Role of CYP2A5 and 2G1 in acetaminophen metabolism and toxicity in the olfactory mucosa of the Cyp1a2(-/-) mouse

Acetaminophen (AP) is a widely-used analgesic agent that has been linked to human liver and kidney disease with prolonged or high-dose usage. In rodents, the target organs that are affected include liver, kidney, and the olfactory mucosa. AP toxicity requires cytochrome P450(CYP)-mediated metabolic activation, and the isozymes CYP1A2, 2E1, and 3A are known to activate AP in the human. In the present study, we determined that olfactory mucosal toxicity of AP was not different between the Cyp1a2(+/+) wild-type and the Cyp1a2(-/-) knockout mouse, whereas the hepatic toxicity of AP was significantly diminished in Cyp1a2(-/-) mice. Western blots of olfactory mucosa revealed that CYP2E1 and CYP3A levels are similar between untreated Cyp1a2(+/+) and Cyp1a2(-/-) mice. Diallyl sulfide (DAS), a known inhibitor of CYP2E1 and of CYP2A10/2A11 (the rabbit orthologue of mouse CYP2A5), completely eliminated olfactory toxicity of AP in both the Cyp1a2(-/-) and wild-type mouse olfactory mucosa. We found that heterologously expressed mouse CYP2A5 and CYP2G1 enzymes (known to be present in olfactory mucosa) form 3-hydroxyacetaminophen (3-OH-AP) and 3-(glutathion-S-yl)acetaminophen (GS-AP); CYP2A5 is considerably more active than 2G1. Addition of GSH caused increases in GS-AP proportional to decreases in 3-OH-AP, suggesting that these two metabolites arise from a common precursor or are formed by way of competing pathways. We also found that both CYP2A5 and CYP2G1 are inhibitable by DAS in vitro. These studies provide strong evidence that, in addition to CYP2E1, CYP2A5 and 2G1 are important in AP bioactivation in the mouse olfactory mucosa and that CYP1A2 appears to be of minor importance for AP olfactory toxicity.     

Reduction of acetaminophen toxicity by sodium sulfate in mice

Twenty-one strains of Venezuelan encephalitis (VE) virus isolated from three habitats in Trinidad, W.I. during 1960 to 1972, were subtype III (Mucambo) VE virus by plaque-reduction neutralization tests. Like prototype Mucambo virus, each strain killed 8- to 15-week-old mice inoculated intraperitoneally. If the subtype I strain of VE virus that caused a major outbreak in Trinidad during 1943 to 1944 persisted on the island into the 1960s and early 1970s, it did not become the dominant VE virus in these three enzootic foci.     

Relationship between alcoholism and CYP2E1 genotypes

We amplified and sequenced the rearranged immunoglobulin heavy chain VDJ genomic unit in B-leukemias and used it as a clone-specific marker for the molecular monitoring of the patients during and after therapeutic treatment. The method described is patient-specific rather than disorder-specific, more sensitive and less time-consuming than other conventional techniques for the detection of minimal residual disease. We propose reproducible and quick procedures, from DNA extraction to Southern blotting, that can be easily performed in any clinical laboratory and also applied to other kinds of investigation.     

Effect of cigarette smoke on CYP1A1, CYP1A2 and CYP2B1/2 of nasal mucosae in F344 rats

