Identification and quantitation in human liver of cytochromes P-450 analogous to rabbit cytochromes P-450 forms 4 and 6

1. Polyclonal antibodies raised against rabbit liver cytochrome P-450 isozymes form 4 and 6 have been used to probe human liver microsomes for analogous proteins using the Western blot technique.

2. Anti-Form 4 IgG recognized a protein in human liver microsomes from six subjects of identical molecular weight to purified rabbit liver cytochrome P-450 Form 4.

3. The equivalent content of cytochrome P-450 Form 4 in the same microsomes ranged from 1˙1 to 9˙1 pmol per mg protein.

4. Anti-Form 6 IgG recognized a protein in human liver microsomes from the same six subjects of slightly higher molecular weight than purified rabbit cytochrome P-450 Form 6.

5. The equivalent content of cytochrome P-450 Form 6 in the above microsomes ranged from 1˙6 to 3˙8 pmol per mg protein.

6. No significant correlations were observed between equivalent cytochrome P-450 Forms 4 and 6 content and 2-acetylaminofluorene N-hydroxylase, aminopyrine N-demethylase, benzyprene and aniline hydroxylase activities in liver microsomes from the six subjects tested.     

Identification and quantitation in human liver of cytochromes P-450 analogous to rabbit cytochromes P-450 forms 4 and 6

1. Polyclonal antibodies raised against rabbit liver cytochrome P-450 isozymes form 4 and 6 have been used to probe human liver microsomes for analogous proteins using the Western blot technique. 2. Anti-Form 4 IgG recognized a protein in human liver microsomes from six subjects of identical molecular weight to purified rabbit liver cytochrome P-450 Form 4. 3. The equivalent content of cytochrome P-450 Form 4 in the same microsomes ranged from 1.1 to 9.1 pmol per mg protein. 4. Anti-Form 6 IgG recognized a protein in human liver microsomes from the same six subjects of slightly higher molecular weight than purified rabbit cytochrome P-450 Form 6. 5. The equivalent content of cytochrome P-450 Form 6 in the above microsomes ranged from 1.6 to 3.8 pmol per mg protein. 6. No significant correlations were observed between equivalent cytochrome P-450 Forms 4 and 6 content and 2-acetylaminofluorene N-hydroxylase, aminopyrine N-demethylase, benzyprene and aniline hydroxylase activities in liver microsomes from the six subjects tested.     

Effects of Plasmodium berghei infection on cytochromes P-450 2E1 and 3A2.

Metabolism and disposition of most drugs used to treat malaria are substantially altered in malaria infection. Few data are available that specify effects of malaria infection on drug metabolism pathways in humans or animal model systems. In this report, studies were undertaken to determine the effect of Plasmodium berghei infection on cytochrome P-450 (CYP450) 2E1 and 3A2-mediated metabolism and enzyme expression in rat liver microsomes. Malaria infection (MAL) resulted in significant decreases in total cytochrome P-450 content (56%, P < 0.05) and NADPH cytochrome P-450 reductase activity (32%, P < 0.05) as compared to control (CON) rats. Chlorzoxazone 4-hydroxylase activity (CYP2E1-mediated) showed no significant difference between CON and MAL microsomes while testosterone 6-beta-hydroxylase activity (CYP3A2-mediated) was reduced by 41% (P < 0.05) in MAL. Enzyme kinetic studies and immunoblot analysis indicate that the loss of activity for CYP3A2 in malaria infection is due to significantly decreased CYP3A2 protein expression. The altered expression of CYP450s in malaria infection should be taken into account when treating patients with malaria in order to minimize drug-drug interactions or toxicity.     

Induction of P-450 cytochromes 2E2, 1A1, and 1A2 by imidazole in neonatal rabbits.

