adverse drug reactions.

Therapy with the aromatic anticonvulsants phenytoin, phenobarbital and carbamazepine has been associated with the occurrence of rare idiosyncratic hypersensitivity reactions. These drugs are thought to be activated to potentially reactive arene oxide (epoxide) metabolites by cytochrome P450-dependent monooxygenation, while liver microsomal epoxide hydrolase (mEH) plays a detoxifying role by converting such reactive intermediates to non-toxic dihydrodiols. Evidence from in vitro lymphocyte toxicity tests and enzyme inhibitor studies has suggested that an inherited defect in mEH function may be responsible for the enhanced drug toxicity observed in affected individuals. To test this hypothesis we designed methods to directly compare mEH gene structure in patients presenting with anticonvulsant adverse reactions and in control subjects in which no in vivo or in vitro toxicity to anticonvulsants could be demonstrated. Southern analysis of peripheral lymphocyte DNA using a full-length mEH cDNA as hybridization probe revealed no gross differences in mEH gene structure in selected patients when compared with DNA samples from unaffected control subjects. The human mEH gene was then cloned and characterized from a control individual. Nine exons were identified within a 22 kb region and sequences of selected regions, including all exons, were determined. Single strand conformation polymorphism (SSCP) analysis was performed on all exonic regions in genomic DNA from each of 26 subjects, including six unrelated patients with previous toxicity to anticonvulsant therapy and seven siblings (three of whom had displayed toxicity). Several distinct SSCP patterns could be observed among the subjects tested, each corresponding to a specific point mutation within one of the amplified fragments of the mEH gene. However, none of the SSCP patterns reflecting point mutations was correlated with the occurrence of anticonvulsant toxicity. From these observations we conclude that a genetic defect altering the structure and function of the mEH protein is unlikely to be responsible for predisposing patients to anticonvulsant adverse reactions.

Deficiency of cytosolic arylamine N-acetylation in the domestic cat and wild felids caused by the presence of a single NAT1-like gene.

The purpose of this study was to determine the molecular basis for a relative deficiency in the cat of cytosolic arylamine N-acetyltransferase (NAT), an enzyme family that is important in the metabolism of xenobiotics and that normally consists of at least two related enzymes, NAT1 and NAT2. N-acetyltransferase in feline liver showed high affinity (mean Km = 2.1 microM) for p-aminobenzoic acid, an NAT1 selective substrate in humans and rabbits, but showed a very poor affinity (mean Km > 10 mM) for sulfamethazine, an NAT2 selective substrate in humans and rabbits. Immunoreactive N-acetyltransferase was detected in feline liver, bladder and colon using an NAT1-specific antipeptide antibody, but was not detected in any tissues using an NAT2-specific antibody. Southern blot analysis of genomic DNA demonstrated a single band in domestic cats using each of six restriction digests; single bands were also found on Southern blot analysis of six wild felids. The deduced amino acid sequence of the central portion of feline N-acetyltransferase, obtained by polymerase chain reaction amplification in both domestic cats and seven wild felids (lion, tiger, lynx, snow leopard, bobcat, Asian leopard cat and cheetah), contained three residues, Phe125, Arg127, and Tyr129, which determine NAT1-like substrate specificity in humans. These results support the conclusion that cytosolic arylamine N-acetylation activity is low in the cat because of the presence of a single N-acetyltransferase that has substrate specificity, immunogenicity and sequence characteristics similar to human NAT1, and that the unusual presence of only a single N-acetyltransferase gene appears to be a family wide trait shared by other felids.

Sulfamethoxazole is metabolized to the hydroxylamine in humans.