BACKGROUND: Dobutamine stress echocardiography (DSE) is sensitive and specific in detecting myocardial ischemia of male patients. However, there have been few reports about the use of DSE for the detection of coronary artery disease (CAD) in women. METHODS: DSE was evaluated in 51 consecutive women who underwent concomitant quantitative coronary angiography. Forty-four of the 51 patients received stress thallium-201 single-photon emission computed tomography (SPECT), and 30 of the 51 patients had interpretable results (exercise level > or = 85% of age-predicted maximal heart rate) of treadmill exercise. Twenty-nine patients had angiographically documented CAD defined as > or = 50% diameter stenosis. RESULTS: The overall sensitivity of DSE and stress 201Tl SPECT in detecting CAD was 93% and 79% (p = nonsignificant), and the specificity was 82% and 75% (p = nonsignificant), respectively. A combination of both tests increased the sensitivity (96%) at the expense of some decrease in specificity (60%). The agreement of DSE and 201Tl SPECT was 68% (30 of 44; kappa statistic = 0.35; p < 0.0001). The overall sensitivity, specificity, and accuracy in detecting CAD by treadmill exercise test and DSE were 71% vs 93% (p = nonsignificant), 44% vs 82% (p = 0.036), and 57% vs 88% (p = 0.003). In patients with abnormal results of treadmill exercise testing, the false-positive rate in detecting CAD was 2 (18%) of 11 in patients with abnormal results of DSE and 7 (88%) of 8 in those with normal results of DSE (p = 0.005). In patients with normal results of treadmill exercise testing, the false-negative rate in detecting CAD was 4 (100%) of 4 in patients with abnormal results of DSE and 0 (0%) of 7 in those with normal results of DSE (p = 0.003). CONCLUSION: The diagnostic accuracy of DSE was similar to that of stress 201Tl SPECT in women. DSE was able to stratify female patients with either abnormal or normal results of treadmill exercise testing and to avoid unnecessary cardiac catheterization.     

Purification and characterization of heterologously expressed mouse CYP2A5 and CYP2G1: role in metabolic activation of acetaminophen and 2,6-dichlorobenzonitrile in mouse olfactory mucosal microsomes

The metabolic activation of two known olfactory mucosal (OM) toxicants, acetaminophen (AP) and 2,6-dichlorobenzonitrile (DCBN), was examined with mouse liver and OM microsomes and purified, heterologously expressed mouse CYP2A5 and CYP2G1. In reconstituted systems, both isoforms were active in metabolizing DCBN and AP to metabolites that formed protein adducts. The formation of DCBN- or AP-protein adducts and other AP metabolites, including 3-hydroxy-AP and, in the presence of glutathione, AP-glutathione conjugate, was also detected in OM microsomal reactions and to a much greater extent than in liver microsomes. Evidence was obtained that CYP2A5 and CYP2G1 play major roles in mouse OM microsomal metabolic activation of DCBN and AP. Immunoblot analysis indicated that CYP2A5 and CYP2G1 are abundant P450 isoforms in OM microsomes. OM microsomal AP and DCBN metabolic activation was inhibited by 5- and 8-methoxsalen, which inhibit both CYP2A5 and CYP2G1, and by an inhibitory anti-CYP2A5 antibody that also inhibits CYP2G1. In addition, the roles of CYP1A2 and CYP2E1 in the OM bioactivation of AP and DCBN were ruled out by comparing activities of acetone-treated mice or Cyp1a2(-/-) mice with those of control mice. Thus, CYP2A5 and CYP2G1 may both contribute to the known OM-selective toxicity of AP and DCBN. Further analysis of the kinetics of AP and DCBN metabolism by the purified P450s suggested that CYP2A5 may play a greater role in OM microsomal metabolism of AP, whereas their relative roles in DCBN metabolism may be dose dependent, with CYP2G1 playing more important roles at low substrate concentrations.     

p53 mutation spectrum in relation to GSTM1, CYP1A1 and CYP2E1 in surgically treated patients with non-small cell lung cancer