Cytochrome P-450 2E1 is induced in adult rabbits by treatment with alcohol, imidazole, and a variety of other agents, as shown earlier in this laboratory, but it is not known whether the highly homologous P-450 2E2 is similarly induced. In this study, the effects of imidazole on 2E2 expression were examined in neonatal rabbits, in which 2E1 is not detectable. Treatment of the animals with imidazole on days 8 through 11 after birth caused a 3-fold increase in the content of total P-450 in liver microsomes. In contrast, the microsomal content of cytochrome b5 and NADPH-P450 reductase was not changed. Immunoblot analysis revealed a significant increase in the level of P-450 2E2 (3-fold) as well as 1A1 (> 10-fold) and 1A2 (> 2-fold) in hepatic microsomes from imidazole-treated neonatal rabbits. The rates of microsomal N-demethylation of N-nitrosodimethylamine and O-deethylation of 7-ethoxyresorufin were similarly increased from 1.3 and 0.03 nmol/min/mg protein, respectively, to 5.6 and 0.24 nmol/min/mg protein, respectively, by imidazole treatment. Blot analysis indicated that the levels of 2E2, 1A1, and 1A2 mRNAs are not increased by imidazole treatment and that 2E1 mRNA is not detectable in either untreated or imidazole-treated neonates. The induction of P-450 2E2 was confirmed by NH2-terminal amino acid sequence analysis of immunopurified 2E protein from hepatic microsomes of imidazole-treated neonatal rabbits.(ABSTRACT TRUNCATED AT 250 WORDS)     

Involvement of cytochromes P-450 2E1 and 3A4 in the 5-hydroxylation of salicylate in humans.

Hydroxylation of salicylate into 2,3 and 2,5-dihydroxybenzoic acids (2,3-DHBA and 2,5-DHBA) by human liver microsomal preparations was investigated. Kinetic studies demonstrated that salicylate was 5-hydroxylated with two apparent Km: one high-affinity Km of 606 microM and one low-affinity Km greater than 2 mM. Liver microsomes prepared from 15 human samples catalyzed the formation of 2,5-DHBA at metabolic rate of 21.7 +/- 8.5 pmol/mg/min. The formation of 2, 3-DHBA was not P-450 dependent. Formation of 2,5-DHBA was inhibited by 36 +/- 14% following preincubation of microsomes with diethyldithiocarbamate, a mechanism-based selective inhibitor of P-450 2E1. Furthermore, the efficiency of inhibition was significantly correlated with four catalytic activities specific to P-450 2E1, whereas the residual activity was correlated with three P-450 3A4 catalytic activities. Troleandomycin, a mechanism-based inhibitor selective to P-450 3A4, inhibited by 30 +/- 12% the 5-hydroxylation of salicylate, and this inhibition was significantly correlated with nifedipine oxidation, specific to P-450 3A4. The capability of seven recombinant human P-450s to hydroxylate salicylate demonstrated that P-450 2E1 and 3A4 contributed to 2, 5-DHBA formation in approximately equal proportions. The Km values of recombinant P-450 2E1 and 3A4, 280 and 513 microM, respectively, are in the same range as the high-affinity Km measured with human liver microsomes. The plasmatic metabolic ratio 2,5-DHBA/salicylate, measured 2 h after ingestion of 1 g acetylsalicylate, was increased 3-fold in 12 alcoholic patients at the beginning of their withdrawal period versus 15 control subjects. These results confirm that P-450 2E1, inducible by ethanol, is involved in the 5-hydroxylation of salicylate in humans. Furthermore, this ratio was still increased by 2-fold 1 week after ethanol withdrawal. This finding suggests that P-450 3A4, known to be also inducible by alcoholic beverages, plays an important role in this increase, because P-450 2E1 returned to normal levels in less than 3 days after ethanol withdrawal. Finally, in vivo and in vitro data demonstrated that P-450 2E1 and P-450 3A4, both inducible by alcohols, catalyzed the 5-hydroxylation of salicylate.     

Selective mechanism-based inactivation of cytochromes P-450 2B1 and P-450 2B6 by a series of xanthates.

