Biofilm of Candida albicans: formation, regulation and resistance.

Candida albicans is the most common human fungal pathogen, causing infections that range from mucous membranes to systemic infections. The present article provides an overview of C. albicans, with the production of biofilms produced by this fungus, as well as reporting the classes of antifungals used to fight such infections, together with the resistance mechanisms to these drugs. Candida albicans is highly adaptable, enabling the transition from commensal to pathogen due to a repertoire of virulence factors. Specifically, the ability to change morphology and form biofilms is central to the pathogenesis of C. albicans. Indeed, most infections by this pathogen are associated with the formation of biofilms on surfaces of hosts or medical devices, causing high morbidity and mortality. Significantly, biofilms formed by C. albicans are inherently tolerant to antimicrobial therapy, so the susceptibility of C. albicans biofilms to current therapeutic agents remains low. Therefore, it is difficult to predict which molecules will emerge as new clinical antifungals. The biofilm formation of C. albicans has been causing impacts on susceptibility to antifungals, leading to resistance, which demonstrates the importance of research aimed at the prevention and control of these clinical microbial communities.

Croton sonderianus essential oil samples distinctly affect rat airway smooth muscle.

Plants of the genus Croton have been used extensively in the northeast of Brazil for treating various clinical conditions. Previous studies have demonstrated that the essential oil of some specimens of Croton sp. have a relaxing effect on tracheal smooth muscle. Our study aimed to characterize the effects of Croton sonderianus essential oil samples, collected at 1:00 pm (EO-13) and 9:00 pm (EO-21), on rat tracheal smooth muscle. The two samples were submitted to gas chromatography (GC) and mass spectrometry (MS) analysis to identify their components. Rat tracheal smooth muscle strips were used to assess the biological activity. The major constituents of EO-21 were: spathulenol (18.32%), beta-caryophyllene (14.58%) and caryophyllene oxide (8.54%) and the major constituents of EO-13 were bicyclogermacrene (16.29%), beta-phellandrene (15.42%) and beta-caryophyllene (13.82%). These samples showed an antispasmodic effect on tracheal smooth muscle strips pre-contracted with high K+ concentration (80 mM) or with acetylcholine. EO-21 increased baseline tonus while EO-13 provoked a decrease. These results demonstrated that EO-13 and EO-21 have different chemical composition and showed myorelaxant activity. In conclusion, EO-13 and EO-21 may have potential therapeutic use in the treatment of bronchospasm.

Effect of human renal cationic transporter inhibition on the pharmacokinetics of varenicline, a new therapy for smoking cessation: an in vitro-in vivo study.

Varenicline is predominantly eliminated unchanged in urine, and active tubular secretion partially contributes to its renal elimination. Transporter inhibition assays using human embryonic kidney 293 cells transfected with human renal transporters demonstrated that high concentrations of varenicline inhibited substrate uptake by hOCT2 (IC(50)=890 microM), with very weak or no measurable interactions with the other transporters hOAT1, hOAT3, hOCTN1, and hOCTN2. Varenicline was characterized as a moderate-affinity substrate for hOCT2 (K(m)=370 microM) and its hOCT2-mediated uptake was partially inhibited by cimetidine. Co-administration of cimetidine (1,200 mg/day) reduced the renal clearance of varenicline in 12 smokers, resulting in a 29.0% (90% CI: 21.5%-36.9%) increase in systemic exposure. This increase is not considered clinically relevant, as it should not give rise to safety concerns. Consequently, it can be reasonably expected that other inhibitors of hOCT2 would not cause greater renal interactions with varenicline than that seen with the efficient hOCT2 inhibitor cimetidine.

Effects of essential oil of Alpinia zerumbet on the compound action potential of the rat sciatic nerve.