The oxidation of sulfamethoxazole to its hydroxylamine metabolite was investigated in vitro with human liver microsomes and in vivo by detection in the urine. Sulfamethoxazole was oxidized to the hydroxylamine in an NADPH-dependent process by liver microsomes prepared from two human livers. Three healthy volunteers ingested 1000 mg sulfamethoxazole, and urine was collected for 24 hours. Sulfamethoxazole hydroxylamine constituted 3.1% +/- 0.7% of the drug excreted in the urine in 24 hours. Fifty-four percent of the ingested dose was excreted during this same time period. We conclude that sulfamethoxazole hydroxylamine is an authentic in vivo metabolite in humans, probably formed predominantly by cytochrome P450 in the liver. It could be responsible for mediation of sulfonamide adverse reactions, particularly hypersensitivity reactions.

Glutathione synthetase-deficient lymphocytes and acetaminophen toxicity.

Toxic electrophilic metabolites of acetaminophen are detoxified by conjugation with glutathione. Cellular glutathione content of patients with glutathione synthetase deficiency (5-oxoprolinuria) is 10% to 20% of normal. These patients might be at increased risk for acetaminophen toxicity. The hypothesis was tested by challenging lymphocytes from normals and a patient with glutathione synthetase deficiency in vitro with acetaminophen metabolites generated by a mouse hepatic microsomal drug-metabolizing system. For toxicity to be manifested in normal cells, glutathione content had to be depleted to less than 20% of control values at high acetaminophen concentrations (500 and 1,500 micrograms/ml), concentrations similar to blood levels in massive overdose and associated with hepatotoxicity in vivo. The patient's cells had only 14% of normal glutathione content, and exhibited more toxicity at 12.5 micrograms/ml acetaminophen (within the therapeutic range) as normals at maximum concentrations. The in vitro system may be of value in screening drugs potentially hazardous for glutathione synthetase-deficient patients, for exploring the role of glutathione in the detoxification of xenobiotics, and for examining glutathione protective mechanisms in patients with idiosyncratic cytotoxic drug reactions.

A urinary metabolite ratio that reflects systemic caffeine clearance.

Systemic caffeine clearance and urinary metabolite profiles were determined in 15 subjects with diverse exposure histories to cytochrome P-450 inducers (cigarette smoke) and inhibitors (oral contraceptive steroids). A correlation was observed between caffeine clearance and a urinary ratio based on the molar recovery of paraxanthine 7-demethylation products relative to a paraxanthine 8-hydroxylation product (r = 0.91; P less than 0.001). Analysis of urinary metabolites was undertaken in a larger population to assess the effects of gender, age, oral contraceptives, and smoking on the ratio. No gender differences were observed in either adults or children; children (n = 21) showed a higher (P less than 0.001) mean metabolite ratio than adults (n = 61), oral contraceptive users (n = 9) had lower (P less than 0.05) ratios than women not taking oral contraceptives (n = 30), and smokers (n = 26) had higher (P less than 0.001) ratios than nonsmokers (n = 61). The data indicate that a urinary metabolite ratio based on paraxanthine 7-demethylation/8-hydroxylation products reflects systemic caffeine clearance and likely monitors cytochrome P-450 activity inducible by polycyclic aromatic hydrocarbons.

Prominence of slow acetylator phenotype among patients with sulfonamide hypersensitivity reactions.

Delayed hypersensitivity reactions are among the most severe adverse effects of the sulfonamides in current clinical use. These reactions appear to occur because of differences in the metabolism and detoxification of reactive metabolites of the sulfonamides. N-Acetylation is a major metabolic pathway for the sulfonamides. Slow acetylation phenotype might be a risk factor for the development of these reactions. We determined the acetylation phenotype of 21 patients who had suffered hypersensitivity reactions to the sulfonamides. There were 11 females and 10 males in the group, with a mean age of 15 years (age range, 1.8 to 50 years). Their acetylator phenotype was determined by determining the ratio of urinary caffeine metabolites (1-methylxanthine to 5-amino-6-formylmethyluracil after an oral dose of 50 mg caffeine). Nineteen (90%) of the patients were slow acetylators compared to a 55% incidence of slow acetylators in a race-matched control population (p less than 0.008). This suggests that a slow acetylator phenotype is a risk factor for the development of sulfonamide hypersensitivity reactions and provides further support for the role of imbalances in genetically determined pathways of metabolism and detoxification of the sulfonamides in the pathogenesis of these reactions.

