Concentrations Of Mitomycin C Research Articles

Effect of modified trabeculectomy on the treatment of glaucoma secondary in uveitis

Objective To investigate the effect of trabeculectomy combined with segmental iridectomy, mitomycin C (MMC) and viscoelastic agents usage on the treatment of glaucoma secondary in uveitis. Methods According to the age, degree of inflammation and the condition of Tenon capsule of patients, differ-ent concentration of MMC (0.25-0.33 mg/ml) was used during the operation, with separation of the anterior and posterior synechia, resection of pupillary organization membrane using viscoelastic agents. Segmental iridec-tomy and releasable sutures were also performed on the patients. The visual acuity of preoperation and postoper-ation, intraocular pressure, inflammation and the complication were record. Results Forty-two eyes of 38 cases with glaucoma secondary in uveitis were studied, the mean follow-up time was (12.01±3.56) months. The postoperative visual acuity improved in 14 eyes, didn't change in 28 eyes. The postoperative inflammation of anterior chamber disappeared in 35 eyes, relieved in 7 eyes. And the average postoperative intraocular pres-sure (15.20± 4.64) mmHg was significantly lower than the preoperative intraocular pressure (38.37±12.93) mmHg (t = 8.255, P = 0.000). The total success rate was 92.9%. There were no severe complication. Conclusion Trabeculeetomy combined with MMC, viscoelastic agents usage, separation of anterior and poste-rior syneehia, segmental iridectomy and releasable suture could increase the success rate of operation on pa tients with glaucoma secondary in uveitis, decrease the complication and inflammation reaction of operation, and the recurrence of uveitis. Key words: Uveitis; Glaucoma; Trabeculectomy; Microsurgical operation

Long-term Efficacy of Maintenance Bacillus Calmette-Guérin versus Maintenance Mitomycin C Instillation Therapy in Frequently Recurrent TaT1 Tumours without Carcinoma In Situ: A Subgroup Analysis of the Prospective, Randomised FinnBladder I Study with a 20-Year Follow-up

BackgroundThe long-term prospective data on bacillus Calmette-Guérin (BCG) and mitomycin C (MMC) instillation therapy are limited. ObjectiveTo compare the long-term benefit of BCG and MMC maintenance therapy in patients with recurrent bladder carcinoma. Design, setting, and participantsEighty-nine patients with frequently recurrent TaT1 disease without carcinoma in situ (CIS) were eligible. Originally, the patients were enrolled in the prospective FinnBladder I study between 1984 and 1987 and randomised to receive BCG or MMC. Both regimens involved five weekly instillations, followed by monthly instillations for 2 yr. Because of alkalinising the urine and adjusting the dose to bladder capacity, the average concentration of MMC was low: 30–40mg in 150–200ml of phosphate buffer. Overall median follow-up time was 8.5 yr, whereas the median follow-up time of the patients who were still alive was 19.4 yr. MeasurementsPrimary end points were time to first recurrence and overall mortality. Secondary end points were progression and disease-specific mortality. Results and limitationsThirty-six of 45 patients (80.0%) in the MMC group experienced recurrence in contrast to 26 of 44 patients (59.1%) in the BCG group. This finding was reflected in significantly lower cumulative incidence estimates in the BCG group (p=0.005). There was a weak trend for fewer progressions (p=0.1) and cancer-specific deaths (p=0.2) in the cumulative incidence analysis, as 4 patients versus 10 patients progressed and 4 patients versus 9 patients died from the disease in the BCG group versus the MMC group, respectively. No difference existed in the overall mortality. The study population, however, was too small for conclusive evidence about progression or survival. ConclusionsAn intensive intravesical BCG immunotherapy results in a sustained and significant long-term reduction in recurrence in frequently recurrent bladder carcinoma. The relatively low progression rate during the long follow-up suggests that it may be difficult to show significant differences in overall mortality with a substantially larger but otherwise similar study population. Trial registrationRegistration was not considered to be necessary at this stage of the follow-up because the study was initiated as early as 1984 and the last randomisation took place in July 1987, that is, long before the current requirements concerning study registrations were implemented.

