[The application of computer assisted credit managing systems in nurses' postgraduate education and continuing education].
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Accounting↗
Biomedical subjects
S S Pan
Publications and source records attributed to S S Pan.
NAD(P)H:quinone oxidoreductase expression and mitomycin C resistance developed by human colon cancer HCT 116 cells.
An association between the resistance to mitomycin C (MMC) and a decrease of NAD(P)H:quinone oxidoreductase (NQO1) activity was reported for a MMC-resistant subline, HCT 116-R30A, derived from MMC-sensitive HCT 116 cells. Eight NQO1 cDNA clones were isolated from these two sublines by reverse transcription-PCR. Two clones, pDT9 from HCT 116 and pDT20 from HCT 116-R30A, are the full length of 274 amino acids. These two clones differ by a T to C substitution at nucleotide 464, which results in a replacement of arginine 139 by tryptophan in the enzyme. NQO1 of pDT9 and pDT20 was expressed in Escherichia coli, purified, and shown to have a protein subunit of M(r) 30,000. The change of amino acid 139 resulted in a shift of isoelectric pH from 9.5 to 8.35 and a 60% decrease of activity in reducing MMC. All of the other six clones differ from pDT9 by a deletion of exon 4. On Northern blot, we detected two mRNA species of NQO1 (1.2 and 2.7 kilobases) due to alternative polyadenylation in all sublines. MMC-resistant sublines showed 75-90% mRNA expression relative to HCT 116 cells. Reverse transcription-PCR amplification of cDNA fragment of nucleotide 298-617 revealed two full-length mRNAs in HCT 116 cells but only one full-length mRNA in HCT 116-R30A cells. An exon 4 deletion mRNA was detected in both sublines. The two full-length mRNAs may be from either alleles or chimeras of the same gene and the exon 4 deletion mRNA is a result of alternative splicing. On Western blot, we detected only one M(r) 30,000 protein in all sublines. A substantial decrease of this protein in MMC-resistant sublines (5% of HCT 116) explained the 95% decrease of their NQO1 activity. Transcriptional regulation and posttranscriptional modification may be responsible for the disparity of gene expression of NQO1 and the low concentration of NQO1 protein in MMC-resistant sublines. Reversal of MMC resistance and the recovery of NQO1 in two revertants further supports the hypothesis that cellular control of NQO1 can modulate the cytotoxicity of MMC.
Base Sequence↗
The role of NAD(P)H:quinone oxidoreductase in mitomycin C- and porfiromycin-resistant HCT 116 human colon-cancer cells.
A mitomycin C (MMC)- and porfiromycin (PFM)-resistant subline of the HCT 116 human colon-cancer cell line was isolated after repeated exposure of HCT 116 cells to increasing concentrations of MMC under aerobic conditions. The MMC-resistant subline (designated HCT 116-R30A) was 5 times more resistant than the parent cells to MMC and PFM under aerobic conditions. Both the MMC-resistant cells and the parent HCT 116 cells accumulated similar amounts of PFM by passive diffusion, but levels of macromolecule-bound PFM were about 50% lower in the resistant cell line, implying a decrease in PFM reductive activation in the resistant cells. The finding that microsomes from either sensitive or resistant cells showed an equal ability to reduce MMC and PFM indicated that the activity of NADPH cytochrome P-450 reductase (EC 1.6.2.4) was not changed in the resistant subline. Soluble extracts of HCT 116 cells reduced MMC and PFM more effectively at pH 6.1, and NADH and NADPH were utilized equally well as electron donors under both aerobic and anaerobic conditions. These data suggest that quinone reductase (EC 1.6.99.2; DT-diaphorase) in soluble extracts is responsible for the reduction of MMC. Quinone reductase activities in soluble extracts of HCT 116-R30A cells for the reduction of dichlorophenol indophenol (DCPIP) and menadione-cytochrome c at optimal pHs were decreased by 95% as compared with those obtained in parent cells. However, the MMC-reducing activity of HCT 116-R30A soluble extracts was only 50% lower than that of the parent cell extracts. The kinetic constants (Km, Vmax) found for quinone reductase in the two cell lines with respect to the substrates DCPIP and menadione differed. Two species of mRNA for quinone reductase (2.7 and 1.2 kb) were detected in both cell lines, and there was no detectable difference between parent and resistant cells in the steady-state level of either of these mRNA species. Furthermore, incubation with the quinone reductase inhibitor dicoumarol rendered HCT 116 cells more resistant to MMC. Alteration of the quinone reductase activity in HCT 116-R30A cells appears to be the mechanism responsible for their resistance to MMC and PFM.
