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Background: Polymorphous light eruption (PLE) is the most common chronic and idiopathic photodermatosis. PLE is assumed to represent an immunological hypersensitivity reaction to a radiation-induced cutaneous antigen involving reactive oxygen species (ROS) on the basis of a genetic predisposition. Among others, cellular protection against ROS is provided by glutathione S-transferases (GSTs). Different variants of the GST enzymes may influence the activity and efficiency of detoxification and biotransformation of unknown UV-induced skin-antigens and other factors that may play an important role in the pathogenesis of PLE., Methods: In this study the relationship between isoenzymes of the GST genes GSTM1, GSTT1 and GSTP1 and possible protective or predisposing effects on PLE was examined in 29 patients and 144 controls. Diagnosis of PLE was based on the presence of characteristic clinical features., Results: No association between the functional polymorphisms of the GST gene family and PLE was found. Prevalence of certain GST isoenzymes or polymorphisms in patients with PLE did not differ from healthy controls., Conclusion: Our data do not support prevalence of GST isoenzymes or polymorphisms as a protective effect against PLE. Especially a higher carrier frequency of GSTP1 Val(105) as a protective factor against PLE which has been published before could not be proved. The GST genotypes GSTM1, GSTT1 and GSTP1 (including SNPs) seem to have no relevant association with PLE., (© 2012 The Authors. Journal of the European Academy of Dermatology and Venereology © 2012 European Academy of Dermatology and Venereology.)

Ketamine is an anesthetic and analgesic regularly used in veterinary patients. As ketamine is almost always administered in combination with other drugs, interactions between ketamine and other drugs bear the risk of either adverse effects or diminished efficacy. Since cytochrome P450 enzymes (CYPs) play a pivotal role in the phase I metabolism of the majority of all marketed drugs, drug-drug interactions often occur at the active site of these enzymes. CYPs have been thoroughly examined in humans and laboratory animals, but little is known about equine CYPs. The characterization of equine CYPs is essential for a better understanding of drug metabolism in horses. We report annotation, cloning and heterologous expression of the equine CYP2B6 in V79 Chinese hamster fibroblasts. After computational annotation of all CYP2B genes, the coding sequence (CDS) of equine CYP2B6 was amplified by RT-PCR from horse liver total RNA and revealed an amino acid sequence identity of 77% and a similarity of 93.7% to its human ortholog. A non-synonymous variant c.226G>A in exon 2 of the equine CYP2B6 was detected in 97 horses. The mutant A-allele showed an allele frequency of 82%. Two further variants in exon 3 were detected in one and two horses of this group, respectively. Transfected V79 cells were incubated with racemic ketamine and norketamine as probe substrates to determine metabolic activity. The recombinant equine CYP2B6 N-demethylated ketamine to norketamine and produced metabolites of norketamine, such as hydroxylated norketamines and 5,6-dehydronorketamine. V(max) for S-/and R-norketamine formation was 0.49 and 0.45nmol/h/mg cellular protein and K(m) was 3.41 and 2.66μM, respectively. The N-demethylation of S-/R-ketamine was inhibited concentration-dependently with clopidogrel showing an IC(50) of 5.63 and 6.26μM, respectively. The functional importance of the recorded genetic variants remains to be explored. Equine CYP2B6 was determined to be a CYP enzyme involved in ketamine and norketamine metabolism, thus confirming results from inhibition studies with horse liver microsomes. Clopidogrel seems to be a feasible inhibitor for equine CYP2B6. The specificity still needs to be established with other single equine CYPs. Heterologous expression of single equine CYP enzymes opens new possibilities to substantially improve the understanding of drug metabolism and drug interactions in horses., (Copyright © 2012 Elsevier Inc. All rights reserved.)