Enhanced differential diagnosis of anticonvulsant hypersensitivity reactions by an integrated Bayesian and biochemical approach.

OBJECTIVE: The differential diagnosis of hypersensitivity reactions associated with anticonvulsants requires accuracy because of the many implications for patient management. We tested an integrated Bayesian and biochemical diagnostic approach. METHODS: The patients were analyzed clinically by two tests. One test, the Bayesian Adverse Reaction Diagnostic Instrument (BARDI), calculates the posterior probability of a drug being the cause based on epidemiologic and case data. The other, the lymphocyte toxicity assay, is an in vitro rechallenge that determines the percentage of cell death attributable to a drug's toxic metabolites. The setting for the study was an adverse drug reaction clinic at Sunnybrook Health Science Centre and the Hospital for Sick Children, Toronto, Ontario, Canada. Fifty-one patients who had hypersensitivity reactions after receiving aromatic anticonvulsants were tested. Four of these patients had more than one reaction reported, with different anticonvulsants generating 56 distinct events. RESULTS: Compared to the lymphocyte toxicity assay, BARDI had 94% sensitivity, 93% accuracy, and 50% specificity. When lymphocyte toxicity assay data were incorporated into BARDI, agreement rose from 93% to 100%. BARDI also identified which drug was a more likely cause for 11 patients receiving multiple anticonvulsants. CONCLUSION: These findings show that BARDI and the lymphocyte toxicity assay have high concordance and, when used in an integrated approach, these tests can improve the diagnostic accuracy and enhance the management of patients with hypersensitivity reactions.

Drug-induced hypothyroidism: the thyroid as a target organ in hypersensitivity reactions to anticonvulsants and sulfonamides.

Inherited defects in detoxification of reactive metabolites of drugs predispose patients to "hypersensitivity" reactions. Covalent interaction of metabolites with cell macromolecules leads to cytotoxic and immunologic outcomes, manifested clinically by multisystem syndromes with variable organ involvement. Hypothyroidism developed in 5 of 202 patients (age range, 1 to 81 years) we investigated for hypersensitivity reactions to anticonvulsants or sulfonamides shortly after their reaction. None had previous personal or family histories of autoimmune disease. All had low thyroxine levels, elevated levels of thyroid stimulating hormone, and autoantibodies including antimicrosomal antibodies. Patients were 2 to 18 years of age at presentation, and two were male. All returned to a euthyroid state within a year of presentation, and all remain well. The demographics, clinical presentation, and course of the patients is atypical of idiopathic lymphocytic thyroiditis. We investigated the pathogenesis of thyroid toxicity using the hydroxylamine metabolite of sulfamethoxazole as a model. The hydroxyalmine was toxic to thyroid cells in vitro, which did or did not express thyroid peroxidase activity, whereas the parent sulfonamide was toxic only to cells with active thyroid peroxidase. The purified enzyme converted sulfamethoxazole to the hydroxylamine. Formation of reactive drug metabolites by thyroid peroxidase in a host who is genetically unable to detoxify the metabolites may lead directly to cytotoxicity. Covalent binding to macromolecules, including thyroid peroxidase, also may lead to expression of neoantigens and formation of autoantibodies. Patients who have sustained hypersensitivity reactions to drugs should be investigated for possible involvement of the thyroid.

Familial occurrence of hypersensitivity to phenytoin.