2,6-Dichloroindophenol↗
Germ-cell deficient (gcd), an insertional mutation manifested as infertility in transgenic mice.
A genetic analysis is necessary to gain a greater understanding of the complex developmental processes in mammals. Toward this end, an insertional transgenic mouse mutant has been isolated that results in abnormal germ-cell development. This recessive mutation manifests as infertility in both males and females and is specific for the reproductive organs, since all other tissues examined were histologically normal. A developmental analysis of the gonadal tissues demonstrated that the germ cells were specifically depleted as early as day 11.5 of embryonic development, while the various somatic cells were apparently unaffected. Therefore, the mutated locus must play a critical role in the migration/proliferation of primordial germ cells to the genital ridges of developing embryos. In addition, females homozygous for the mutation could potentially be a valuable animal model of a human syndrome, premature ovarian failure. This mutation has been named germ-cell deficient, gcd.
Animals↗
Interaction of N,N',N''-triethylenethiophosphoramide and N,N',N''-triethylenephosphoramide with cellular DNA.
The antineoplastic agents N,N',N''-triethylenethiophosphoramide (thioTEPA) and N,N',N''-triethylenephosphoramide (TEPA) were studied for their interaction with the DNA of L1210 cells in the presence and absence of rat hepatic microsomes and NADPH. Alkaline elution was used to study 3 types of DNA lesions. When L1210 cells were incubated with thioTEPA alone, or with thioTEPA in the presence of microsomes and NADPH, no single-strand breaks were detected. However, incubation of L1210 cells for 2 h with thioTEPA, at concentrations greater than or equal to 100 microM, caused a dose-dependent increase in interstrand cross-linking that reached a maximum by 2 h after drug exposure. In the presence of rat hepatic microsomes and NADPH, this cross-linking was eliminated, but a different DNA lesion, alkali-labile sites, was produced. These alkali-labile sites were partially reparable with maximum repair achieved by 2 h after removal of drug. ThioTEPA was greater than 85% consumed by the microsomal incubation conditions employed, and TEPA was the only product of the microsomal metabolism of thioTEPA. Alkaline elution studies of L1210 cells that had been incubated with TEPA, alone or in the presence of microsomes and NADPH, demonstrated an elution pattern identical to that produced by thioTEPA in the presence of microsomes and NADPH. Lymphoblastoid cell lines derived from patients with Fanconi's anemia were far more sensitive to thioTEPA and mechlorethamine hydrochloride than were lymphoblasts derived from normal humans, but this hypersensitivity was not noted with TEPA or bleomycin. This is consistent with the known hypersensitivity of cells from patients with Fanconi's anemia to agents that produce interstrand cross-links and with the alkaline elution studies described above. In contrast, lymphoblastoid cell lines derived from patients with ataxia telangiectasia were no more sensitive to thioTEPA than were lymphoblasts derived from normal humans but were far more sensitive to bleomycin. One of these cell lines proved hypersensitive to TEPA, whereas the other was no more sensitive to TEPA than were lymphoblasts from normal humans. Our data imply that thioTEPA produces interstrand cross-links but that TEPA, the primary metabolite of thioTEPA, produces DNA lesions that are alkali labile.