Transconjunctival penetration of mitomycin C

The study was performed to estimate transconjunctival penetration of mitomycin C (MMC) to Tenon's tissue following application over the intact conjunctiva before routine trabeculectomy. Institution-based case series. In 41 eyes of 41 patients, MMC (0.4 mg/ml for 3 min) was applied over the intact conjunctiva before beginning trabeculectomy. Tenon's capsule directly beneath the site of application was excised during trabeculectomy and was homogenized, centrifuged and MMC concentrations were analyzed using high-performance liquid chromatography (HPLC). Statistical analysis was performed using STATA 8.0 version software (STATA Corporation, Houston, TX, USA). In this study, P -values less than 0.05 were considered as statistically significant. The average weight of the sample of Tenon's tissue excised was 5.51+/-4.42 mg (range: 0.9-17.1) and the average estimated MMC concentration found to be present in Tenon's tissue using HPLC was 18.67+/-32.36 x 10(-6) moles/kg of the tissue (range: 0.38-197.05 x 10(-6)). In 36 of the 41 patients (87.80%), the MMC concentration reached above 2 x 10(-6) moles/kg of the tissue concentration required to inhibit human conjunctival fibroblasts. Mitomycin C does permeate into the subconjunctival tissue after supraconjunctival application for 3 min. Application of MMC over the conjunctiva may be a useful alternative to subconjunctival or subscleral application during routine trabeculectomy and as an adjunct for failing blebs.

Comparison of Standard (0.02%) and Low Dose (0.002%) Mitomycin C in the Prevention of Corneal Haze Following Surface Ablation for Myopia

To retrospectively compare the safety and efficacy of lower dose mitomycin C (MMC) (0.002%) to that of the standard dose (0.02%) in eyes treated with photorefractive keratectomy (PRK) for myopia. The clinical efficacy of 95 eyes receiving myopic PRK with a standard concentration of MMC (0.02%) is sequentially compared to 126 eyes receiving low dose MMC (0.002%). The topical exposure times for MMC varied between 30 seconds and 2 minutes in both groups, and direct contralateral eye comparison of these two exposure times was analyzed in a prospective subset of 39 patients from among the low dose group. Patients were examined preoperatively and postoperatively at 1, 3, 6-9, and 9-12 months. Haze, visual acuity, and efficacy ratio outcomes were analyzed. The preoperative findings were overall statistically similar, except for higher spherical equivalent refractive error (P = .007) and best spectacle-corrected visual acuity (P = .007) in the standard MMC group. Postoperatively, haze levels ranged from 0 to 4+. With multivariable analysis, significantly less haze was noted among the standard dose MMC eyes for high myopia and higher ablation depth at all postoperative time points. In contrast, the haze levels were statistically similar for moderate myopia and lower ablation depths at the latter postoperative time points. The subset of contralateral eyes randomly receiving low dose MMC (0.002%) at either 30 seconds or 2 minutes exposure showed no significant difference in haze between these exposure times. The standard concentration of topical MMC (0.02%) is more effective than low dose MMC (0.002%) in preventing postoperative haze following surface ablation for myopia > or = -6.00 D and deeper ablation depth > or = 75 microm. However, for moderate myopia and shallow depth, low dosing appears to be equally effective. The duration of MMC exposure appears to be less important than its concentration.

Corneal Opacity Caused by LASEK with Improper High-concentrated Mitomycin-C

Purpose: To report a case of corneal opacity with postoperative changes of topographic features and visual acuity after laser subepithelial keratomileusis (LASEK) treated with an improper concentration of mitomycin-C. Case summary: A 25-year-old female patient who experienced corneal opacity after LASEK treated with mitomycin-C was evaluated for changes in corneal opacity by photography, central corneal power by Orb scan Ⅱz, and best corrected visual acuity (BCVA) over the period of 1 year. The corneal epithelial defect was completely healed 2 weeks after the LASEK operation. Subepithelial and stromal opacity decreased over time, but her cornea did not return to normal. BCVA was 20/25 in the right eye and 20/20 in the left eye at postoperative 1 year, but stromal opacity remained. Conclusions: An improper concentration of mitomycin-C during LASEK could result in corneal opacity. Long time is needed to recover visual acuity and a normal corneal structure.