Alpinia zerumbet, known popularly as "colônia" in Northeastern Brazil, is a medicinal plant that has been used widely in folk medicine as teas and infusions for the treatment of intestinal and cardiovascular diseases, including arterial hypertension. Our previous studies have demonstrated that the essential oil of A. zerumbet (OEAZ) is very active on excitable tissues, such as smooth muscle, and in this study we verified its effects on the compound action potential (CAP) of rat sciatic nerve. EOAZ induced a dose-dependent blockade of the CAP. Control peak-to-peak amplitude and conduction velocity of CAPs were 7.6 +/- 0.43 mV and 80.6 +/- 3.19 m/s, respectively. At 60 microg/ml, EOAZ induced no demonstrable effect. Conduction velocity was significantly reduced at 180 min of preparation exposure to 100 microg/ml of EOAZ. At 300, 600 and 2000 microg/ml doses of EOAZ, the peak-to-peak amplitudes of CAPs following 180 min exposure of the nerve to the drug were reduced significantly, to 75.3 +/- 7.36%, 50.45 +/- 2.17% and 0% respectively, of control value. Conduction velocity was reduced significantly by 300, 600 and 2000 microg/ml of EOAZ, at 180 min, to 83.61 +/- 3.28%, 64.06 +/- 8.21% and 22.7 +/- 5.79%, respectively, of control value. All these effects developed slowly and were reversible upon a 180-min wash.

A new genetic defect in human CYP2C19: mutation of the initiation codon is responsible for poor metabolism of S-mephenytoin.

The 4'-hydroxylation of the S-enantiomer of the anticonvulsant drug mephenytoin exhibits a genetic polymorphism in humans. This polymorphism shows marked interracial heterogeneity, with the poor metabolizer (PM) phenotype representing 2 to 5% of Caucasian and 13 to 23% of Asian populations. Two defective CYP2C19 alleles, CYP2C19*2 and CYP2C19*3, have been described which account for approximately 87% of Caucasian and > 99% of Oriental PM alleles. The present study identifies a new allele (CYP2C19*4) in Caucasian PMs which contains an A-->G mutation in the initiation codon. A new polymerase chain reaction-restriction fragment length polymorphism genotyping test was developed, and the incidence of this allele was examined in a European Caucasian population which had been phenotyped for mephenytoin metabolism. One of nine putative PMs was heterozygous for CYP2C19*2/CYP2C19*4, which suggests that CYP2C19*4 represents a defective allele. Six of the seven remaining putative PMs available for genotyping were explained by CYP2C19*2. The frequency of the CYP2C19*4 allele in Caucasians was 0.6%. An additional Caucasian PM from a separate study was also heterozygous for CYP2C19*2 and CYP2C19*4. To verify that CYP2C19*4 represented a defective CYP2C19 allele, the initiation codon of the normal CYP2C19*1 cDNA was mutated to a GTG, and both cDNAs were expressed in yeast. Recombinant CYP2C19 protein was detected by Western blot analysis of colonies transformed with CYP2C19*1 cDNA, but not in those transformed with CYP2C19*4 cDNA. The two cDNAs were also used in an in vitro coupled transcription/translation assay. CYP2C19 protein was translated only from the CYP2C19*1 allele. These data indicate that CYP2C19*4 represents a new PM allele.

Frequencies of the defective CYP2C19 alleles responsible for the mephenytoin poor metabolizer phenotype in various Oriental, Caucasian, Saudi Arabian and American black populations.

The 4'-hydroxylation of S-mephenytoin is polymorphic in man. The poor metabolizer (PM) phenotype exhibits a lower frequency in Caucasians (2-5%) compared to Oriental populations (13-23%). Previous studies from our laboratory have described two mutations (CYP2C19m1 and CYP2C19m2) which account for approximately 100% of Oriental and approximately 85% of Caucasian PM alleles. The present study examined whether the genotype predicted the phenotype in Japanese, Filipino and Saudi Arabian populations, and compared the frequencies of the defective CYP2C19 alleles in these populations with those found in European-Americans, Chinese-Taiwanese, and African-Americans from North Carolina. Among 53 Japanese, 15% were PMs and among 52 Filipinos 23% were PMs. Among 97 Saudi Arabians, only two were PMs. There was a complete concordance between genotype and phenotype in all three populations. The incidence of CYP2C19m1 was 0.23 (95% confidence limits 0.15-0.31) in Japanese, 0.39 (95% confidence limits 0.29-0.48) in Filipinos, 0.32 (95% confidence limits 0.26-0.38) in Chinese-Taiwanese, 0.15 (95% confidence limits 0.10-0.20) in Saudi Arabians, 0.13 (95% confidence limits 0.08-0.17) in European-Americans, and 0.25 in African-Americans from North Carolina (95% confidence limits (0.14-0.31). The incidence of CYP2C19m1 in Saudi Arabians was similar to that found in European-Americans, and significantly lower than that found in Oriental populations or African-Americans (p < 0.05). CYP2C19m2 was not found in European-Americans, Saudi Arabians or African-Americans (95% confidence limits 0-0.014). The incidence of CYP2C19m2 in the three Oriental populations ranged from 0.10 (95% confidence limits 0.05-0.17) in Japanese and 0.08 (95% confidence limits 0.03-0.13) in Filipinos to 0.06 (95% confidence limits 0.03-0.08) in Chinese-Taiwanese.