OBJECTIVE: The purpose of this study was to assess the feasibility of using a prototype split-specimen design to assess integrity of a portion of the total testing process in medical clinics and laboratories. DESIGN: Two or three tubes of venous blood were collected from 177 patients for analysis of one of three analytes (serum potassium, serum total cholesterol, and whole-blood hemoglobin). Patients were seen at one of the nine clinics participating in this study. In all cases, one tube of blood from each patient was sent to a commercial referral laboratory, and the other tube(s) forwarded to the laboratory that routinely tested specimens for the clinic (participating laboratory) for analysis. Each participating laboratory removed a preanalysis and sometimes a post-analysis aliquot from each specimen and forwarded these to the referral laboratory for analysis. SETTING: The study was conducted in six physician office laboratories (three serving 1 to 4 [mean, 2.7] internists and three serving 3 to 24 [mean, 12] family physicians) and three hospital laboratories (serving hospitals with 100 to more than 700 beds). PATIENTS: Study patients were voluntary participants and provided informed consent. Patient age ranged from 18 to 80 years, and for all the laboratory test was specifically ordered for clinical reasons. Patients who were unable or unwilling to provide informed consent, those for whom testing would require that they provide more than 100 mL of blood, those whose blood was being collected by fingerstick, and those with results that were part of a laboratory test profile were excluded. MAIN OUTCOME MEASURES: Two main outcome measures were assessed: (1) percent differences between split-specimen results exceeding the maximum allowable imprecision level, which was based on published biological variation data (defined as one-half of the intraindividual percent coefficient of variation), for each analyte (result discrepancies); and (2) all "problems" (defined as departures from standard operating procedures) that could be documented by retrospective review of all relevant medical and laboratory records. RESULTS: The rate of result discrepancies was 1 in 20 (5%) for patients in whom hemoglobin was analyzed, 12 in 57 (21%) for patients in whom potassium was analyzed, and 1 in 60 (2%) for patients in whom total cholesterol was analyzed. Results of samples obtained during the aliquoting and storage phases of the total testing process were subject to study-induced problems and were generally not useful in tracing problems to specific stages of the testing process. A total of 28 problems (involving 26 patients) were documented, but only 6 problems were due to routine testing processes. CONCLUSIONS: The feasibility and limitations of a split-specimen design to detect result discrepancies were demonstrated. Most documented problems (22 of 28, or 79%) were study induced. To assess integrity of the total testing process, such problems need to be avoided.     

Differential in vivo effects of alpha-naphthoflavone and beta-naphthoflavone on CYP1A1 and CYP2E1 in rat liver, lung, heart, and kidney

Tenascin is a large extracellular matrix glycoprotein expressed in neural and non-neural tissues. In the central nervous system, tenascin is synthesized by astrocytes during development and wound healing, forming barriers and affecting neurite outgrowth. In this study we examined tenascin expression in optic nerve heads of normal and glaucomatous eyes and found that there is upregulation of tenascin mRNA and protein in reactive astrocytes from human glaucomatous optic nerve heads compared to normal age-matched controls. In the prelaminar region there was a band of tenascin immunoreactivity around the blood vessels of glaucomatous, but not in normal eyes. However, tenascin mRNA was only localized to astrocytes, suggesting that astrocytes are the cellular source of tenascin. In the lamina cribrosa, tenascin immunoreactivity and gene expression were localized to astrocytes in the cribriform plates and inside the nerve bundles. In the post-lamina region, tenascin immunoreactivity and gene expression were localized to astrocytes lining the pial septum immediately adjacent to the lamina cribrosa. In normal optic nerve heads, tenascin expression at the mRNA and protein levels was confined to clusters of astrocytes at the level of Bruch's membrane in the prelaminar optic nerve head. In glaucoma, enhanced expression of tenascin may be protective to the axons of the retinal ganglion cells by providing a barrier for humoral and/or blood-borne factors that may cause further neural damage. However, the precise role of tenascin in glaucomatous optic neuropathy is not yet elucidated.     

Imipramine demethylation in vivo: impact of CYP1A2, CYP2C19, and CYP3A4

Nephron loss leads to increased production of reactive oxygen intermediates. We measured the effect of carvedilol, a beta-blocking drug with radical scavenging properties, on renal function, glomerulosclerosis, antioxidant enzyme status and in vivo hydrogen peroxide (H2O2) production in rats with chronic renal failure caused by 5/6 nephrectomy (remnant kidney) and compared results to data obtained with propranolol, a beta-blocking drug without scavenging characteristics. Carvedilol and propranolol were administered during 11 weeks following reduction of nephron number. Kidneys were examined using enzymatic and histological techniques. Both carvedilol and propranolol decreased systolic blood pressure. Compared to propranolol, carvedilol offered some additional beneficial effects on renal function, particularly with regard to glomerulosclerosis. Lipid peroxidation, evaluated by malonaldehyde and 4-hydroxynonenal concentration in cortex homogenates, was decreased in carvedilol-treated rats only. Superior beneficial effect of carvedilol treatment is not linked to a significant up-regulation of the activities of the remnant kidney antioxidant enzymes (catalase, glutathione peroxidase and superoxide dismutase) or to a decreased in vivo H2O2 production.     