Investigations on Permeation of Mitomycin C Through Double Layers of Natural Rubber Gloves

Treating peritoneal carcinomatosis by the aggressive cytoreductive surgery with the hyperthermic intraoperative intraperitoneal chemotherapy (HIPEC) surgeons expose their gloved hands for up to 90 min to a peritoneal dialysis solution (PDS) containing mitomycin C (MMC). We investigated the permeation of MMC through the material of three different natural rubber gloves under conditions similar to the in-use during HIPEC as well as under worst-case exposure scenario. Two different methods, a two-chamber diffusion cell and a single-chamber glass chamber method, were used to demonstrate the permeation capability. The permeation of MMC dissolved in 0.9% NaCl solution and PDS through double natural rubber glove material was tested over 2 h using four concentrations (c = 0.004, 0.008, 0.016 and 0.4 mg ml(-1)) and three receptor fluids (0.9% NaCl solution, PDS and a novel artificial sweat). In none of four glass chamber experiments and in only one of 40 diffusion cell experiments was permeation through glove material detected. The permeation occurred between 15 and 30 min under worst-case exposure scenario at a approximately 100-fold higher MMC concentration than under in-use conditions during HIPEC. The double-layer natural rubber gloves tested were effective to prevent a permeation of MMC in vitro under HIPEC-similar exposure. Our results support the glove wearing procedure in our university hospital. However, occupational exposure to antineoplastic drugs should be minimized, since there is insufficient knowledge regarding harmful effects from a long-term exposure to low doses.

Electrochemical investigation of interaction between mitomycin C and DNA in a novel drug-delivery system

A novel drug-delivery system was developed by loading the anticancer drug, mitomycin C (MC) into an oil/water system with the aim of investigation by electrochemical sensing the interaction between the drug and DNA in microemulsion phase. The physical and physicochemical properties (droplet size, pH, viscosity, conductivity and refractive index) of this microemulsion were examined. The electrochemical detection of the interaction between MC and double-stranded DNA (dsDNA) in microemulsion phase was performed by using differential pulse voltammetry (DPV) in combination with a disposable sensor, pencil graphite electrode (PGE). The magnitude of guanine oxidation signal was monitored before and after interaction between MC and dsDNA. The effect of different experimental parameters, such as MC concentration, MC interaction time with dsDNA, and dsDNA concentration were also studied to find the optimum analytical performance based on electrochemical detection of this interaction in microemulsion phase.

Mitomycin-C Concentration in Cornea and Aqueous Humor and Apoptosis in the Stroma After Topical Mitomycin-C Application

To evaluate the effects of the applied mitomycin-C (MMC) concentration and application time on the aqueous MMC concentration and apoptosis in the corneal stroma. New Zealand white rabbits underwent mechanical epithelium debridement of the central 7.5 mm of the cornea. A sponge soaked in MMC solution was placed on the denuded corneal stroma. The effect of the exposure times ranging from 15 to 120 seconds and the different MMC concentrations ranging from 0.005% to 0.04% on the aqueous MMC concentration and the apoptosis in the stromal cells were evaluated. The aqueous concentration of MMC increased linearly with increasing exposure time and MMC concentration. The correlation between the aqueous MMC concentration and the applied concentration (r = 0.809, P < 0.001) was higher than the correlation between the aqueous MMC concentration and the application time (r = 0.693, P < 0.001). Terminal deoxyribonucleotidyltransferase-mediated dUTP-digoxigenin nick end labeling (TUNEL)-positive cells were detected in the superficial stroma of the central denuded cornea. The numbers of TUNEL-positive cells increased linearly with increasing concentrations, and the increase was statistically significant (P = 0.026). However, the numbers of TUNEL-positive cells increased only slightly with an increasing application time, and the increase was not statistically significant (P = 0.928). Reducing the applied concentration or decreasing the exposure time might be a good modality for reducing the potential MMC toxicity. The applied MMC concentration had greater effects on the aqueous MMC concentration and apoptosis in the stromal cells than the exposure time.

Concentrations of Mitomycin C in Rabbit Corneal Tissue and Aqueous Humor After Topical Administration

To study the aqueous and corneal pharmacokinetics of mitomycin C (MMC) after single topical administration to the central cornea and to evaluate the effects of different concentrations and different application times on the aqueous concentration of MMC. Mechanical epithelium debridement of the central 7.5 mm of the cornea was performed in New Zealand white rabbits, and a sponge soaked in 0.02% MMC solution was placed on the denuded corneal stroma for 2 minutes. Aqueous fluid and central corneal tissues samples were taken at 0.5, 1, 2, and 3 hours thereafter. MMC concentration of the samples was analyzed by high-performance liquid chromatography and evaluated at different exposure times (range: 15-120 seconds) and concentrations of applied MMC (range: 0.005%-0.04%). Peak corneal concentration was 3.728 +/- 2.547 microg/g at 30 minutes after topical administration. Maximum aqueous concentration was 0.380 +/- 0.038 microg/mL at 1 hour after topical application. The aqueous concentration of MMC increased in a dose-dependent manner with increasing exposure time and application concentration. Aqueous MMC concentration increased at a higher rate with change of applied concentration than with exposure time. Good penetration of MMC through central bare cornea may be noxious to endothelial cells. Reducing concentration or decreasing exposure time seems a good modality to reduce potential MMC toxicity.