History and Admission Findings: Seventeen East-European workers with a suspected lead-intoxication presented themselves to the Department of Toxicology. All of them had worked on the renovation of pylons of a high-tension line. The old paint, known to contain lead was removed with needle descalers. The patients had blood lead concentrations between 325 and 1124 microg/l, but no specific symptoms. The workers neglected the protective measures at their working-place., Investigations: 12 of 17 workers had lead-concentrations above 400 microg/l (Reference < 90 microg/l). 10 of 17 patients showed an increased level of free protoporphyrins and all workers showed a decreased activity of delta-aminolaevulinacid-dehydratase (ALAD)., Treatment and Course: Patients with lead-concentration above 700 microg/l were treated with the chelating agent meso-2,3-dimercaptosuccinic acid (DMSA) 3 x 200 mg/d for nine days. The patients with lead concentrations between 400 and 700 microg/l were treated which DMSA 3 x 100 mg/d. After the DMSA-treatment the lead-concentrations had dropped (p < 0.001). During the DMSA-therapy one patient had to be treated in the hospital because of a generalised allergic exanthema., Conclusion: We report seventeen patients with high lead concentration in their blood due to occupational exposure. The high blood lead levels showed that the workers had not been protected adequately. This examplifies that occupational lead exposure still occurs, also in Germany. By patients with unspecific symptoms connected with lead exposure a biomonitoring for lead is necessary.

Hybridomas were isolated that produce 13 monoclonal antibodies (mAbs) that are specific and highly inhibitory to members of the human P450 2C subfamily, 2C8, 2C9, 2C9*2, and 2C19. Many of the mAbs to P450 2C8, 2C9, and 2C19 are specific and exhibit potent inhibitory activity (85-95%). mAb 281-1-1 specifically binds, immunoblots, and strongly inhibits the activity of P450 2C8. mAb 763-15-5 specifically binds and strongly inhibits the activity of P450 2C9. mAb 1-7-4-8 specifically binds and strongly inhibits the activity of P450 2C19. The other mAbs bind and inhibit sets and subsets of the P450 2C family. The single and the combinatorial use of the mAbs can "reaction phenotype", i.e., determine the metabolic contribution and interindividual variation of a P450 isoform for the metabolism of a drug or nondrug xenobiotic in human liver microsomes. The utility of the mAb-based analytic system was examined with the model substrates Taxol (paclitaxel), diazepam, tolbutamide, diclofenac, mephenytoin, and imipramine. The mAb system can identify drugs metabolized by a common P450 or several P450s and polymorphic P450s. The mAb system identifies drugs or drug metabolic pathways that are catalyzed by a single P450 and thus may be used for in vivo phenotyping. The mAb system can identify whether a particular drug is metabolized by a single P450 that may exhibit polymorphic expression in humans. The mAb system offers large potential for studies of cytochrome P450 function useful in drug discovery and reduces the possibility of adverse drug reactions due to polymorphisms and drug interactions.

Humans are exposed to polycyclic aromatic hydrocarbons (PAHs) through many environmental pollutants, especially cigarette smoke. These chemicals cause a variety of tumors and immunotoxic effects, as a consequence of bioactivation by P-450 cytochromes to dihydrodiol epoxides. The recently identified cytochrome P4501B1 (CYP1B1) bioactivates PAHs but is also a physiological regulator, as evidenced by linkage of CYP1B1 deficiency to congenital human glaucoma. This investigation demonstrates that CYP1B1 null mice are almost completely protected from the acute bone marrow cytotoxic and preleukemic effects of the prototypic PAH 7,12-dimethylbenz[a]anthracene (DMBA). CYP1B1 null mice did not produce the appreciable amounts of bone marrow DMBA dihydrodiol epoxide DNA adducts present in wild-type mice, despite comparable hepatic inductions of the prominent PAH-metabolizing P-450 cytochrome, CYP1A1. Wild-type mice constitutively expressed low levels of bone marrow CYP1B1. These findings suggest that CYP1B1 is responsible for the formation of DMBA dihydrodiol epoxides in the bone marrow. Furthermore, this study substantiates the importance of DMBA dihydrodiol epoxide generation at the site of cancer initiation and suggests that tissue-specific constitutive CYP1B1 expression may contribute to cancer susceptibility in the human population.