Benzene-induced genotoxicity in mice in vivo detected by the alkaline comet assay: reduction by CYP2E1 inhibition

Acute, severe injury of the rabbit spinal cord, induced by the weight-drop method, causes alterations of the enzyme activities related to cholinergic and energy metabolism. Morphological examinations at the trauma site show degenerative processes in neurons 0.5 hr posttrauma and a marked decrease in the number of living cells 24 hrs later. Both biochemical and cytochemical findings show that the tissue metabolic and morphologic derangement, caused by severe spinal cord injury, is mostly confined to the gray matter at an early stage (0.5 hr), whereas 24 hrs later the white matter is also involved. The decrease in choline acetyl-transferase and acetylcholinesterase activities in the gray matter parallels the impairment of complex IV (cytochrome c oxidase) of the respiratory chain and the presence of morphological alteration in neurons. The dramatic drop in the enzyme activities, observed 24 hrs after the induction of the severe trauma is clearly associated with the loss of cells.     

Distinction of CYP1A1 and CYP1A2 activity by selective inhibition using fluvoxamine and isosafrole

A one step competitive Enzyme-Linked Immunosorbent assay (ELISA) method was developed to detect mycobacterial antigen in cerebrospinal fluid (CSF) for the diagnosis of tuberculous meningitis and compared with a standard competitive ELISA method. Indigenously prepared soluble extract of Mycobacterium tuberculosis H37 Rv was used as antigen. The study was conducted using CSF of 230 clinically diagnosed cases of tuberculous meningit is and 208 control subjects. A cutoff value of 0.57 ng/ml by the one step ELISA and 0.5 ng/ml by the standard ELISA method were determined. The specificity of both methods were 100% and positivity was 68.26% and 70.43% respectively. A follow up study was conducted in 63 cases at various interval of time after starting anti-tubercular therapy i.e. at 3 weeks (63 cases), 6 weeks (27 cases) and > or = 4-12 months (13 cases). It was observed that antigen levels decreased gradually, but were much above the cutoff range. Indigenously prepared antigen was compared with antigen prepared in other laboratories and standard molecular weight markers using SDS PAGE (Sodium Do-decyl Sulphate Polycrylamide Gel Electrophoresis).     

Detection and localization of CYP1A1/CYP1A2 in murine liver: electrophoresis, immunoblotting and immunocytochemistry

Multiple forms of cytochrome P450 exist some of which are selectively inducible by exposure of the organism to a variety of foreign compounds. In this study, a monoclonal antibody specific for 3-methyl-cholanthrene-inducible cytochrome P450, Mab 1-7-1, was used to detect, localize and quantify CYP1A1/CYP1A2 in livers of C57BL/6 mice. Mab 1-7-1 recognized a faint band in the range between 45-66 Kd in Western immunoblots of liver microsomes from control mice, and a strong band in the same range, in liver microsomes from beta-naphthoflavone-treated mice. Microsome from control liver contained minimal levels of CYP1A1/CYP1A2; pretreatment with beta-naphthoflavone caused an increase in their expression. Immunoelectron microscopy was used to demonstrate the cellular localization and quantification of these isozymes in the liver. The immunolabeling procedure confirmed the endoplasmic reticulum as the primary site of CYP1A1/CYP1A2 induction in hepatocytes. This organelle showed the highest labeling density after treatment with beta-naphthoflavone. Increase in CYP1A1/CYP1A2 was 33.4-fold by morphometric analysis in induced hepatocytes in comparison to non-induced cells. In conclusion, CYP1A1/CYP1A2 is highly induced by beta-naphthoflavone in C57BL/6 mouse liver, and the cellular site of expression is the endoplasmic reticulum.     