PURPOSE: Therapy with anticonvulsants such as phenytoin, phenobarbital, and carbamazepine can be complicated by severe hypersensitivity reactions. Previous work has suggested that the predisposition to such reactions is based on an inherited abnormality in the detoxification of reactive metabolites of the drugs. However, there are no reports of familial occurrence of the reactions in the literature. In the current study, we examined a family in which three siblings developed hypersensitivity reactions to phenytoin, confirming the inheritance of a predisposition to the reactions. Detoxification of reactive metabolites of the anticonvulsants was studied in cells from the patients and their siblings. PATIENTS AND METHODS: Three siblings from a family of 12 siblings developed hypersensitivity reactions to phenytoin characterized by fever, rash, lymphadenopathy, and anicteric hepatitis. All recovered completely after discontinuation of treatment. One sibling tolerated phenobarbital without toxic sequelae. Peripheral blood mononuclear cells from the three patients and five additional siblings who had never taken anticonvulsants were exposed to oxidative metabolites of phenytoin, phenobarbital, and carbamazepine generated by a hepatic microsomal drug-metabolizing system in vitro. The toxicity of metabolites in the cells from the siblings was compared with that in cells from control subjects. RESULTS: Cells from each of the patients who had experienced a hypersensitivity reaction exhibited increased toxicity from metabolites of phenytoin and carbamazepine, while the cellular response to metabolites of phenobarbital was within normal limits. Cells from four of the other siblings showed an abnormal response to phenytoin metabolites, while cells from the final sibling detoxified phenytoin metabolites normally. CONCLUSION: Our observations on the patients confirm the inherited nature of phenytoin hypersensitivity reactions in vivo. In vitro studies demonstrated abnormal metabolite detoxification in the patients and several of their siblings. The detoxification defect included metabolites of phenytoin and carbamazepine but not of phenobarbital. A family history of a drug hypersensitivity reaction should alert physicians to the probability of a markedly increased risk of an adverse reaction in family members. In vitro assays to confirm adverse reaction risks may ultimately be able to provide individualized risk assessment for patients who must take anticonvulsants.

Psychologic and behavioral effects of antiepileptic drugs in children: a double-blind comparison between phenobarbital and valproic acid.

Traditional clinical monitoring of children with epilepsy does not appear to be sufficiently sensitive to cognitive functioning and behavioral problems. Although subtle, these changes may alter a child's ability to perform well in school and in society. Physicians must prevent seizures without producing intolerable side effects, and ways of more appropriately assessing these side effects must be developed. In this double-blind, counter-balanced, crossover study of 21 children, the effects of phenobarbital and valproic acid on cognitive functioning and behavior were measured. There was no difference in seizure control between the drugs, and each medication was maintained in the therapeutic range for 6 months (mean phenobarbital level, 21.2 micrograms/mL; mean valproic acid level, 94.1 micrograms/mL). Children were treated with each drug for 6 months. Differences between the drugs were seen on measurements of cognitive function and behavior. On four tests of neuropsychologic function, children performed significantly less well while receiving phenobarbital (P less than .01). There was no evidence that the patients were sedated or less able to perform continuous performance tasks while receiving phenobarbital. Parental assessment of behavior indicated significantly worse behavior with the phenobarbital regimen for three items (P less than .01) and children were measurably more "hyperactive" (P less than .05). Routine clinical assessment of the patients did not reveal differences between the drugs with respect to routine laboratory measurements or side effects as assessed by history or physical examination. Although children may appear to tolerate a medication without clinically apparent problems, subtle but significant changes in intellectual function and behavior may be occurring. Additional, more sensitive, methods of monitoring patients while receiving these drugs is necessary.

Glutathione transferase mu deficiency is not a marker for predisposition to sulphonamide toxicity.

Glutathione transferase mu activity, a marker for susceptibility to lung cancer and chemically induced cytogenetic damage, is not a predictive index for the predisposition to sulphonamide hypersensitivity reactions. However, considering the functional diversity and broad, overlapping substrate specificity of GSH-dependent enzymes, it is conceivable that an as yet unidentified deficiency in another GST isozyme or GSH-related enzyme may be a marker for sulphonamide toxicity. In addition, heterogeneity in cellular repair mechanisms and the diversity of the human immune response [22] may also contribute to the manifestation of the toxic effects of sulphonamides. Experiments are currently in progress to determine which of this myriad of variables is predominantly responsible for inter-individual susceptibility to the idiosyncratic reactions produced by these antibacterial agents.