Animals↗
Mechanisms for the modulation of alkylating activity by the quinone group in quinone alkylating agents.
Previous studies have demonstrated that the quinone group may play an important role in modulating the alkylating activity of quinone alkylating agents. Introduction of a quinone moiety markedly increased the alkylating activity and cytotoxic activity of the model quinone alkylating agents benzoquinone mustard and benzoquinone dimustard. However, the cytotoxic and DNA-damaging activity of benzoquinone mustard was considerably greater than that of benzoquinone dimustard. In this study, we have investigated the role of the quinone group as a modulator of alkylating activity in these antitumor agents, using extracellular assays to eliminate differences due to cellular drug uptake and metabolism. Evidence was obtained that the alkylating activities of both benzoquinone mustard and benzoquinone dimustard were enhanced by reduction of the quinone group. In addition, when these agents were reduced, they displayed equal alkylating activity. This finding suggests that the difference in the activity of these agents in cells is not due to intrinsic differences in alkylating activities of the activated forms of these agents. Electrochemical studies revealed that benzoquinone dimustard has a lower redox potential than benzoquinone mustard and, thus, is less easily reduced. Inactivation and spectroscopic studies suggested that a major reason for the differences in activity between benzoquinone mustard and benzoquinone dimustard may be the rapid inactivation of the dimustard before its reduction. This effect may be enhanced by the lower redox potential of benzoquinone dimustard, compared with benzoquinone mustard. These findings support the hypothesis that the quinone group can modulate the alkylating activity of quinone alkylating agents; however, the mechanisms by which this modulation occurs may vary for different antitumor agents.
Alkylating Agents↗
Porfiromycin disposition in oxygen-modulated P388 cells.
The cytotoxicity, metabolism, and DNA alkylation of porfiromycin (PFM) under aerobic and hypoxic conditions were evaluated in P388 murine leukemia cells. Clonogenic assays showed that the IC50 value for a 1-h exposure to PFM was 4 microM for aerobic cells and 0.5 microM for hypoxic cells. After a 1-h exposure to concentrations of 1, 5, and 10 microM [14C]-PFM, the accumulation of total radioactivity in hypoxic cells was 10 to 20 times that in aerobic cells. The disposition of radioactivity in cells that had been treated for 1 h with 5 microM PFM under aerobic or hypoxic conditions showed that (a) under either condition, internal free-PFM concentration equalled the external drug concentration; (b) DNA-, RNA-, and protein-bound radioactivity were at least 10 times greater in hypoxic cells than in aerobic cells; and (c) known metabolites and unidentified radioactive products were also generated in greater amounts in hypoxic cells than in aerobic cells. Thus, the increased amounts of radioactivity accumulated by hypoxic P388 cells after exposure to [14C]-PFM resulted from the accumulation of nonexchangeable protein and nucleic-acid adducts and metabolites rather than free PFM. Determinations of DNA adducts formed in P388 cells revealed five possible adducts: (1) N2-(2'-deoxyguanosyl)-7-methylaminomitosene, (2) a second monofunctional PFM-guanine adduct, (3) a PFM cross-linked dinucleotide, (4) possibly a nucleoprotein-related adduct, and (5) an unknown. We conclude that the enhancement of PFM-induced cytotoxicity by hypoxia appears to be primarily due to increased alkylation of macromolecules.
Aerobiosis↗
Cellular transport and accumulation of thiotepa.