Dose-response relationships for polyhalogenated dioxins and dibenzofurans following subchronic treatment in mice. I. CYP1A1 and CYP1A2 enzyme activity in liver, lung, and skin

The dose-response relationships for induction of liver, lung, and skin ethoxyresorufin-O-deethylase (EROD) activity and liver acetanilide-4-hydroxylase (ACOH) activity following subchronic exposure to either 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 1,2,3,7,8-pentachlorodibenzo-p-dioxin, 2,3,7,8-tetrabromodibenzo-p-dioxin, 2,3,7,8-tetrachlorodibenzofuran (TCDF), 1,2,3,7,8-pentachlorodibenzofuran (1-PeCDF), 2,3,4,7,8-pentachlorodibenzofuran (4-PeCDF), or octachlorodibenzofuran (OCDF) were determined in female B6C3F1 mice in order to estimate the relative enzyme inducing potency of these chemicals in three different tissues. The relative potencies were calculated based on tissue concentrations as well as administered dose. A dose-dependent induction of EROD activity in liver, lung, and skin and of ACOH activity in liver was found for all seven chemicals. When based on administered dose, the relative potencies for specific congeners did not vary substantially among tissues. The relative potencies for TCDF and 1-PeCDF, congeners which have much shorter half-lives than TCDD, increased for all enzymes when estimated from tissue concentrations. The relative potency of OCDF, which is poorly absorbed, was greater when estimated from tissue concentrations than when estimated from administered dose. 4-PeCDF is highly sequestered in hepatic tissue and when the relative potency was estimated based on tissue concentration, its potency for skin enzyme induction increased. These data indicate that the relative potency of these chemicals is influenced not only by the relative binding affinity to the Ah receptor, but also by differences in pharmacokinetic properties of these chemicals. In addition, it may be useful to derive two sets of toxic equivalency factor values, one used for estimating intake equivalents and the other for estimating tissue equivalents.     

ACTH receptor, CYP11A1, CYP17 and CYP21A2 genes are expressed in skin

Evidence is provided that mRNA for ACTH (MC-2) receptor and mRNAs for three obligatory enzymes of steroid synthesis including cytochromes P450scc, P450c17 and P450c21 are expressed in normal and pathologic human skin. Thus, molecular elements of the distal loop of the "pituitary-adrenal axis" such as the MC-2, CYP11A1, CYP17 and CYP21A2 genes are expressed in the skin.     

Insulin effects on CYP2E1, 2B, 3A, and 4A expression in primary cultured rat hepatocytes

Although hyperketonemia and/or altered growth hormone secretion caused by diabetes have been implicated in enhanced CYP2E1, 2B, 3A and 4A expression, the effect of insulin on hepatic P450 expression, in the absence of associated metabolic/hormonal alterations, remains unknown. Primary cultured rat hepatocytes have been shown (Zangar et al., Drug Metab. Dispos., 23:681, 1995) to express stable and inducible CYP2E1 mRNA and protein levels, and provide an excellent system for mechanistic examination of the effect of insulin on CYP2E1, 2B, 3A and 4A expression. Maintaining primary rat hepatocytes in culture in the absence of insulin for 48, 72, or 96 h increased CYP2E1 mRNA levels 5-, 11-, and 4-fold, respectively, relative to cells maintained in the presence of the standard concentration of 1 microM insulin. In contrast, CYP2B mRNA levels increased only approximately 2-fold in the absence of insulin, when compared with the presence of 1 microM insulin. CYP2E1 and 2B protein levels were increased 6.7- and 3.8-fold, respectively, in cells cultured for 96 h in the absence of insulin as compared with those cultured in medium containing 1 microM insulin. Concentration-response studies revealed that decreasing the concentration of insulin below 10 nM (i.e. 1 nM, 0.1 nM, no insulin) increased CYP2E1 mRNA levels 4-, 7-, and 11-fold, respectively. In contrast, no such concentration-dependence was observed for CYP2B mRNA expression. As CYP3A and 4A expression is also elevated in diabetic rats, the effects of insulin on these P450s was also examined. CYP3A mRNA levels were unaltered and CYP4A mRNA levels were decreased marginally (approximately 50%) by the absence of insulin relative to levels in cells cultured in the presence of 1 microM insulin over 96 h in culture. The results of this study provide evidence that insulin itself, in the absence of other diabetes-induced metabolic or hormonal alterations, affects CYP2E1 and 2B, but not CYP3A or 4A, expression in primary cultured rat hepatocytes. Furthermore, CYP2E1 expression is differentially regulated by insulin relative to CYP2B, 3A or 4A. This study also demonstrates that decreasing the concentration of insulin in the culture medium provides a method by which CYP2E1 levels can be increased in primary cultured hepatocytes to facilitate mechanistic studies on the regulation of CYP2E1 expression.     