High Incidence of Hematopoietic Stem Cell Mosaicism in Fanconi Anemia.

Determination of the degree of somatic mosaicism providing functional correction of Fanconi anemia (FA) hematopoiesis has direct implications for gene therapy for FA: it may help assess the percentage of FA hematopoietic cells corrected by gene therapy approaches that are needed to achieve clinically meaningful effects. Hypersensitivity to DNA interstrand cross-linking agents, such as diepoxybutane (DEB) and mitomycin C (MMC), is a cellular marker for diagnosis of FA. However, in some FA patients a population of DEB-resistant PHA-stimulated lymphoblasts (PHA-L) was observed, and this population sometimes varied over time. To assess the significance of this finding on hematopoietic function, we evaluated the MMC sensitivity of bone marrow mononuclear cells (BMMC) and DEB sensitivity of PHA-L and cultured lymphoblastoid cell lines (LCL) in 42 consecutive FA patients referred to the University of Minnesota. In cases where LCL were DEB-resistant, cultured fibroblasts were also studied. BMMC were cultured in the presence of increasing concentrations of MMC. PHA-L and LCL were cultured in DEB at 0.1 mcg/ml. Wild type BM progenitors (N = 17 subjects) proliferated regardless of increasing MMC concentrations (albeit at decreased efficiency at the highest concentrations) as follows: 0 MMC (normalized to 100%), 5 nM MMC (99% [standard deviation, SD, 16%]), 10 nM MMC (90% [SD 22%]), 25 nM MMC (77% [SD21%]), and 50 nM MMC (44% [SD 30%]). Of the 42 FA patients, BMMC failed to proliferate at 0 nM MMC in 10 patients and at 5 nM MMC in 20 patients. Twelve FA patients had MMC resistant BMMC: cells cultured in 5, 10, 25 and 50 nM MMC grew 44% (SD 28%), 35% (SD 24%), 24% (SD 30%) and 17% (SD 32%) of colony numbers in MMC free culture, respectively. Six of these 12 subjects were PHA-L mosaics as determined by DEB sensitivity testing. Four patients with no growth of BMMC at 0 or 5 nM MMC were also somatic mosaics in their PHA-L and LCL. Thus there was no clear correlation between somatic mosaicism as demonstrated by DEB testing in peripheral blood and sensitivity of BMMC to growth in MMC. Clinically, two patients with hematopoietic somatic mosaicism developed severe marrow aplasia, one of which received hematopoietic stem cell transplantation. Four of the mosaic patients had normal or near normal peripheral blood counts with one patient having clonal hematopoiesis by HUMARA assay and only low levels of metaphases with multiple breaks in multiple DEB studies. While patients with hematopoietic somatic mosaicism had mixed populations of DEB sensitive cells in their peripheral blood, all their fibroblast cultures were DEB sensitive. In summary, these data show that the presence of somatic mosaicism per se does not necessarily prevent bone marrow failure. Moreover, the data suggest that patients with stigmata of FA may have chromosomal breakage studies showing few cells (or no cells) with the characteristic changes of FA; in these cases, skin fibroblasts should be tested as well.