Because the transport and accumulation of N,N',N''-triethylenethiophosphoramide (thiotepa) by cells has not been characterized, these processes were investigated with [14C]thiotepa and cultured L1210 or freshly obtained human or avian RBCs. The octanol: phosphate-buffered saline (PBS) partition coefficient of thiotepa was 2.4 +/- 0.1 (n = 8). With this value, the permeability coefficient (Ps) for thiotepa was estimated to be between 2.8 x 10(-4) and 1.81 x 10(-3) cm/sec, and the half-life of accumulation of thiotepa by L1210 cells was estimated to be 0.063 to 0.40 seconds. Thiotepa accumulation by cells was measured after incubation of cells with [14C]thiotepa and subsequent harvesting of cells by centrifugation through silicone fluid. Thiotepa accumulation by L1210 cells was biphasic. The initial phase was rapid and essentially complete by 10 seconds. The amount of cell-associated 14C increased linearly with increasing extracellular concentrations of thiotepa or with increasing size of the cell pellet. The absolute amount of cell-associated 14C was consistent with that expected if the [14C]thiotepa had been evenly distributed in the incubation medium and a volume equal to that of the cell pellet had been sampled and counted. This rapid phase of thiotepa accumulation was not slowed when cells were incubated on ice. The second phase of [14C]thiotepa accumulation occurred at a rate much slower than that of the initial phase. This slower phase of drug accumulation was linear for at least 5 hours. The rate of 14C accumulation increased progressively over a range of extracellular thiotepa concentrations from 5 to 100 nmol/mL and could not be saturated under acceptable tissue culture conditions. The slower rate of 14C accumulation was ablated by incubating cells on ice and was reduced by 30% to 50% in the presence of 1mM of sodium azide or 2,4-dinitrophenol. The slow rate of accumulation of 14C reflected summation of a relatively stable or constant amount of exchangeable 14C and an amount of nonexchangeable 14C that increased linearly from almost undetectable levels at the start of the experiment to amounts equal to 64 +/- 11% of total cellular radioactivity after 5 hours. The initial association of [14C]thiotepa with both human and avian RBCs was also very rapid. Avian RBCs also exhibited a slow rate of 14C accumulation that was linear for at least 5 hours but that was 15% to 20% that of L1210 cells.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
Mitomycin antibiotic reductive potential and related pharmacological activities.
Relationships of reductive potential, kinetics of enzymatic reduction, augmented oxygen consumption, and cytotoxicity were determined for seven clinically relevant mitomycin antibiotics. Potentials for one-electron reduction were obtained by cyclic voltammetry analysis in dimethyl sulfoxide with 0.1 M tetraethyl-ammonium perchlorate. These potentials were -0.55 V for N7-acetylmitomycin C, -0.61 V for mitomycin A, -0.75 V for N7-(p-hydroxyphenyl)mitomycin C, -0.79 V for N7-(dimethylamino-methylene)mitomycin C, -0.81 V for N7-(2-(4-nitrophenyldithio)-ethyl)-mitomycin C, -0.81 V for mitomycin C, and -0.89 V for porfiromycin. All seven antibiotics were reduced by xanthine oxidase and NADPH-cytochrome P450 reductase, but the rate of reduction varied for each antibiotic and each enzyme. The less negative the reductive potential of an antibiotic, the more easily that antibiotic was reduced enzymatically. These seven mitomycin antibiotics also augmented oxygen consumption by rat liver microsomes. As with their reduction by xanthine oxidase and NADPH-cytochrome P450 reductase, the less negative the reductive potential of an antibiotic, the more it augmented oxygen consumption. Cytotoxicity of each antibiotic was assessed by defining the IC50 against HCT 116 human colon carcinoma cells. A relationship between the reductive potential of these antibiotics and their cytotoxicity against HCT 116 cells was also observed.
Animals↗
Cellular transport and accumulation of thiotepa in murine, human, and avian cells.