A general strategy for the expression of recombinant human cytochrome P450s in Escherichia coli using bacterial signal peptides: expression of CYP3A4, CYP2A6, and CYP2E1

Heterologous expression of unmodified recombinant human cytochrome P450 enzymes (P450s) in Escherichia coli has proved to be extremely difficult. To date, high-level expression has only been achieved after altering the 5'-end of the native cDNA, resulting in amino acid changes within the P450 protein chain. We have devised a strategy whereby unmodified P450s can be expressed to high levels in E. coli, by making NH2-terminal translational fusions to bacterial leader sequences. Using this approach, we initially tested two leader sequences, pelB and ompA, fused to CYP3A4. These were compared with an expression construct producing a conventional NH2-terminally modified CYP3A4 (17alpha-3A4). Both leader constructs produced spectrally active, functional protein. Furthermore, the ompA-3A4 fusion gave higher levels of expression, and a marked improvement in the recovery of active P450 in bacterial membrane fractions, when compared with 17alpha-3A4. We then tested the ompA leader with CYP2A6 and CYP2E1, again comparing with the conventional (17alpha-) approach. As before, the leader construct produced active enzyme, and, for CYP2E1 at least, gave a higher level of expression than the 17alpha-construct. The ompA fusion strategy thus appears to represent a significant advance for the expression of P450s in E. coli, circumventing the previous need for individual optimization of P450 sequences for expression.     

Kinetic characterization of CYP2E1 inhibition in vivo and in vitro by the chloroethylenes

Trans- and cis-1,2-dichloroethylene (DCE) isomers inhibit their own metabolism in vivo by inactivation of the metabolizing enzyme, presumably the cytochrome P450 isoform, CYP2E1. In this study, we examined cytochrome P450 isoform-specific inhibition by three chloroethylenes, cis-DCE, trans-DCE, and trichloroethylene (TCE), and evaluated several kinetic mechanisms of enzyme inhibition with physiological models of inhibition. Trans-DCE was more potent than cis-DCE, and both were much more effective than TCE in inhibiting CYP2E1. The kinetics of in vitro loss of p-nitrophenol hydroxylase (pNP-OH) activity (a marker of CYP2E1) in microsomal incubations and of the in vivo gas uptake results were most consistent with a mechanism in which inhibition of the metabolizing enzyme (CYP2E1) was presumed to be related to interaction of a reactive DCE metabolite with remaining substrate-bound, active CYP2E1. The kinetics of inhibition by TCE, a weak inhibitor in vitro, were very different from that of the dichloroethylenes. With TCE, parent compound concentrations influenced enzyme loss. Trans-DCE was a more potent inhibitor of CYP2E1 than cis-DCE based on both in vivo and in vitro studies. Quantitative differences in the inhibitory properties of the 1,2-DCE isomers may be due to the different stability of epoxides formed from bioactivation by CYP2E1. Epoxide intermediates of DCE metabolism, reacting by water addition, would yield dialdehyde, a potent cross-linking reagent.     

Protection by extracellular glutathione against sulfur mustard induced toxicity in vitro

BACKGROUND: Salmeterol xinafoate is a highly selective beta2-adrenoceptor for the maintenance treatment of asthma in adults and children. OBJECTIVE: To review the pharmacokinetics, clinical pharmacology, and therapeutic properties of a recently introduced, long acting antiasthmatic drug. METHODS: Recent English-language publications were selected using Medline as database. RESULTS: Salmeterol's pharmacokinetics, clinical pharmacology, and therapeutic properties are reviewed and aspects related to salmeterol's unusual duration of action, its high potency, beta2-selectivity, possible antiinflammatory actions, its interaction with other drugs, low systemic adverse effects, dosage, and administration are also discussed. CONCLUSION: Salmeterol is a safe long-acting beta2-agonist very useful for maintenance treatment of asthma.