Genetic analysis of the S-mephenytoin polymorphism in a Chinese population.

The 4'-hydroxylation of S-mephenytoin exhibits a polymorphism in humans, with the poor metabolizer phenotype exhibiting a lower frequency in white (3% to 5%) than in Oriental populations (13% to 23%). Two mutations in CYP2C19 (CYP2C19m1 and CYP2C19m2) have recently been described that account for approximately 85% of white and 100% of Japanese poor metabolizers. This study examines whether these mutations account for the poor metabolizer phenotype in the Chinese population. The metabolism of S-mephenytoin exhibited a bimodal distribution in 244 unrelated Chinese subjects, although the distribution of the two phenotypes overlapped. In 75 selected Chinese subjects, CYP2C19 genotype analysis predicted the phenotype with 100% accuracy. The frequency of the poor metabolizer phenotype was approximately 11% (95% confidence interval 7% to 15%). The frequency of the CYP2C19m1 allele was 0.289, whereas that of CYP2C19m2 was 0.044. Homozygous extensive metabolizers had slightly lower ratios of S/R-mephenytoin compared with heterozygous extensive metabolizers, showing a gene-dosage effect. These data show the advantages of genotype analysis in investigations of the mephenytoin phenotype in Oriental subjects.

A multifamily study on the relationship between CYP2C19 genotype and s-mephenytoin oxidation phenotype.

It has recently been shown that the most common mutation (named m1) in both Caucasian and Japanese poor metabolizers (PM) of S-mephenytoin is a single base pair mutation (G-->A) in exon 5 of the CYP2C19 gene. In Japanese, a second defective allele of CYP2C19 named m2 consists of a G-->A mutation in exon 4. In the present study, we have investigated the inheritance of the CYP2C19 wild type allele (wt) and the two defective alleles (m1 and m2) in families of 11 Danish PM probands. The study was carried out for two principal reasons. First, we wanted to confirm the autosomal recessive inheritance of the defective alleles, and second, we wanted to examine the specificity and sensitivity of the CYP2C19 genotyping test. Individuals were phenotyped by measuring the ratio of S/R mephenytoin excreted in the urine after administration of mephenytoin, and genotyping was carried out by a PCR-based DNA amplification procedure. The genotypes of nine of the 11 probands were consistent with their phenotypes. Eight were homozygous m1/m1, and one was heterozygous m1/m2. The genotypes of two putative PM probands (wt/m1) were not consistent with their phenotypes. On the basis of extended phenotyping (additional late urine collections (24-36 h) and acidification of urine), one of these could probably be reclassified as an extensive metabolizer (EM) while the other was considered to be a true PM. This suggests the presence of an additional unknown mutant allele in the latter. Seven of the 41 phenotyped relatives in the 11 families were phenotyped as PMs, and with the exception of the father of family 10, their genotypes (m1/m1) were consistent with their phenotypes. Extended phenotyping (acidification of urine) suggested that the father of family 10 in fact is an EM and hence that his genotype (wt/m1) is concordant with his phenotype. Thus, the specificity of genotyping tests for PM was 100%, while the sensitivity was 15/16 or 94%. Our study provides unequivocal evidence for autosomal recessive inheritance of the PM trait.

Biochemistry and molecular biology of the human CYP2C subfamily.