Cellular toxicity of sulfamethoxazole reactive metabolites--I. Inhibition of intracellular esterase activity prior to cell death.

Reactive metabolites produced by oxidative metabolism of the parent compound are considered responsible for the toxicity of a number of drugs, including idiosyncratic reactions to sulfonamide antibiotics. Using sulfamethoxazole hydroxylamine (SMX-HA) as a model compound, we report the use of a pH-sensitive fluorescent probe, 2',7'-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF), to identify early subcellular targets of chemically synthesized, toxic drug metabolites in peripheral blood mononuclear cells. When toxicity was assessed with this probe immediately after a 2-hr drug challenge, SMX-HA produced a concentration-dependent decrease in cellular fluorescence which was not accompanied by the development of compromised cell membrane integrity until 18 hr later. Dissipation of pH gradients across the cell membrane with nigericin and monensin demonstrated that decreased intracellular pH was only a small component of SMX-HA-induced toxicity. Loading cells with BCECF 30 min prior to SMX-HA challenge produced only a 3% decrease in cellular fluorescence at an SMX-HA concentration of 1 mM, whereas addition of BCECF after drug challenge resulted in a 71% decrease in fluorescence, consistent with a direct drug effect on cellular esterase activity. This was confirmed by monitoring BCECF cleavage in cell lysates in the presence and absence of SMX-HA. These studies demonstrate that inhibition of cellular esterase activity accounted for the observed loss of cellular fluorescence after drug exposure. Since changes in cellular fluorescence at 2 hr correlated well with cell death at 18 hr, we conclude that SMX-HA inhibition of intracellular esterase activity is an early event in the process that terminates in metabolite-induced cell death.

Cellular toxicity of sulfamethoxazole reactive metabolites--II. Inhibition of natural killer activity in human peripheral blood mononuclear cells.

Based on the identification of intracellular esterase activity as one early target of sulfamethoxazole hydroxylamine (SMX-HA), we wished to determine if the metabolite affected immune functions which involve esterases. The natural killer (NK) activity of human peripheral blood mononuclear cells (PBMC) was assessed with a cell concentration fluorescence technique following exposure to SMX-HA. When K562 target cells were incubated (4 hr/37 degrees) with various ratios of untreated PBMC effector to K562 target cells (E:T), NK activity increased from 17.8 +/- 3.1% (mean +/- SEM; N = 12) at an E:T ratio of 5:1 to 46.2 +/- 2.0% at an E:T ratio of 40:1. Pretreatment of fresh PBMC with 0.1 to 1.0 mM SMX-HA produced a concentration-dependent inhibition of NK activity (E:T ratio 40:1) reaching approximately 80% at 1 mM SMX-HA. Maximum suppression of NK activity was completed within a 60-min pretreatment period with measurable inhibition detected within 30 min. The viability of effector cells was not affected by the metabolite during the pretreatment period. Therefore, the SMX-HA effects could not be directly attributed to decreased viability of the effector cells; they were irreversible and could be prevented by the inclusion of exogenous reduced glutathione (GSH) in a concentration-dependent manner. Given the important roles of NK cells in immune responsiveness and host resistance, our findings of rapid functional inactivation of the cytolytic effector function provide a possible link between idiosyncratic drug toxicity and drug effects directly on components of the immune system.

Cytosolic arylamine N-acetyltransferase (NAT) deficiency in the dog and other canids due to an absence of NAT genes.