EFFECTS OF TOPICAL APPLICATION OF MITOMYCIN-C AND 5-FLUOROURACIL ON MYRINGOTOMY IN RATS

In Reply: We appreciate Dr. Eken's critical letter to the editor regarding our recent publication and the good review of laser myringotomy. First of all, simple myringotomy and laser myringotomy were not compared in our study. We used mitomycin C (MMC) and 5-fluorouracil with simple myringotomy. As a result, we have found that MMC may prolong myringotomy patency time for about 6 weeks. As we mentioned in our article, Estrem and Batra (1) perforated the tympanic membranes of 60 rats by potassium-titanyl-phosphate laser, applied 2 mg/mL topical MMC, and divided rats into 4 groups according to exposure time and repeat application. The median patency time of the single 10-minute application was 6.5 weeks; of the single 20-minute application, 5.5 weeks; of the biweekly 10-minute-application, 6.5 weeks; and of the biweekly application, for 20 and 10 minutes, was 8.5 weeks. The median patency time of the control group was determined to be 1.5 weeks. We think that this article has proven that the patency time produced by KTP laser alone is very close to that of simple myringotomy, and increased exposure time or repeat application of MMC did not statistically alter the patency rate. Therefore, we think that usage of KTP laser in myringotomy is not effective in prolonging the patency time, and that topical MMC application in appropriate dosage may lengthen the patency time up to 6.5 weeks. When we reviewed studies about myringotomy, mean closure time is mostly between 1 and 3 weeks in laser myringotomy (2,3), and 1 and 3 weeks in thermal myringotomy (4). However, when we quickly checked literature about MMC application with simple myringotomy or myringotomy by using laser-thermal equipments, mean closure time is more than 5 weeks (Table 1). Along with the literature, we think that MMC is superior to laser and thermal myringotomy. A novel study that was conducted on 180 ears of 96 patients showed that MMC can prolong myringotomy closure time in patients with otitis media with effusion (mean, 5.3 wk) and in patients with recurrentacute otitis media (mean, 7 wk) (5). Ragab (5) and Jasir et al. (6) found that the dose of 0.4 mg/mL MMC is almost the optimal dose, and the novel study used the same dose in patients. There is a concentration-response curve for topical MMC when used for prolonging myringotomy patency in concentrations up to 0.4 mg/mL. Higher concentrations do not appear to prolong patency and are usually associated with otorrhea, suggesting middle ear toxicity. Also, Jang et al. (7) reported that topical application of MMC in 0.4 mg/mL concentration for 10 minutes has the highest antiproliferative effect on cultured human tympanic membrane fibroblasts. Myringotomy closure time after MMC application depends on some parameters: size of myringotomy, shape of myringotomy, MMC concentration, MMC application way, MMC application duration, status of middle ear (healthy or not), and the species on which the study was applied. Therefore, all studies should be analyzed under the guidance of these parameters. So, our results were expected to be longer because we used topical application of MMC in 0.4 mg/mL concentration. CO2 laser myringotomy experience in our clinic on rats showed that it may prolong myringotomy patency time at least 2.5 weeks (8). However, our results with MMC were longer compared with laser myringotomy. As a result, our mean duration of patency time with MMC is similar to other studies using MMC either with laser or not, and there is no contradiction with previous reports. We have to emphasize that there are a few studies using only MMC after myringotomy, and there are a lot of studies using only laser. In most of these, laser prolongs myringotomy closure time by approximately 2 weeks. In Dr. Eken's literature list, we may easily see that argon and KTP lasers were used with MMC. Therefore, the results are probably dependent on MMC. Our idea is that when MMC is used in proper dosage and duration after simple myringotomy, it may prolong myringotomy patency time as much as that of MMC after laser myringotomy.TABLE 1: Patency duration of myringotomy with different techniquesThanks for your kind interest. Engin Çekin, M.D. Hakan Cincik, M.D. Gülhane Military Medical Academy Haydarpasa Training Hospital Department of Ear, Nose, and Throat Istanbul, Turkey

The effect of mitomycin C in reducing epidural fibrosis after lumbar laminectomy in rats

Extensive epidural fibrosis after lumbar spine surgery might be an important underlying cause of failed-back syndrome. Based on previously obtained data, the effect of mitomycin C (MMC) in a concentration of 0.1 mg/ml on spinal epidural fibrosis in a rat laminectomy model was investigated in a large series. Eighty adult Wistar rats underwent lumbar laminectomy. In 40 rats, MMC in a concentration of 0.1 mg/ml was locally applied to the laminectomy sites. No similar treatment was performed in the other 40 rats. At intervals from one to 12 weeks after laminectomy, both macroscopic and histological evaluations were performed. For radiological investigation, 10 rats underwent magnetic resonance (MR) imaging at 6 weeks postoperatively. Furthermore, the concentration of MMC in cerebrospinal fluid (CSF) and serum was determined 12 hours postoperatively in seven rats. Due to ease of absorption, high levels of MMC were rapidly detectable in serum, whereas the values obtained from the CSF were markedly lower. In the majority of MMC-treated laminectomy sites, epidural scarring was significantly reduced and dural adhesions were absent, in comparison with control sites (p < 0.001), as confirmed by MR images. Accordingly, the macroscopic dissection of epidural fibrous tissue to reexpose the dura mater was performed more easily and without severe bleeding in these rats. The healing of skin and the lumbar fascia was not affected, and dural leakage was not observed. All control sites showed dense epidural fibrosis with marked dural adherence. In this experimental model, it was shown that locally applied MMC in a concentration of 0.1 mg/ml effectively reduces epidural fibrosis and dural adherence without side effects in rats that underwent lumbar laminectomy.