The cytochromes P450 (CYP) are a superfamily of hemoproteins which metabolize foreign chemicals as well as a number of endogenous compounds such as steroids. The human CYP2C subfamily appears to principally metabolize a number of clinically used drugs. Four members of this subfamily have been identified in humans: CYP2C8, CYP2C9, CYP2C18, and CYP2C19. CYP2C9 is important in the metabolism certain of therapeutically used drugs including the anticoagulant drug warfarin and a number of nonsteroidal antiinflammatory drugs. A number of allelic variants of CYP2C9 exist in humans, but the effects of these allelic variants on metabolism in vivo remain to be determined. A well-characterized genetic polymorphism occurs in the 2C subfamily which is associated with the metabolism of the anticonvulsant drug mephenytoin. In population studies, individuals can be segregated into extensive and poor metabolizers of mephenytoin. Poor metabolizers are unable to 4'-hydroxylate the S-enantiomer of mephenytoin. There are marked interracial variations in the frequency of the poor metabolizer phenotype which represents 3-5% of Caucasians, but 18-23% of Oriental populations. The mechanism of this polymorphism has been recently elucidated. The enzyme responsible for S-mephenytoin metabolism has been shown to be CYP2C19, and two defects in the CYP2C19 gene have been described in poor metabolizers. The first defect, CYP2C19m1, consists of the creation of an aberrant splice site in exon 5. This defect accounts for approximately 75-85% of Caucasian and Japanese poor metabolizers. A second defect, CYP2C19m2, has been found only in Oriental populations and accounts for the remaining 25% of poor metabolizers in Japanese populations. The availability of genotyping tests for this polymorphism will enhance the assessment of the role of this pathway in clinical studies.

Identification of a new genetic defect responsible for the polymorphism of (S)-mephenytoin metabolism in Japanese.

A genetic polymorphism in the metabolism of the anticonvulsant drug (S)-mephenytoin has been well documented in humans. There are marked interracial differences in the frequency of the poor metabolizer phenotype, which comprises 2-5% of Caucasian but 18-23% of Asian populations. We have recently reported that the principal genetic defect responsible for the poor metabolizer phenotype is a single-base pair mutation in exon 5 of CYP2C19 (CYP2C19m), which accounts for approximately 75-83% of the defective alleles in both Japanese and Caucasians subjects. In the present study, we have identified a new mutation (CYP2C19m2) in Japanese poor metabolizers, consisting of a guanine to adenine mutation at position 636 of exon 4 of CYP2C19, which creates a premature stop codon. Genotyping of seven Japanese poor metabolizers who were not homozygous for the previously described CYP2C19m defect (now designated CYP2C19m1) indicated that they were either homozygous for the new defect (CYP2C19m2/CYP2C19m2) or heterozygous (CYP2C19m1/CYP2C19m2) for the two defects. CYP2C19m1 accounts for 25 of 34 alleles in Japanese poor metabolizers, whereas CYP2C19m2 accounts for the remaining nine alleles. Hence, CYP2C19m1 and CYP2C19m2 explain 100% of the available Japanese poor metabolizers (34 alleles). In contrast, the CYP2C19m2 defect was not detected in nine Caucasian poor metabolizers (83% of available poor metabolizer alleles were CYP2C19m1), indicating the existence of another, as yet unidentified, mutation. Genetic testing of the families of two Japanese poor metabolizer probands showed that coinheritance of the CYP2C19m1 and CYP2C19m2 alleles was concordant with the autosomal recessive inheritance of the poor metabolizer phenotype.

The major genetic defect responsible for the polymorphism of S-mephenytoin metabolism in humans.

The metabolism of the anticonvulsant drug mephenytoin exhibits a genetic polymorphism in humans, with the poor metabolizer trait being inherited in an autosomal recessive fashion. There are large interracial differences in the frequency of the poor metabolizer phenotype, with Oriental populations having a 5-fold greater frequency compared to Caucasians. Impaired metabolism of mephenytoin and a number of other currently used drugs results from a defect in a cytochrome P450 enzyme recently identified as CYP2C19. Attempts over the past decade to define the molecular genetic basis of the polymorphism have, however, been unsuccessful. We now report that the principal defect in poor metabolizers is a single base pair (G-->A) mutation in exon 5 of CYP2C19, which creates an aberrant splice site. This change alters the reading frame of the mRNA starting with amino acid 215 and produces a premature stop codon 20 amino acids downstream, which results in a truncated, non-functional protein. We further demonstrate that 7/10 Caucasian and 10/17 Japanese poor metabolizers are homozygous for this defect, indicating that this is the major defect responsible for the poor metabolizer phenotype. Finally, the familial inheritance of the deficient allele was found to be concordant with that of the phenotypic trait.

Photoaffinity labeling of the Ah receptor with 3-[3H]methylcholanthrene and formation of a 165-kDa complex between the ligand-binding subunit and a novel cytosolic protein.