Because the transport and accumulation of thiotepa by cells has not been characterized, these process were investigated with [14C]thiotepa and cultured L1210 or freshly obtained human or avian RBC. The octanol:phosphate buffered saline partition coefficient of thiotepa was 2.4 +/- 0.1 (n = 8). With this value, the permeability coefficient (P) for thiotepa was estimated to be between 2.8 X 10(-4) and 1.81 X 10(-3) cm/s and the half-life of accumulation of thiotepa by L1210 cells was estimated to be 0.063-0.40 s. Thiotepa accumulation by cells was measured after incubation of cell with [14C]thiotepa and subsequent harvesting of cells by centrifugation through silicone fluid. Thiotepa accumulation by L1210 cells was biphasic. The initial phase was rapid essentially complete by 10 s. The amount of cell-associated 14C increased linearly with increasing extracellular concentrations of thiotepa or with increasing size of the cell pellet. The absolute amount of cell-associated 14C was consistent with that expected if the [14C]thiotepa had been evenly distributed in the incubation medium and a volume equal to that of the cell pellet had been sampled and counted. This rapid phase of thiotepa accumulation was not slowed when cells were incubated on ice. The second phase of [14C]thiotepa accumulation occurred at a rate much slower than that of the initial phase. This slower phase of drug accumulation was linear for at least 5 h. The rate of 14C accumulation increased progressively over a range of extracellular thiotepa concentrations between 5 and 100 nmol/ml and could not be saturated under acceptable tissue culture conditions. The slower rate of 14C accumulation was ablated by incubation cells on ice and was reduced by 30-50% in the presence of 1 mM sodium azide or 2,4-dinitrophenol. The slow rate of accumulation of 14C reflected summation of a relatively stable or constant amount of exchangeable 14C an an amount of nonexchangeable 14C which increased linearly from almost undetectable levels at the start of the experiment to amounts approximately equal to those of exchangeable radioactivity after 5 h. The initial association of [14C]thiotepa with both human and avian RBCs was also very rapid. Avian RBCs also exhibited a slow rate of 14C accumulation which was linear for at least 5 h which was 15-20% that of L1210 cells. Human RBCs did not exhibit a slower rate of 14C accumulation and essentially all of the 14C associated with human RBCs was exchangeable for the 5 h duration of the experiment.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
Mechanism of transport and intracellular binding of porfiromycin in HCT 116 human colon carcinoma cells.
The mechanism of uptake and efflux of porfiromycin (PFM) by HCT 116 human colon carcinoma cells or freshly obtained human RBC was investigated. The time course of uptake of radioactivity upon exposure of HCT 116 cells to [14C]PFM showed one fast and one slow phase of linear increase. The initial phase of PFM uptake was not saturable with external drug concentrations from 2 to 100 microM. PFM accumulation was temperature dependent with a temperature coefficient (Q10 24-37 degrees C) of 2.3 +/- 0.3. PFM uptake was not affected either by individual inhibitors such as 1 mM 2,4-dinitrophenol, sodium azide, iodoacetic acid, ouabain, 0.02 mM oligomycin, p-hydroxylmercuribenzoate, 0.2 mM N-ethylmaleimide, or by combinations of inhibitors. PFM uptake did not demonstrate competitive inhibition by unlabeled PFM and mitomycin C. Efflux of cellular radioactivity was not affected by the above mentioned inhibitors or by verapamil, diltiazem, or trifluoperazine. Only aliphatic alcohols accelerated the initial influx rate. The RBC, however, only exhibited the initial fast accumulation of [14C]PFM, and all the 14C accumulated by RBC was exchangeable. These data demonstrate that the uptake and the efflux of PFM in HCT 116 cells and RBC comprise a passive diffusion process.
Animals↗
Liquid chromatography-thermospray mass spectrometry of DNA adducts formed with mitomycin C, porfiromycin and thiotepa.
High-performance liquid chromatography (HPLC) and thermospray mass spectrometry were combined for the analysis of DNA adducts formed from the interaction of the anticancer drugs mitomycin C, porfiromycin and thiotepa with calf thymus DNA. The adducts formed from reaction of mitomycin C and porfiromycin with DNA were separated from unmodified nucleosides by HPLC on a C18 column and identified by thermospray mass spectrometry. Thiotepa DNA adducts readily depurinated from DNA and were chromatographed and identified by thermospray liquid chromatography-mass spectrometry as the modified bases without the ribose moiety attached. The utility of thermospray mass spectrometry for the identification of microgram quantities of nucleoside adducts and depurinated base adducts of these anticancer drugs was demonstrated.