Fifteen xanthates with carbon chains of different lengths or substitutions, including the antiviral compound D609 (O-tricyclo[5.2. 1.0(2,6)]dec-9-yl-dithiocarbonate), were tested for their ability to inactivate cytochromes P-450 (P-450s) 2B1 and 2B6. All of the xanthates tested were found to inactivate P-450 2B1 in a time- and concentration-dependent manner. The rates of inactivation at 30 degrees C ranged from 0.22 min-1 to 0.02 min-1. The concentrations required for half-maximal inactivation were between 2.4 and 69 microM. A general trend in the inactivation kinetics could be observed with an increasing chain length of the xanthates. Longer carbon chains resulted in slower rates of inactivation with longer half-times of inactivation and higher partition ratios. For P-450 2B1, the most effective inactivators were xanthates with substitutions of intermediate length. The best inactivator for P-450 2B1 was the C8 xanthate, with an inactivation potency (KI) of 2.4 microM, a rate of inactivation of 0.07 min-1, and a partition ratio of 4. Four xanthates were further examined for their effect on the 7-ethoxy-4-(trifluoromethyl)coumarin activity of P-450 2B6. The C8 xanthate was again the most effective inactivator, with a KI of 1 microM. Although the KI values were generally lower than those found with P-450 2B1, the rates of inactivation for P-450 2B6 with the various xanthates were 3- to 5-fold slower. In addition, the isozyme selectivity of xanthates was tested with P-450s 2E1, 1A1, 3A2, 3A4, 2C9, and 2D6. P-450 2E1 was inactivated by xanthates at concentrations 15- to 100-fold higher than those required to inactivate either P-450 2B1 or 2B6. P-450 1A1 was not inactivated by xanthates. However, all of the xanthates tested were able to inhibit the enzymatic activity of P-450 1A1 to a different extent, depending on the length of the xanthate carbon chain. Virtually no inactivation of P-450s 2D6 or 2C9 was seen, except that C8 and D609 were inhibitory at high concentrations (0.2-0.6 mM). None of the xanthates studied had any effect on the activities of P-450s 3A2 or 3A4.     

Altered expression of hepatic cytochromes P-450 in mice deficient in one or more mdr1 genes

We hypothesized that the drug efflux protein P-glycoprotein (Pgp), the product of the multidrug resistance gene MDR1, might influence hepatic expression of CYP3A or other cytochromes P-450 (P-450s) because Pgp can transport endogenous regulators of these cytochromes. We began with variants of a CF-1 mouse strain containing a defective mdr1a gene that is inherited in a Mendelian fashion. The amount of CYP3A protein in liver was inversely related to the gene dose of the normal mdr1a allele in these mice. mdr1a knockout mice of either mixed (FVB x 129/Ola) or pure FVB genetic background and housed in Amsterdam display an increased expression of CYP2B and CYP3A proteins. However, because mdr1a ablation causes a compensatory increase in hepatic mdr1b (which can efflux intracellular glucocorticoids), we reasoned that mdr1b might mask the overall effect of mdr1a absence on P-450 gene expression. Targeted inactivation of the mdr1b gene increased P-450 expression, but the effect was modest compared with mdr1a ablation. Mice nullizygous for both mdr1a and mdr1b-type Pgps and kept in Amsterdam had dramatically increased levels of CYP3A protein as well as other P-450s examined and of the electron donor P-450 reductase. Consistent with the protein results, CYP3A catalytic activity measured as midazolam 1'- and 4-hydroxylation in liver microsomes from these knockout mice revealed a rank order of activities with mdr1a/1b > mdr1a > mdr1b > (+/+) mice. In contrast to results in mice housed in Amsterdam, in the genetically identical mdr1a or mdr1a/1b (-/-) male mice housed in the United States, hepatic P-450 expression was unaffected by mdr1 genotype or actually showed a slight decrease in mdr1a (-/-) mice. These results provide a revealing picture of mdr1-type Pgp as an upstream regulator of hepatic P-450 expression, and demonstrate that these pharmacologically relevant phenotypes in knockout mice depend not only on the genetic make-up of the mice but also on the environment.     

Temperature effect on the functional expression of human cytochromes P450 2A6 and 2E1 inEscherichia coli

Human cytochromes P450 (CYP) 2A6 and 2E1 are of great interest because of their important roles in the oxidation of numerous drugs and carcinogens. Bacterial expression systems, especially Escherichia coli cells, have been widely used for the production of various CYP enzymes in order to obtain high yield of proteins. The expression methods usually employ longer culture time (30-72 h) at lower temperature (usually under 30 degrees C). Expression levels of CYPs 2A6 and 2E1 at 37 degrees C were compared to those at 280 degrees C, which is a usual temperature used in most bacterial expression systems for human CYP expression. Within 18 h the expression levels of CYPs 2A6 and 2E1 reached up to 360 and 560 nmol per liter culture at 37 degrees C, respectively, which are compatible with those of 36 h culture at 280 degrees C. The activities of CYPs expressed at 37 degrees C were also comparable to those expressed at 28 degrees C. The present over-expression system can be useful for rapid production of large amounts of active human CYPs 2A6 and 2E1 in E. coli.     