The aromatic hydrocarbon (Ah) receptor is a cytosolic protein that binds halogenated ligands such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and nonhalogenated ligands such as 3-methylcholanthrene (MC) and benzo[a]pyrene. The best characterized biological response mediated by the Ah receptor is induction of cytochrome P4501A1 (CYP1A1). Photoaffinity labeling of the Ah receptor has been reported only with halogenated ligands such as TCDD and some of its iodinated derivatives. In this study, photolabeling of the Ah receptor was achieved with the nonhalogenated aromatic hydrocarbon [3H]MC. Sources of Ah receptor were the mouse hepatoma cell line Hepa-1c1c9 and the human colon adenocarcinoma line LS180. Cytosolic fractions either were used in a crude form or were enriched by glycerol density gradient centrifugation. These then were incubated with [3H]MC, irradiated with UV light (> 300 nm), precipitated with acetone, and analyzed by SDS-polyacrylamide gel electrophoresis. The yield of photoadduct formation was lower with [3H]MC (approximately 1%) compared with [3H]TCDD (3.5%) in Hepa-1c1c9 cells. The same was true in LS180 cells, i.e. the yield was 0.2% for [3H]MC versus 5.48 +/- 0.26% for [3H]TCDD. The relative molecular mass of the [3H]MC-labeled receptor estimated by SDS-polyacrylamide gel electrophoresis was 94,600 +/- 2,400 (mean +/- S.E.) for Hepa-1c1c9 cells and 113,600 +/- 3,200 for LS180 cells; these are the same molecular masses as determined by photolabeling with [3H]TCDD. In velocity sedimentation assays of mouse cytosol, [3H]MC binds specifically to two cytosolic proteins: the 4 S carcinogen-binding protein and the Ah receptor (9 S). However, no photolabeling of the 4 S protein was detected in our experiments. [3H]MC photolabeling of the human Ah receptor from LS180 cells was detected only in experiments using enriched cytosolic preparations. In addition to the 95-kDa ligand-binding subunit, a specifically radiolabeled protein of 164,900 +/- 5,800 kDa was also detected in Hepa-1c1c9 cytosol photolabeled with [3H]MC, suggesting cross-linking, by MC, of another subunit of the multimeric Ah receptor complex to the ligand-binding subunit. Immunochemical analysis showed that the ligand-binding subunit of the Ah receptor is one component of the 165-kDa complex. The other protein in the complex could not be identified with antibodies to the heat shock proteins hsp90 or hsp70 or with antibodies to the p59 protein or Ah receptor nuclear translocator protein. The identity and function of the protein that becomes cross-linked to the ligand-binding subunit require further investigation.

Gene structure and upstream regulatory regions of human CYP2C9 and CYP2C18.

There is a genetic polymorphism in humans in the metabolism of S-mephenytoin which has been suggested to be mediated by either CYP2C18 or CYP2C9. We have isolated genomic clones for CYP2C9 and CYP2C18 from the liver of an individual phenotyped in vitro as an extensive metabolizer of S-mephenytoin. Analysis of the genes reveals nine coding exons spanning approximately 55 kb. The intron-exon organization was similar to that of other members of the CYP2C subfamily. Analysis of 2200 bp of 5' upstream sequence for CYP2C9 and 1300 bp 5' upstream sequence for CYP2C18 reveals canonical TATA boxes situated 57 bp upstream from the first codon, multiple consensus sequences for glucocorticoid regulatory elements, and identification of a 15 base sequence with high homology to a 5'-flanking sequence responsible for barbiturate-inducible expression of P450BM-3 in Bacillus megaterium. The upstream region for CYP2C9 was highly homologous (75%) to that of human CYP2C8 through most of the 2200 bp sequenced, but the upstream region of CYP2C18 was similar to CYP2C8 and CYP2C9 for only the first 200 bases. The availability of the sequences of the upstream regions and intron-exon junctions of CYP2C9 and CYP2C18 will allow future analysis of these genes in humans which differ in their ability to metabolize S-mephenytoin and other drugs.

Correlation of human cytochrome P4502C substrate specificities with primary structure: warfarin as a probe.