We previously demonstrated that murine bone marrow stromal cells express high levels of cytochrome P4501B1 (CYP1B1) that metabolizes 7,12-dimethylbenza[a]anthracene (DMBA), and that DMBA activates the Ah receptor (AhR) in these cells in vitro. More recently, we reported that CYP1B1 is required for DMBA-induced lymphoblastoma formation in vivo. In this study, we addressed the hypothesis that bone marrow stromal cell CYP1B1, and not AhR activation, is required for DMBA-induced pre-B-cell apoptosis. Although DMBA did not directly cause apoptosis in pre-B cells, dose-dependent apoptosis of pre-B cells was observed when they were cocultured with a bone marrow stromal cell line. The DMBA 3,4-dihydrodiol metabolite was more potent in effecting pre-B-cell apoptosis than DMBA, whereas the potent AhR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin was inactive. Both pre-B cells and bone marrow stromal cells contained DMBA-diol-epoxide DNA adducts, indicating that reactive metabolites were transferred from stromal cells to pre-B cells. DMBA caused apoptosis when cocultured with primary bone marrow stromal cells isolated from AhR-null mice but not CYP1B1-null mice. When cocultured with AhR-null primary bone marrow stromal cells, DMBA induced approximately 50% of the pre-B-cell apoptosis seen with stromal cells from AhR-heterozygous mice. This reduced level of apoptosis parallels the decreased CYP1B1 expression in AhR-null mouse bone marrow stromal cells. These findings provide convincing evidence that bone marrow stromal cell CYP1B1 metabolism of DMBA, but not AhR activation, is required for DMBA-induced pre-B-cell apoptosis.

CYP1B1-null mice, created by targeted gene disruption in embryonic stem cells, were born at the expected frequency from heterozygous matings with no observable phenotype, thus establishing that CYP1B1 is not required for mouse development. CYP1B1 was not detectable in cultured embryonic fibroblast (EF) or in different tissues, such as lung, of the CYP1B1-null mouse treated with the aryl hydrocarbon receptor agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin whereas the equivalent wild-type EF cells express basal and substantial inducible CYP1B1 and lung expresses inducible CYP1B1. CYP1A1 is induced to far higher levels than CYP1B1 in liver, kidney, and lung in wild-type mice and is induced to a similar extent in CYP1B1-null mice. 7,12-dimethylbenz[a]anthracene (DMBA) was toxic in wild-type EFs that express CYP1B1 but not CYP1A1. These cells effectively metabolized DMBA, consistent with CYP1B1 involvement in producing the procarcinogenic 3,4-dihydrodiol as a major metabolite, whereas CYP1B1-null EF showed no significant metabolism and were resistant to DMBA-mediated toxicity. When wild-type mice were administered high levels of DMBA intragastrically, 70% developed highly malignant lymphomas whereas only 7.5% of CYP1B1-null mice had lymphomas. Skin hyperplasia and tumors were also more frequent in wild-type mice. These results establish that CYP1B1, located exclusively at extrahepatic sites, mediates the carcinogenicity of DMBA. Surprisingly, CYP1A1, which has a high rate of DMBA metabolism in vitro, is not sufficient for this carcinogenesis, which demonstrates the importance of extrahepatic P450s in determining susceptibility to chemical carcinogens and validates the search for associations between P450 expression and cancer risk in humans.

Acetaminophen (APAP) hepatotoxicity is due to its biotransformation to a reactive metabolite, N-acetyl-p-benzoquinone imine (NAPQI), that is capable of binding to cellular macromolecules. At least two forms of cytochrome P450, CYP2E1 and CYP1A2, have been implicated in this reaction in mice. To test the combined roles of CYP1A2 and CYP2E1 in an intact animal model, a double-null mouse line lacking functional expression of CYP1A2 and CYP2E1 was produced by cross-breeding Cyp1a2-/- mice with Cyp2e1-/- mice. Animals deficient in the expression of both P450s developed normally and exhibited no obvious phenotypic abnormalities. Comparison of the dose-response to APAP (200-1200 mg/kg) indicated that double-null animals were highly resistant to APAP-induced toxicity whereas the wild-type animals were sensitive. Administration of 600 to 800 mg/kg of this drug to male wild-type animals resulted in increased plasma concentrations of liver enzymes (alanine aminotransferase, sorbitol dehydrogenase), lipidosis, hepatic necrosis, and renal tubular necrosis. In contrast, when APAP of equivalent or higher dose was administered to the double-null mice, plasma levels of liver enzymes and liver histopathology were normal. However, administration of 1200 mg of APAP/kg to the double-null mice resulted in infrequent liver lipidosis and mild kidney lesions. Consistent with the protection from hepatotoxicity, the expected depletion of hepatic glutathione (GSH) content was significantly retarded and APAP covalent binding to hepatic cytosolic proteins was not detectable in the double-null mice. Likewise, in vitro activation of APAP by liver microsomes from the double-null mice was approximately one tenth of that in microsomes from wild-type mice. Thus, the protection against APAP toxicity afforded by deletion of both CYP2E1 and CYP1A2 likely reflects greatly diminished production of the toxic electrophile, NAPQI., (Copyright 1998 Academic Press.)