The purpose of this study was to determine the molecular basis in the dog for an unusual and absolute deficiency in the activity of cytosolic N-acetyltransferase (NAT), an enzyme important for the metabolism of arylamine and hydrazine compounds. NAT activity towards two NAT substrates, p-aminobenzoic acid and sulfamethazine, was undetectable in dog liver cytosol, despite substrate concentrations ranging from 10 microM to 4 mM and a wide range of incubation times. Similarly, no protein immunoreactive to NAT antibody was evident on western blot analysis of canine liver cytosol. Southern blot analysis of genomic DNA from a total of twenty-five purebred and mixed bred dogs, and eight wild canids, probed with a full-length human NAT2 cDNA, suggested an absence of NAT sequences in all canids. Polymerase chain reaction amplification of genomic DNA using degenerate primers designed to mammalian NAT1 and NAT2 consensus sequences generated products of the expected size in human, mouse, rabbit, and cat DNA, but no NAT products in any dog or wild canids. These results support the conclusion that cytosolic NAT deficiency in the domestic dog is due to a complete absence of NAT genes, and that this defect is shared by other canids.

Role of polymorphic and monomorphic human arylamine N-acetyltransferases in determining sulfamethoxazole metabolism.

Sulfonamides are associated with a variety of adverse reactions, some of which have been linked with the classical acetylator phenotypes. Although the slow acetylator phenotype has been identified as a risk factor for hypersensitivity reactions to sulfamethoxazole (SMX), the disposition of this compound appears not to be affected by the acetylation polymorphism in vivo in humans. We therefore investigated the acetylation of SMX by monomorphic (NAT1) and polymorphic (NAT2) arylamine N-acetyltransferases in humans with the objective of determining their role in the metabolism of SMX. SMX was acetylated by both NAT1 and NAT2. Km values determined in hepatic cytosol for NAT1- and NAT2-mediated acetylation of SMX were 1.2 mM and approximately 5 mM, respectively, at an acetyl coenzyme A concentration of 100 microM. Mononuclear leukocytes, which contain only NAT1, had a Km value of 1.2 mM. Km values determined with recombinant NAT1 and NAT2 proteins expressed in Escherichia coli were 1.5 mM and approximately 15 mM, respectively. The higher affinity of NAT1 for SMX indicates that acetylation by this enzyme will predominate at therapeutic plasma concentrations, in agreement with the observed in vivo monomorphic acetylation of SMX. NAT1 may be the primary determinant of SMX systemic metabolic clearance. However, in the hepatocyte NAT2 variation may be an important competitive pathway which influences the extent of oxidative metabolism of SMX to its reactive hydroxylamine metabolite. Therefore, variation in both monomorphic and polymorphic N-acetyltransferases may play a role in determining susceptibility to sulfamethoxazole toxicity.

The caffeine breath test and caffeine urinary metabolite ratios in the Michigan cohort exposed to polybrominated biphenyls: a preliminary study.

A field biochemical epidemiology study was conducted using the Michigan cohort consisting of 51 rural residents exposed to polybrominated biphenyls (PBB). The study had three major objectives: a) to determine the serum half-life of the major PBB congener, hexabromobiphenyl (HBB), in the human, b) to determine if the PBB-exposed subjects had elevated cytochrome P-450I function as determined by the caffeine breath test (CBT) and the caffeine urinary metabolite ratio (CMR), and c) to determine the applicability of the CBT and CMR in field studies. PBB serum levels were detected in 36 of the 51 PBB-exposed subjects. The serum half-life of HBB was determined by comparing the current serum HBB values to the subject's previous serum values obtained 5 to 8 years earlier. The median HBB half-life was 12 years (range 4-97 years). The CBT and CMR were elevated in the subjects exposed to PBBs as compared to the values obtained from urban nonsmokers and were similar to those found in adults who smoke. A gender effect was seen in the PBB-exposed subjects, the median CBT and CMR values of the females being lower than the values of males. There was a correlation between the CBT and the HBB serum values (r2 = 0.2, p = 0.01) but not between CMR and HBB serum values. The CBT and CMR were easily conducted in the field and appear to be useful metabolic probes of cytochrome P-450I activity in human environmental toxicology.