The regio- and stereoselectivity of warfarin metabolism have been used to assess structure-function relationships of human P4502C subfamily members. Metabolism was investigated using a yeast cDNA expression system in which full length cDNAs for P4502C8, -2C9 (alleles Arg144 Tyr358 Ile359 Gly417 and Arg144 Tyr358 Leu359 Gly417), -2C18 (alleles Thr385 and Met385), and -2C19 were expressed. Additionally, two mutations reported in other P4502C9/2C10 alleles were individually introduced into P4502C9 by site-directed mutagenesis, to yield Cys144 Tyr358 Ile359 Gly417, Arg144 Tyr358 Ile359 Asp417, and Arg144 Cys358 Ile359 Gly417, which were expressed in yeast; their ability to metabolize warfarin was then studied. Warfarin metabolism by purified preparations of P4502C9 allele Arg144 Tyr358 Ile359 Gly417 and its Leu359 mutant was also investigated in reconstituted systems. Both alleles of P4502C18 were regioselective for 4'-hydroxywarfarin, without any significant stereoselectivity. Both also metabolized warfarin at the 6-position, but to a lesser extent, and metabolism at this site was stereoselective for (R)-warfarin. P4502C8 metabolized warfarin at the 7-position and was stereospecific for (R)-warfarin. It also metabolized warfarin to a lesser extent at the 4'-position, and metabolism at this site was stereoselective for (R)-warfarin. P4502C19 was regioselective for 6- and 8-hydroxywarfarin and was stereoselective for (R)-warfarin. The highly conservative mutation of Ile359 to Leu359 in P4502C9 profoundly altered the regio- and stereoselectivity of warfarin metabolism, from regioselective for 7-hydroxywarfarin, with stereospecificity for (S)-warfarin, to regioselective for 4'-hydroxywarfarin, with stereoselectivity for (R)-warfarin, which was confirmed in a reconstituted system using purified recombinant enzymes. In contrast, individual mutations of P4502C9 of Arg144 to Cys, Tyr358 to Cys, and Gly417 to Asp did not markedly affect the regio- or stereoselectivity of warfarin metabolism, although the overall rates of warfarin metabolism were apparently increased by these changes. We conclude that residue 359 is at the substrate binding site of P4502C9, whereas residues 144, 358, and 417, and residue 385 of P4502C18, are not.

Biotransformation and toxicity of acetaminophen in congenic RHA rats with or without a hereditary deficiency in bilirubin UDP-glucuronosyltransferase.

Acetaminophen is eliminated primarily by glucuronidation, thereby avoiding cytochrome P450-catalyzed bioactivation to a toxic reactive intermediate. Previous studies have shown that UDP-glucuronosyltransferase-deficient Gunn rats are more susceptible to acetaminophen toxicity than normal Wistar controls, from which the Gunn strain was derived. However, the Gunn and Wistar strains are not congenic, and differences in toxicologic susceptibility could be due in part to genetic differences other than UDP-glucuronosyltransferase activity. Accordingly, acetaminophen (750 mg/kg, ip) was administered to congenic RHA rats with normal (homozygous, RHA/++), moderately deficient (heterozygous, RHA/j+), and severely deficient (homozygous jaundiced, RHA/jj) activities of bilirubin UDP-glucuronosyltransferase. Acetaminophen metabolites were measured by high-performance liquid chromatography and production of the acetaminophen glucuronide conjugate was quantified by the area under plasma concentration-time curve (AUC) from 0 to 2 hr, standardized by the AUC value for acetaminophen in the same animal (glucuronidation ratio = AUC acetaminophen glucuronide/AUC acetaminophen). The 0- to 2-hr time period for AUC calculations was necessitated by the accumulation at later time points of glucuronide and sulfate conjugates in the plasma of animals experiencing severe nephrotoxicity. Acetaminophen bioactivation was quantified by the 24-hr urinary recovery of glutathione-derived conjugates. Hepatotoxicity and nephrotoxicity were assessed respectively by the peak concentrations of plasma alanine aminotransferase (ALT) and blood urea nitrogen (BUN). Glucuronidation of acetaminophen in RHA/jj rats (0.065 +/- 0.005) (mean +/- SE) was reduced 63% compared to the RHA/++ controls (0.17 +/- 0.01) (p < 0.05). RHA/jj rats demonstrated respective 230- and 7-fold increases in the peak plasma concentrations of ALT (17144 +/- 1014 vs 75 +/- 10) and BUN (128 +/- 23 vs 18.4 +/- 0.2) compared to congenic normal controls (RHA/++) (p < 0.05). Heterozygous animals (RHA/j+) demonstrated intermediary toxicity for both parameters (ALT = 2029 +/- 1581, BUN = 41 +/- 16, p < 0.05). Decreased glucuronide production correlated with elevations in ALT (r = -0.86, p < 0.001), while increased acetaminophen bioactivation correlated directly with both elevated ALT (r = 0.93, p < 0.001) and BUN (r = 0.83, p = 0.001). These results using congenic controls demonstrate that the enhanced susceptibility of UDP-glucuronosyltransferase-deficient rats to acetaminophen toxicity is due to decreased glucuronidation resulting in enhanced bioactivation, rather than to other unappreciated genetic differences.