Heterologous expression using baculovirus vectors has become a popular method for the production of catalytically active cytochrome P450s (CYPs). We have systematically optimized the multiplicity of infection (MOI) for a coinfection approach for the coexpression of CYP2A6 (viral vector designated v2A6) and NADPH-P450 oxidoreductase (OR; viral vector designated vOR) using Sf9 insect cells. A 3000-fold range of MOI was examined in stationary culture and stirred suspension culture. Surprisingly, our results indicate that the best CYP2A6 catalytic activity (850-1300 pmol/ min/mg total lysate protein as measured by coumarin 7-hydroxylase activity) was obtained only when using a low MOI of v2A6 (1.5-3 x 10(-2)) and a vOR of 10- to 20-fold less. This activity was approximately 7- to 11-fold higher than the best activity obtained when infecting cells with v2A6 alone. At this level of coinfection, the P450 content ranged from 180 to 250 pmol/mg total lysate protein, and the NADPH cytochrome c reductase activity ranged from 350 to 520 nmol/min/mg total lysate protein. Increasing the MOI of both viruses to 50-fold higher resulted in lower overall activity with the optimum (250 pmol/min/mg total lysate protein) being seen earlier postinfection (60 vs. 72 hr). Increasing the MOI of vOR to levels comparable with those of v2A6, decreased coumarin 7-hydroxylase activity 14-fold. These results suggest that the best CYP2A6 catalytic activity depends on properly posttranslationally modified proteins accumulating in a right ratio as a result of primary, secondary, and possibly tertiary infection of both viruses. These results also suggest that high OR expression results in degradation of P450.

The kinetics of CO binding to human cytochrome P450 1A1 was used to probe the interaction of polycyclic aromatic hydrocarbons (PAHs) with the membrane-bound P450 expressed in baculovirus-infected SF9 insect cells. Biexponential kinetics was observed, indicating that P450 1A1 is composed of at least two kinetically distinguishable species. To define the substrate specificity of the individual species, we evaluated the effect of a series of PAHs of varying sizes and shapes on the CO binding kinetics of P450 1A1. The overall rate of CO binding was increased in the presence of the tricyclic PAHs phenanthrene and anthracene and the tetracyclic PAHs pyrene and 1,2-benzanthracene, but was decreased by the pentacyclic PAHs benzo[a]pyrene and 1,2:3,4-dibenzanthracene. A kinetic difference method was applied to kinetically define the individual P450 1A1 species. Two species differing in their PAH specificities were identified: a slowly reacting species sensitive to the smaller PAHs, and a rapidly reacting species responsive to larger PAHs. Upon PAH binding, CO binding to these species was accelerated and decelerated, respectively. The results furthermore suggest that the two species are interconvertable. In addition to PAHs, the interactions of P450 1A1 with 7-ethoxy- and 7-pentoxyresorufin were likewise examined for their effect on the CO binding kinetics. These compounds interacted with and decreased the rate of the rapidly and slowly reacting P450 1A1 species, respectively. The markedly variable effects of these PAHs and resorufins on the CO binding kinetics indicate differential modes of interaction with the two target P450 1A1 species, resulting in differential modulation of their conformations. These results demonstrate that multiple P450 1A1 species with distinct conformations and substrate recognition profiles coexist in a biological membrane and are resolvable using a rapid kinetic technique.