Ligand-complex formation between cytochromes P-450 and P-448 and methylenedioxyphenyl compounds

1. The formation of ligand complexes between hepatic microsomal cytochrome P-450 and safrole, isosafrole and other methylenedioxyphenyl compounds was studied in vivo and in vitro in rats pretreated with either phenobarbital or 3-methylcholanthrene.

2. Both the phenobarbital-induced cytochrome P-450 and the 3-methylcholanthrene-induced cytochrome P-448 metabolically convert safrole, isosafrole, and those metabolites possessing an intact methylenedioxy group, to reactive metabolites which then interact with the cytochromes to form ligand complexes. Formation of these ligand complexes was accompanied by loss of mixed-function oxidase activities, and dissociation of the complexes with the type I substrate biphenyl restored activities.

3. Safrole and, to a lesser extent, 1′-hydroxysafrole formed complexes in vivo when administered to phenobarbital-pretreated rats; none was obtained with epoxysafrole. However, when administered to 3-methylcholanthrene-pretreated animals all three compounds formed complexes, safrole being the least effective.

4. Epoxysafrole and 1′-hydroxysafrole administered to phenobarbital-pretreated rats resulted in slight inhibition of the type I binding of safrole to liver microsomal P-450 in vitro; in contrast, with 3-methylcholanthrene-pretreated animals marked competitive inhibition was observed.

5. This study shows that oxidation of the allyl chain of safrole analogues enhances their affinity for cytochrome P-448, but not for cytochrome P-450, and further demonstrates that these cytochromes possess distinctly different binding sites.     

Ligand-complex formation between cytochromes P-450 and P-448 and methylenedioxyphenyl compounds

The formation of ligand complexes between hepatic microsomal cytochrome P-450 and safrole, isosafrole and other methylenedioxyphenyl compounds was studied in vivo and in vitro in rats pretreated with either phenobarbital or 3-methylcholanthrene. Both the phenobarbital-induced cytochrome P-450 and the 3-methylcholanthrene-induced cytochrome P-448 metabolically convert safrole, isosafrole, and those metabolites possessing an intact methylenedioxy group, to reactive metabolites which then interact with the cytochromes to form ligand complexes. Formation of these ligand complexes was accompanied by loss of mixed-function oxidase activities, and dissociation of the complexes with the type I substrate biphenyl restored activities. Safrole and, to a lesser extent, 1'-hydroxysafrole formed complexes in vivo when administered to phenobarbital-pretreated rats; none was obtained with epoxysafrole. However, when administered to 3-methylcholanthrene-pretreated animals all three compounds formed complexes, safrole being the least effective. Epoxysafrole and 1'-hydroxysafrole administered to phenobarbital-pretreated rats resulted in slight inhibition of the type I binding of safrole to liver microsomal P-450 in vitro; in contrast, with 3-methylcholanthrene-pretreated animals marked competitive inhibition was observed. This study shows that oxidation of the allyl chain of safrole analogues enhances their affinity for cytochrome P-448, but not for cytochrome P-450, and further demonstrates that these cytochromes possess distinctly different binding sites.     

酒精性肝损伤大鼠细胞色素P450 CYP2E1和细胞色素P450 CYP3A的代谢活性

目的观察酒精性肝损伤对大鼠细胞色素P450CYP3A(CYP3A)和细胞色素P450CYP2E1(CYP2E1)代谢活性的影响。方法采用ig给予白酒制备大鼠酒精性肝损伤模型,检测血清中谷丙转氨酶(GPT)和谷草转氨酶(GOT)活性,采用HE染色法光镜下观测酒精对肝脏损伤程度。大鼠ip给予CYP3A探针药物咪达唑仑10mg·kg-1或ig给予CYP2E1探针药物氯唑沙宗50mg·kg-1后,采用高效液相色谱法测定不同时间点大鼠血浆中咪达唑仑和氯唑沙宗的血药浓度,并应用3P87软件计算其药代动力学参数,以考察CYP2E1和CYP3A的代谢活性的变化。大鼠ig给予氯唑沙宗80mg·kg-1后,热板方法测定大鼠添足次数和添足反射潜伏期。结果酒精性肝损伤可致大鼠肝小叶结构不清,肝索排列紊乱,肝细胞体积增大,呈弥漫性中度水变性,肝窦受压,大部分肝细胞胞浆内见大小不等的脂肪空泡;与正常对照组相比,酒精性肝损伤组大鼠GPT和GOT活性分别增加了16.0%和20.0%(P<0.05,P<0.01)。酒精性肝损伤致大鼠CYP2E1对探针药物氯唑沙宗的代谢活性增强,AUC,t1/2和cmax分别降低了38.0%,30.5%和35.0%(P<0.05);酒精肝损伤组大鼠氯唑沙宗镇痛效果明显降低;酒精性肝损伤致大鼠CYP3A对探针药物咪达唑仑的代谢活性增强,AUC,t1/2和cmax分别降低了122.6%,54.9%和56.9%(P<0.01,P<0.05)。结论酒精性肝损伤可使大鼠CYP2E1和CYP3A代谢活性增强。     