Decreased glucuronidation and increased bioactivation of acetaminophen in Gilbert's syndrome.

Gilbert's syndrome occurs in 5%-7% of the human population and is caused by an inherited deficiency in the glucuronidation of endogenous bilirubin, resulting in its accumulation and jaundice. The authors of the present study have previously shown that rats with a similar deficiency in bilirubin glucuronidation (Gunn rats) had reduced glucuronidation and enhanced susceptibility to the toxicity of the widely used analgesic, acetaminophen. Acetaminophen is eliminated primarily by glucuronidation, which prevents its cytochrome P-450-catalysed bioactivation to a hepatotoxic reactive intermediate. The purpose of this study was to determine whether people with Gilbert's syndrome had reduced glucuronidation and enhanced bioactivation of acetaminophen. Therefore, the biotransformation of acetaminophen, 20 mg/kg IV, was investigated in six subjects with Gilbert's syndrome (total bilirubin, 41 +/- 6 mumol/L; mean +/- SE) and six normal controls (total bilirubin, 11 +/- 2 mumol/L; P less than 0.01). Formation of the acetaminophen glucuronide conjugate measured by high-performance liquid chromatography was quantified by the ratio of the area under the plasma concentration-time curve (AUC) from 0 to 2 hours for the acetaminophen glucuronide divided by the AUC for acetaminophen. Acetaminophen bioactivation was quantified by the molar percentage of acetaminophen excreted in the urine during 24 hours as glutathione-derived conjugates (cysteine and mercapturic acid). Acetaminophen glucuronide formation in subjects with Gilbert's syndrome was 31% lower than that in normal controls (0.27 +/- 0.05 vs. 0.39 +/- 0.03; P less than 0.05), and bioactivation was 1.7-fold higher (3.5% +/- 0.4% vs. 2.1% +/- 0.3%; P less than 0.05). One control subject with normal bilirubin glucuronidation had substantially decreased acetaminophen glucuronide formation (0.20) and enhanced bioactivation (4.8%). Among all subjects, glucuronidation correlated inversely with bioactivation (r = -0.84; P less than 0.001), indicating that a decrease in a major pathway of elimination can shunt more drug through the toxifying route. Thus, a deficiency in bilirubin UDP-glucuronosyltransferase, evidenced by jaundice, can be paralleled by a deficiency in glucuronidation of other compounds. In these cases, jaundice can be a phenotypic determinant of enhanced acetaminophen bioactivation. On the other hand, some people with normal bilirubin glucuronidation may have a deficiency in the glucuronidation of acetaminophen; these people are not easily recognized.(ABSTRACT TRUNCATED AT 400 WORDS)

Enhanced acetaminophen toxicity in rats with bilirubin glucuronyl transferase deficiency.

Glucuronidation is the major pathway for elimination of acetaminophen, diverting it from the toxifying pathway catalyzed by cytochromes P-450. A genetic deficiency in bilirubin UDP-glucuronyl transferase may predispose humans and animals to the toxicity of drugs that are extensively glucuronidated, if other glucuronyl transferase isoenzymes are concurrently deficient. Homozygous and heterozygous Gunn rats are, respectively, severely and moderately deficient in glucuronyl transferase. Acetaminophen (500 mg per kg) was administered intraperitoneally to homozygous and heterozygous Gunn rats and to Wistar controls. Hepatic and renal cellular damage was assessed by peak plasma concentrations of ALT and blood urea nitrogen, respectively. Homozygous and heterozygous Gunn rats showed, respectively, 115-fold and 9-fold higher ALT concentrations compared to Wistar controls. Blood urea nitrogen was elevated only in the homozygous Gunn rats (3-fold). Biotransformation of acetaminophen was measured by high-performance liquid chromatography. Acetaminophen glucuronidation was decreased by 72 and 35% (p less than 0.05), respectively, in the homozygous and heterozygous Gunn rats compared with Wistar controls. Production of acetaminophen glucuronide correlated negatively with ALT concentration (r = -0.89, p less than 0.001). Production of glutathione-derived metabolites, reflecting acetaminophen bioactivation, was 2 to 3-fold higher in the Gunn rats (p less than 0.05) and correlated with ALT concentrations (r = 0.90, p less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)