7,12-Dimethylbenz[a]anthracene (DMBA), a potent carcinogen, requires metabolic activation by cytochrome P450s (P450s) to electrophilic metabolites that result in DNA modification, mutagenicity, and carcinogenicity. In this study, we used eight human forms, four rodent forms, and one rabbit form of P450 expressed from recombinant vaccinia or baculovirus vectors to define their specificity for metabolizing DMBA. Of the eight human P450s, 1A1 was the most active (specific activity = 14.7 nmol/min/nmol of P450) in total metabolism of DMBA and showed approximately 6- to 33-fold more activity than other P450s, 2B6, 2C9, and 1A2 were also capable of metabolizing DMBA (2.0-2.5 nmol/min/nmol of P450), whereas 2C8, 2E1, 3A4, and 3A5 exhibited relatively low activities. Among animal P450s, mouse 1A1 exhibited activity similar to that of human 1A1 and had 5.0- to 37-fold more activity than other rodent and rabbit P450s. In regard to enzyme regioselectivity, most human and rodent P450s predominantly formed the 8,9-diol, but human 2B6 and rat 2B1 preferentially formed the 5,6-diol. In the production of monohydroxymethyl metabolites, all the enzymes yielded more 7-hydroxymethyl-12-methylbenz[a]anthracene (7HOM12MBA) than 12-hydroxymethyl-7-methylbenz[a]anthracene (7M12HOMBA), except for human 1A1, which presented the reverse selectivity. Human liver microsomes from 10 organ donors were shown to metabolize DMBA and in most circumstances generated the metabolic profile DMBA trans-8,9-dihydrodiol > 7HOM12MBA > or = DMBA trans-5,6-dihydrodiol > or = 7,12-dihydroxymethylbenz[a]anthracene > 7M12HOMBA > DMBA trans-3,4-dihydrodiol. Thus, the combined activity of hepatic microsomal 2C9, 1A2, and 2B6 may contribute to the metabolic activation and the metabolism of DMBA in normal human liver.

We investigated the involvement of CYP1A2 in the pharmacokinetics and metabolism of caffeine using mice lacking its expression (CYP1A2 -/-). The half-life of caffeine elimination from blood was seven times longer in the CYP1A2 -/- than wild-type mice. The clearance was concomitantly eight times slower. No parameter that could affect the pharmacokinetics differed between CYP1A2-/-and wild-type mice such as creatinine for kidney function; alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase and bilirubin for liver function; or albumin for protein binding. Other P450s CYP2A, 2B, 2C, 2EI, and 3A were also unchanged in the knockout animals. Caffeine 3-demethylated metabolites thought previously to be characteristic of CYP1A2 (especially 1-methylxanthine and I-methylurate) were also found in the urines of the CYP1A2-/-animals, although at 40% of the level found in wild-type mice. These data indicate that the clearance of caffeine in wild-type mice is primarily determined by CYP1A2.

Loss of or mutations in p53 protein have been shown to decrease both radio- and chemosensitivity. The present study assessed the p53 gene status, ability to arrest in G1 of the cell cycle, the functionality of the p53 transduction pathway, and apoptosis following treatment with radiation in a series of drug-resistant human breast cancer cells to determine whether p53 alterations occur during the development of drug resistance. We used 13 sublines derived from MCF-7, ZR75B, and T47D cells, which were resistant to doxorubicin, paclitaxel, vinblastine, cisplatin, etoposide, and amsacrine. Eleven of 12 drug-resistant sublines retained the parental p53 gene status, as determined by sequence analysis and functional yeast assay; only one subline was found to have acquired a mutation in the p53 gene. The MCF-7 TH subline was found to both acquire mutated p53 and to have major changes in p53 protein expression and function. In 12 other drug-resistant sublines, the G1 checkpoint was conserved or only slightly impaired. A normal accumulation of p53, p21Cip1/Waf1, and Mdm2 proteins and hypophosphorylation of Rb protein occurred in response to radiation with only small differences noted in the kinetics of p53 and p21Cip1/Waf1 induction. Increased susceptibility to apoptosis was found in the ZR75B drug-resistant sublines, whereas no evidence for apoptosis was observed in the ZR75B, MCF-7, and T47D parentals and the MCF-7 and T47D drug-resistant sublines. This effect could not be explained by alterations in bcl-2 or bax expression. Our results demonstrate that alterations in: (a) p53 gene status; (b) ability to arrest in G1; (c) induction of p53 protein and p53-dependent genes; and (d) decreased activation of apoptosis is not a requirement for the onset of drug resistance. The function of p53 appears to be dissociated from drug resistance in our model system.

High-level expression of three cloned cytochrome P450 enzymes was accomplished using the baculovirus-insect cell expression system. The amount of enzyme expression was enhanced by cell infections in the presence of medium-supplements containing hemin and by growth in suspension cultures. Human cytochromes P450 2E1 and 2C9 and rat cytochrome P450 2A1 were partially purified from cell extracts using hydrophobic interaction and hydroxyapatite chromatography. The resulting enzymes were of estimated molecular masses similar to those reported previously and analyzed by PAGE. Reconstitution of enzyme activity resulted when the enzymes were incubated together with NADPH-cytochrome P450 reductase, phospholipid, NADPH, and appropriate substrates. The cytochrome P450 activity of the partially purified enzymes was comparable to that of the corresponding enzymes expressed in the vaccinia virus-Hep G2 system. These results provide evidence for a general means of obtaining cytochrome P450 enzymes for mechanistic, immunochemical, and biophysical investigations.