Effect of topical mitomycin C for antrostomy in rabbit with sinusitis

The aim of this study is to evaluate if mitomycin C (MMC), applied topically at the site of maxillary antrostomy in experimentally inflamed maxillary sinuses, may reduce postoperative ostial stenosis without any harmful effect. Twenty-four rabbits with experimentally induced maxillary sinusitis underwent maxillary antrostomy bilaterally. MMC at a concentration of 0.4 or 1 mg/ml was applied at the site of antrostomy on the right whilst the antrostomy site on the left served as the control. Half of each MMC concentration group of animals were killed after 3 weeks, and the remaining half after 6 weeks. For every rabbit, the areas of antrostomies were measured and mucosal specimens around the ostia were obtained for microscopic evaluation at the time of antrostomy and killing. Within each MMC concentration group, MMC-treated antrostomies were significantly wider than those of the controls (P < 0.05), even after 6 weeks. However, comparison of the areas of MMC-treated antrostomies between the two MMC concentration groups did not show any significant difference (P > 0.05). No systemic side effect or permanent mucosal damage was detected. These results suggest that postoperative topical MMC at a concentration of 0.4 mg/ml can be used to prolong the patency of maxillary antrostomy in an inflammed sinus.

Targeted Disruption of FANCC and FANCG in Human Cancer Provides a Preclinical Model for Specific Therapeutic Options

How specifically to treat pancreatic and other cancers harboring Fanconi anemia gene mutations has raised great interest recently, yet preclinical studies have been hampered by the lack of well-controlled human cancer models. We endogenously disrupted FANCC and FANCG in a human adenocarcinoma cell line and determined the impact of these genes on drug sensitivity, irradiation sensitivity, and genome maintenance. FANCC and FANCG disruption abrogated FANCD2 monoubiquitination, confirming an impaired Fanconi anemia pathway function. On treatment with DNA interstrand-cross-linking agents, FANCC and FANCG disruption caused increased clastogenic damage, G2/M arrest, and decreased proliferation. The extent of hypersensitivity varied among agents, with ratios of inhibitory concentration 50% ranging from 2-fold for oxaliplatin to 14-fold for melphalan, a drug infrequently used in solid tumors. No hypersensitivity was observed on gemcitabine, etoposide, 3-aminobenzamide, NU1025, or hydrogen peroxide. FANCC and FANCG disruption also resulted in increased clastogenic damage on irradiation, but only FANCG disruption caused a subsequent decrease in relative survival. Finally, FANCC and FANCG disruption increased spontaneous chromosomal breakage, supporting the role of these genes in genome maintenance and likely explaining why they are mutated in sporadic cancer. Our human cancer cell model provides optimal controls to elucidate fundamental biologic features of individual Fanconi anemia gene defects and facilitates preclinical studies of therapeutic options. The impact of Fanconi gene defects on drug and irradiation sensitivity renders these genes promising targets for a specific, genotype-based therapy for individual cancer patients, providing a strong rationale for clinical trials.

Effects of Mitomycin-C on Normal Dermal Fibroblasts

To evaluate the effects of mitomycin-C on the growth and autocrine growth factor production of human dermal fibroblasts from the face. In vitro study using normal adult dermal fibroblast cell lines in a serum-free model. Cell cultures were exposed to 4 mg/mL, 0.4 mg/mL, 0.04 mg/mL, 0.004 mg/mL, and 0.0004 mg/mL concentrations of mitomycin-C solution. Cell counts were performed, and the cell-free supernatants were collected at 0, 1, 3, and 5 days after the initial exposure. Population doubling times were calculated and supernatants were quantitatively assayed for basic fibroblast growth factor (bFGF) and transforming growth factor (TGF)-beta1. Continuous exposure to mitomycin-C caused fibroblast cell death by day 7 at all tested concentrations. A 4 minute exposure to mitomycin-C at 4 mg/mL caused rapid fibroblast cell death. A 4-minute exposure to mitomycin-C at either 0.4 mg/mL or 0.04 mg/mL resulted in decreased fibroblast proliferation. A 4 minute exposure to mitomycin-C at 0.4 mg/mL resulted in a marked increase in the production of both bFGF and TGF-beta1. A clinically ideal concentration of mitomycin-C would slow fibroblast proliferation yet not cause cell death to allow for a wound healing response. Mitomycin-C 0.4 mg/mL for 4 minutes satisfies the above criteria in vitro.