Animals↗
Involvement of monoamine oxidase and diamine oxidase in the metabolism of the cell differentiating agent hexamethylene bisacetamide (HMBA).
We have previously demonstrated a number of metabolites of hexamethylene bisacetamide (HMBA) in the urine of patients treated with HMBA. These include N-acetyl-1,6-diaminohexane (NADAH), 6-acetamidohexanoic acid (6AcHA), 1,6-diaminohexane (DAH) and 6-aminohexanoic acid (6AmHA). Because these compounds have potential roles in the dose-limiting metabolic acidosis and neurotoxicity associated with HMBA therapy, and are similar in structure to known substrates of monoamine oxidase (MAO) and diamine oxidase (DAO), we investigated the activities of these enzymes in the metabolic interconversion of HMBA metabolites. NADAH (5 mM) was incubated with MAO and aldehyde dehydrogenase. 6AcHA production was verified by gas chromatography-mass spectrometry and quantified by gas chromatography. 6AcHA production was linear for up to 4 hr. Complete inhibition of MAO activity was observed with 2 mM tranyl-cypromine or pargyline. Mouse liver microsomes, which do not contain MAO, did not convert NADAH to 6AcHA and, in control experiments, did not degrade 6AcHA. The HMBA metabolite, DAH, was a substrate for DAO, producing 3,4,5,6-tetrahydro-2H-azepine. Participation of DAO in the metabolism of HMBA implies potential interaction of HMBA and metabolites with polyamine metabolism and may represent a mechanism for HMBA's effects on cellular growth and differentiation. Metabolism of NADAH, also a differentiator, by MAO implies that concurrent use of HMBA and an MAO inhibitor may be clinically useful.
Acetamides↗
Metabolites and DNA adduct formation from flavoenzyme-activated porfiromycin.
Porfiromycin was reductively metabolized by NADPH cytochrome P-450 reductase and xanthine oxidase under anaerobic conditions. The production of metabolites varied with the pH and the contents of the reaction buffer. In Tris buffer, two major metabolites were produced at pH 7.5 and above, whereas one major metabolite was produced at pH 6.5. The three major metabolites were separated and isolated by HPLC. Identification by californium-252 plasma desorption mass spectrometry showed that the two major metabolites from pH 7.5 were (trans) and (cis)-forms of 7-amino-1-hydroxyl-2-methylaminomitosene and the major metabolite from pH 6.5 was 7-amino-2-methylaminomitosene. All three major metabolites showed substitutions at the C-1 position. DNA was alkylated readily by enzyme-activated porfiromycin. Digestion of porfiromycin-alkylated DNA by DNase, snake venom phosphodiesterase, and alkaline phosphatase resulted in an insoluble nuclease-resistant fraction and a soluble fraction. The nuclease-resistant fraction reflected a high content of cross-linked adducts. Upon HPLC analysis, the solubilized fraction contained two monofunctionally linked porfiromycin adducts and a possibly cross-linked dinucleotide. The major adduct was isolated by HPLC and identified by NMR, as N2-(2'-deoxyguanosyl)-7-amino-2-methylaminomitosene. The N2 position of deoxyguanosine appeared as the major monofunctional alkylating site for DNA alkylation by porfiromycin. Thus, mitomycin C and porfiromycin (which differs from mitomycin C only by the addition of a methyl group to the aziridine nitrogen) share the same enzymatic activating mechanism that leads to the formation of the same types of metabolites and the same specificity of DNA alkylation.
Alkylating Agents↗
[Professional morality of nurses in hospitals].
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Attitude of Health Personnel↗
DNA alkylation by enzyme-activated mitomycin C.