We have analysed kinetic parameters of cDNA-derived CYP2D6 proteins derived from the original CYP2D6 cDNA isolate (Gonzalez FJ et al. Nature 1988: 331, 442-446) which contains methionine at position 374 (CYP2D6-Met) and a modified cDNA which contains valine at position 374 (CYP2D6-Val). This latter protein is predicted from the CYP2D6 genomic sequence. Several quantitative differences, but no qualitative differences in metabolism were observed. CYP2D6-Met was found to have a two-fold lower Km and a three-fold lower turnover rate for (R)(+)-bufuralol 1'-hydroxylation as compared to CYP2D6-Val. In contrast, CYP2D6-Met and CYP2D6-Val had a similar Km for debrisoquine 4-hydroxylation while CYP2D6-Val had an 18-fold higher turnover rate. CYP2D6-Val and CYP2D6-Met had similar Kms for metoprolol but CYP2D6-Val showed a three-fold higher capacity for the O-demethylation reaction compared to alpha-hydroxylation which is more similar to that seen in human liver. In the case of sparteine, CYP2D6-Val and CYP2D6-Met showed similar capacities for formation of the 2-dehydrosparteine metabolite but the Km value for CYP2D6-Met was six-fold higher than that for CYP2D6-Val. Kinetic differences between CYP2D6-Met and CYP2D6-Val were further probed by examination of apparent Ki for inhibition of (R,S)(+/-)-bufuralol 1'-hydroxylation. Similar Ki values (within a factor of three) were observed for perhexiline and (R,S)-propranolol while quinidine and dextromethorphan were 8.5-fold and 21-fold more effective inhibitors of CYP2D6-Val relative to CYP2D6-Met. An allele specific polymerase chain reaction assay was developed for the CYP2D6-Met allele. The CYP2D6-Met allele was not found among 83 individuals. In the aggregate, these data indicated that the CYP2D6-Val allele is the more common allele in human populations. The quantitative kinetic differences between these two enzymes appears most pronounced for substrates/inhibitors with rigid structures. CYP2D6-Val more often has a substantially lower Km and/or a substantially higher capacity to metabolize those substrates.

A recombinant baculovirus containing the human CYP3A4 cDNA was constructed and used to express CYP3A4 in SF9 insect cells (0.46 +/- 0.13 nmol/mg protein, 103 +/- 29 nmol/liter, N = 15). The enzyme represented approximately 2-3% of total cellular protein and could be purified by a two-column procedure to a specific content of 12.7 nmol/mg protein. Catalytic activity of the purified enzyme after reconstitution was optimum using molar ratios of CYP3A4 to cytochrome b5 to NADPH-P450 oxidoreductase of 1:3:20, respectively. The enzyme metabolized cortisol, erythromycin, testosterone, and (R)-warfarin. Recombinant baculovirus expresses the highest amounts of all expression systems published to date of catalytically intact CYP3A4. This system is an excellent alternative for the isolation and characterization of P450 forms from human liver.

Antipyrine metabolism depends on at least three isoenzymes of cytochrome P450 forming the main metabolites 3-OH-, 4-OH- and norantipyrine. We investigated to which extent antipyrine clearance and metabolite formation in vivo correlate with metabolite formation by microsomal fractions in vitro. The influence of sex was investigated in two rat strains. Antipyrine clearance in saliva was determined in 10-month-old Sprague-Dawley and Dark Agouti rats of either sex. Antipyrine and its metabolites in urine and microsomes were measured by a new HPLC method after solid phase or liquid extraction. Antipyrine clearance was 46% higher in males than in female rats. This was associated with a 40% higher urinary excretion of 3-OH-antipyrine in the male rats, the other metabolites being excreted to a similar extent. This higher production of 3-OH-antipyrine in vivo was paralleled by a higher intrinsic clearance in vitro while no sex difference in intrinsic clearance for the formation of the other metabolites was seen. The correlation between in vivo and in vitro metabolic clearance for 3-OH-antipyrine was good (r = 0.75) but unconvincing for 4-OH- (r = 0.49) and norantipyrine (r = 0.01). This could be due to further metabolism of 4-OH- and norantipyrine.