196 EFFECT OF MITROMYCIN-C ON VIABILITY AND CELL PROLIFERATION OF DOMESTIC CAT AND MOUSE EMBRYONIC FIBROBLASTS

Nondomestic cat cloned embryos created by intergeneric nuclear transfer (ig-NT) have lower rate of blastocyst development than that observed in cloned embryos produced by inter-species NT. One promising technique for increasing the efficiency of ig-NT is to use embryonic stem cells (ESC) derived from the pluripotent inner cell mass (ICM) of pre-implantation blastocysts. Mouse embryonic fibroblasts (MEF) are commonly used as feeder cells to support the growth of human and mouse ESC, but they do not always maintain human ESC in an undifferentiated state and carry the risk of transmitting mouse retroviral virus and other pathogens. Thus, it is important to determine whether domestic cat embryonic fibroblasts (DCEF) are able to support cat ESC growth. Proliferation of MEF cells is inhibited before their use as feeder layers by exposure to gamma irradiation or Mitomycin-C. Therefore, in the present study, we determined what concentration of Mitomycin-C was required to inhibit proliferation of DCEF and MEF cells without affecting viability. Embryonic fibroblasts were generated from fetuses collected from a single pregnant domestic cat and mouse at 30 and 14 days of gestation, respectively. Tissue was minced and cultured in DMEM for 7 to 10 days before freezing and storage at −196°C. After thawed DCEF and MEF cells reached 80 to 100% confluence, proliferation was inhibited by exposure to 10, 30, or 40 µg of Mitomycin-C for 2.5 or 5 h. Cells were then washed and labeled with BrdU to measure DNA synthesis as a specific marker for replication. For cytotoxicity, cells were labeled with a dual fluorescent stain to evaluate membrane integrity and esterase activity. The amounts of DNA-labeled BrdU as well as calcein and ethidium homodimer-1 incorporated into the cells were quantified by flow cytometry (see Table 1). Proliferation of DCEF and MEF cells was affected similarly by both concentration and exposure time to Mitomycin-C, with the highest inhibition of DCEF cells occurring after treatment with 40 μg for 5 h (92% not replicating). Cell viability was influenced by both concentration and exposure time to Mitomycin-C, with the highest survival of DCEF cells occurring after exposure to 30 μg for 5 h (90%) and in MEF cells after exposure to 40 μg for 2.5 h (88% viable). In summary, these results indicate that cell proliferation of DCEF can be inhibited by Mitomycin-C, but a higher dosage and longer exposure time should be considered for use compared to the dosage commonly employed for inhibiting cell proliferation of MEF. High concentrations of Mitomycin-C did not significantly increase cytotoxicity of cells in either species. Table 1.

Effect of Topical Mitomycin C on the Healing of Surgical and Laser Wounds: A Hint on Clinical Application

Mitomycin C (MMC) is used in otolaryngological surgery to reduce complications from postoperative scarring. However, the effects of MMC on wound healing at different doses and on different wound types have not been fully described. The aim of this study was to evaluate the effects of topical MMC at 2 different doses on the healing of surgical and laser wounds. This was a randomized study using 20 rabbits. Six full-thickness wounds were made by excision or laser vaporization on the flank skin. The wounds were randomly treated by topical MMC of 0.4 or 1.0 mg/mL or saline for 5 minutes. Re-epithelialization and contraction of the original wounds were significantly delayed by the use of MMC (P < 0.01). The delayed healing effect of MMC was more prominent in the laser wounds than in the excision wounds. MMC inhibited collagen deposition and fibroblast proliferation of wounds on histological analysis. The 2 different concentrations of MMC showed no difference in gross or histological wound healing characteristics. Our results showed that MMC delays the healing of wounds as a result of the inhibition of fibrosis, especially for laser wounds.