After anaerobic reductive activation by either NADPH cytochrome P-450 reductase (EC 1.6.2.4) or xanthine oxidase (EC 1.2.3.2), mitomycin C readily alkylated DNA. When the mitomycin C-alkylated DNA is digested by DNase, snake venom phosphodiasterase, and alkaline phosphatase, only partial release of the monofunctionally linked mitomycin C nucleotide adduct occurs. Cross-linked adducts are not released into dinucleotides but resist nuclease digestion and remain in oligonucleotides and insoluble precipitates. Kinetic analyses show that the nuclease-resistant fraction which is indicative of DNA cross-linking by mitomycin C takes place quite readily. This nuclease-resistant fraction is particularly significant when the amount of total bound mitomycin C is less than 15 mumol/mmol of DNA. The cross-linked mitomycin C product accounts for more than half of the total alkylation under all pH conditions tested. Our data suggest that particular DNA sites are available for DNA cross-linking by mitomycin C, and these sites are probably the preferred and immediate alkylating targets. Furthermore, DNA cross-links by mitomycin C are not the secondary product of monofunctional adducts. Activity of both flavoenzymes is pH dependent, hence, mitomycin C activation and the rate of DNA alkylation are pH dependent. At elevated mitomycin C alkylation of DNA, the highest amount of cross-linking occurs at neutral pH. High pressure liquid chromatographic separation of the nuclease-digested DNA detected one major and two less prominent mitomycin C adducts. These were verified to be mononucleotide mitosene types by UV spectra showing maximum absorbance at 312 and 250 nm. The major adduct was purified and identified as O6-(2'-deoxyguanosyl)-2,7-diaminomitosene by NMR, indicating that the O6 position of guanine is a preferred site in DNA for at least monofunctional linkage formation.
Alkylating Agents↗
Reductive activation of mitomycin C and mitomycin C metabolites catalyzed by NADPH-cytochrome P-450 reductase and xanthine oxidase.
Under anaerobic conditions and with proper electron donors, NADPH-cytochrome P-450 reductase (EC 1.6.2.4) and xanthine oxidase (EC 1.2.3.2) similarly reductively metabolized mitomycin C. Reversed phase high performance liquid chromatography was used to separate, detect, and isolate several metabolites. Three metabolites were identified by mass spectrometry and thin layer chromatography as 1,2-cis- and trans-2,7-diamino-1-hydroxymitosene and 2,7-diaminomitosene. Three metabolites were phosphate-dependent, and two of them were identified to be 1,2-cis- and trans-2,7-diaminomitosene 1-phosphate. The amounts of the five identified metabolites generated during the reduction of mitomycin C varied with pH and nucleophile concentration. At pH 6.5, 2,7-diaminomitosene was essentially the only metabolite formed, whereas from pH 6.8 to 8.0, trans- and cis-2,7-diamino-1-hydroxymitosene increased in quantity as 2,7-diaminomitosene decreased. The disappearance of mitomycin C and the production of metabolites were enzyme and mitomycin C concentration-dependent. Substrate saturation was not reached for either enzyme up to 5 mM mitomycin C. Electron paramagnetic resonance studies demonstrated the formation of mitomycin C radical anion as an intermediate during enzymatic activation. Our results indicate that either enzyme catalyzed the initial activation of mitomycin C to a radical anion intermediate. Subsequent spontaneous reactions, including the elimination of methanol and the opening of the aziridine ring, generate one active center at C-1 which facilitates nucleophilic attack. Simultaneous generation of two reactive centers was not observed. All five primary metabolites were metabolized further by either flavoenzyme. The secondary metabolites exhibited similar changes in their absorbance spectra and were unlike the primary metabolites, suggesting that a second alkylating center other than C-1 was generated during secondary activation. We propose that secondary activation of monofunctionally bound mitomycin C is probably a main route for the bifunctional binding of mitomycin C to macromolecules and that the cytotoxic actions of mitomycin C result from multiple metabolic activations and reactions.
Animals↗
[The length of digestive tract intubation under fibrogastroscopic guidance].
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Adolescent↗