Proteasome-dependent degradation of cytochromes P450 2E1 and 2B1 expressed in tetracycline-regulated HeLa cells

The degradation of ethanol-inducible cytochrome P450 2E1 (CYP2E1) and phenobarbital-inducible cytochrome P450 2B1 (CYP2B1) expressed in tetracycline (Tc)-inducible HeLa cell lines was characterized. A steady-state pulse-chase analysis was used to determine a half-life of 3.8 h for CYP2E1 while the half-life of CYP2B1 was 2.3-fold greater in the same cell line. In contrast, NADPH cytochrome P450 reductase which is constitutively expressed in Tc-HeLa cells had a half-life of about 30 h. Lactacystin and other selective proteasome inhibitors including N-benzyloxycarbonyl-leucyl-leucyl-leucinal (MG132) and N-benzyloxycarbonyl-L-leucyl-L-leucyl-L-norvalinal (MG115) significantly inhibited both CYP2E1 and CYP2B1 degradation. The turnover of CYP2E1 was slightly inhibited by calpain inhibitors while CYP2B1 turnover was not altered. Inhibitors of lysosomal proteolysis had no effect on the degradation of either protein. Treatment of cells with brefeldin A did not alter the degradation of either P450 which suggested the degradation occurred in the endoplasmic reticulum (ER). Even in the presence of proteasome inhibitors high molecular weight ubiquitin conjugates were not observed. Mutagenesis of two putative ubiquitination sites (Lys 317 and 324) did not alter the degradation of CYP2E1. The role of ubiquitination in the degradation of CYP2E1 was also examined in a Chinese hamster mutant cell line E36ts20 that contains a thermolabile ubiquitin-activating enzyme (E1). The turnover of CYP2E1 was not significantly different at the nonpermissive temperature in the ts20 when compared to the control E36 cells. Furthermore, the addition of the hsp90 inhibitors geldanamycin, herbimycin, and radicicol had no effect on the turnover of CYP2E1, differentiating the degradation of CYP2E1 from other substrates for proteasome-dependent degradation.     

Cellular distribution of cytochromes P-450 in the rat kidney.

The distribution of several cytochrome P-450 (P-450) isoenzymes between proximal tubular (PT) and distal tubular (DT) cells of the rat kidney was determined. Western blot analysis of microsomes prepared from liver and kidney cortical homogenates revealed that CYP2E1 protein was expressed in rat kidney microsomes at approximately 10% of hepatic levels. Microsomes from renal cortical, PT, and DT cells all expressed CYP2E1, with DT microsomes expressing slightly higher levels than PT microsomes. In contrast, chlorzoxazone hydroxylation activity was markedly higher in microsomes from PT cells than in those from DT cells. Northern blot analysis of total RNA from PT and DT cells exhibited a pattern of CYP2E1 mRNA distribution similar to that of CYP2E1 protein. CYP2C11 protein expression in renal cortical microsomes was approximately 10% of that in liver microsomes but was significantly higher in microsomes from PT cells than in those from DT cells. CYP3A1/2 was not detected in microsomes from either cortical, PT, or DT cells, but was detected in microsomes isolated from total liver or kidney cortical homogenates. CYP2B1/2 expression was detected in all tissues tested. The peroxisomal proliferator clofibrate enhanced the level of CYP2B1/2 in microsomes from both total liver and kidney cortical homogenates but not in microsomes from cortical, PT, or DT cells. CYP4A2/3 protein and CYP4A mRNA expression were detected in microsomes from total liver and kidney cortical homogenates and from renal cortical, PT, and DT cells using Western and Northern blot analyses, respectively. Lauric acid hydroxylation activity, an indicator of CYP4A, was comparable in PT and DT cells. Clofibrate elevation of CYP4A in cortical, PT, and DT microsomes was not as great as that detected in total kidney cortical microsomes. These results establish the distribution of several P-450 isoenzymes between different cell populations of the rat kidney. Furthermore, these results present evidence that the level of induction of certain P-450 isoenzymes in the kidney is cell type-specific.     

Identification of the rabbit and human cytochromes P-450IIIA as the major enzymes involved in the N-demethylation of diltiazem

Oxidative metabolism of diltiazem (DTZ), a calcium channel blocker, was investigated in rabbit and human liver microsomes as well as in primary cultures of human hepatocytes. DTZ N-demethylation, the major metabolic pathway in man, was strongly increased by treatment of animals, patients, and hepatocyte cultures with rifampicin and other inducers of the P-450IIIA subfamily. In a reconstituted system with purified forms of P-450 and NADPH cytochrome P-450 reductase, P-450IIIA7 exhibited the highest DTZ N-demethylase activity. In both rabbit and human liver microsomes, this activity was highly correlated with erythromycin demethylase, a characteristic substrate of P-450IIIA, or with an immunoquantitated level of P-450IIIA, and was specifically inhibited by anti-P-450IIIA7 polyclonal and monoclonal antibodies. Cyclosporin A, another specific substrate of P-450IIIA in rabbit and human, competitively inhibited DTZ N-demethylase in both species. In primary cultures of human hepatocytes treated with various inducers, including rifampicin, dexamethasone, phenobarbital, phenylbutazone or beta-naphthoflavone, the rate of release of N-demethyl-DTZ in the extracellular medium was highly correlated with the intracellular level of P-450IIIA, which appeared to be strongly induced by rifampicin and phenobarbital and to a lesser extent by dexamethasone and phenylbutazone. In aggregate, these results are consistent with the view that in both rabbit and human, cytochromes P-450 from the P-450IIIA subfamily are the major enzymes involved in the N-demethylation of DTZ. Accordingly, drugs which may be specific substrates or inducers of this P-450 are likely to influence both the side effects and the efficacy of this molecule.     

Exposure to various benzene derivatives differently induces cytochromes P450 2B1 and P450 2E1 in rat liver

Benzene (B), toluene (T), ethylbenzene (EB), styrene (S) and xylene isomers (oX, mX, pX) are important environmental pollutants and B is a proved human carcinogen. Their inhalation by male Wistar rats (4 mg/1,20 h/day, 4 days) caused cytochrome P450 (P450) induction. The degree of P450 2B1 induction increased and that of 2E1 decreased in the series B, T, EB, S, oX, mX and pX, as estimated by Western blots, while neither solvent was as effective for 2B1 induction as phenobarbital and B was more effective for 2E1 than ethanol. The levels of several other P450s decreased after exposure to these solvents, B being most effective. Exposure to these solvents increased in vitro hepatic microsomal oxidation of B and aniline (AN) (2E1 substrates) 3 to 6-fold, indicating induction of this P450. T oxidation was increased 2 to 4-fold and chlorobenzene (ClB) oxidation 3-fold. Sodium phenobarbital (PB, 80 mg/kg/day, 4 days, i.p.) did not increase ethylmorphine (EM) and benzphetamine (BZP) demethylation (2B1 substrates), neither of the B derivatives did so, and oX decreased it; however, pentoxyresorufin O-dealkylation was well related to the immunochemically detected 2B1 levels in control, PB and B microsomes. PB did not increase B, but increased T and C1B oxidation 2–4 and 3-fold, respectively, indicating possible 2B1 role in their oxidation. B oxidation after various inducers was related to immunochemical 2E1 levels, T and C1B oxidation to both 2B1 and 2E1 and AN oxidation to 2E1 and 1A2 levels. Very efficient B oxidation by 2E1 at low B levels indicates that induction of 2E1 may contribute to B myelotoxicity in vivo more than any other P450 enzyme tested, especially considering the fact that B is the most efficient inducer of its metabolism.