Pharmacokinetics of Mitomycin C after Resection of Peritoneal Carcinomatosis and Intraperitoneal Chemohyperthermic Perfusion

Over the last few years surgery on patients with abdominal malignancies has become more aggressive but the majority of patients present locoregional recurrence as peritoneal dissemination. Cytoreductive surgery followed by intraperitoneal chemohyperthermic perfusion (ICHP) has been described for treatment and prevention of locoregional cancer spread from various origins. This paper reports our study of the pharmacokinetics of mitomycin C (MMC) administered by intraperitoneal chemohyperthermic perfusion (ICHP) in patients with peritoneal carcinomatosis. 28 patients received MMC 20 mg/m2 intraperitoneally as a perfusion over 60 min. MMC was determined in perfusate, plasma and urine samples with a UVHPLC method. A compartmental model was used to fit the drug concentrations in plasma and perfusate.Our results showed a mean maximum plasma concentration (Cmax) of 0.14±0.086 μg/ml with a peak time (Tmax) of 48..7 ± 5.61 min. The mean area under the curve (AUC) and terminal half-life (t1/2 ) were 15.8 ± 9.8 mg x min / L and 83.7 ± 31.74 min respectively. Clearance (CL) was estimated by fitting the data by a compartmental model and the mean value was 72 ± 66 L/h. The percent of the dose absorbed was very variable and ranged between 14 and 57% (mean 37 ± 14%). The mean percentage of dose recovered unchanged in the urine during 24 hours was 7.21 ± 3.73%.We conclude that ICHP in patients with peritoneal surface malignancies seems to have clinical value since it gives high peritoneal and tumor MMC concentrations with limited systemic exposure and toxicity.

Single Perivascular Delivery of Mitomycin C Stimulates p21 Expression and Inhibits Neointima Formation in Rat Arteries

Mitomycin C (MMc) is an antibiotic that exerts a potent antiproliferative effect in tumor cells. Because the proliferation of vascular smooth muscle cells (VSMCs) plays a prominent role in the development of restenosis after percutaneous coronary interventions, the present study examined the effect of MMc on VSMC proliferation and on neointima formation after arterial balloon injury. Treatment of cultured rat aortic VSMCs with MMc (1 nmol to 30 micromol/L) inhibited VSMC proliferation in a concentration-dependent manner. Whereas high concentrations of MMc (1 to 30 micromol/L) induced VSMC apoptosis, as reflected by DNA laddering and caspase-3 activation, lower concentrations of MMc (1 to 300 nmol/L) directly inhibited VSMC growth by arresting cells in the G2/M phase of the cell cycle. The antiproliferative action of MMc was associated with a selective increase in the expression of the cyclin-dependent kinase inhibitor p21, and with a decrease in cyclin B1-cyclin-dependent kinase-1 complex activity. Finally, the local perivascular delivery of MMc immediately after balloon injury of rat carotid arteries induced p21 expression and markedly attenuated neointima formation. These studies demonstrate that MMc exerts a potent inhibitory effect on VSMC proliferation and neointima formation after arterial injury. MMc represents a potentially new therapeutic agent in treating and preventing vasculoproliferative disease.

Studies on the action of mitomycin C and bleomycin on telomere lengths of human lymphocyte chromosomes

In order to address the problem of the action of cytostatics on chromosome ends, telomere length was measured in human lymphocyte cultures exposed to mitomycin C (MMC) and bleomycin (BLM). Telomere-specific PNA probes were used for the quantitative estimation of the relative telomere length of each individual chromosome by fluorescence in situ hybridization. A high inter-cellular and inter-individual variability of relative telomere lengths was found throughout all experiments. Different responses could be observed with respect to the action of the examined mutagens: The total average fluorescence intensity of labeled telomere repeats was decreased under the action of MMC in two of the experiments, while two revealed no significant alteration. BLM caused no significant change of total average telomeric signal intensity in four, a clear decrease in one of the six experiments, and an increase in another. Although all chromosome ends contributed to the observed trends, single telomeres were affected in a very distinct way. The highest concentration of MMC (1 microg/ml) induced significant shortening of telomeres of the chromosome arms; 2q, 3p, 5q, 7p, 10q, 11p, 13q, 17p, 18p&q, and 21q in two independent experiments. In one BLM experiment with 8 microg/ml, the most distinct decrease (p< or =0.005) of telomeric fluorescence was found at the ends of chromosome arms; 1q, 6p, 17p, 20p&q, and 22q. The increase of telomeric signal intensity affected the telomeres of some individual chromosome arms more than others, e.g. 4q, 6p, 7p, 8p, 13p, and 18q. Although the telomere length of the individual chromosome arms varied widely, clear trends could be observed with respect to the rank which was occupied by telomeric length of the various chromosome arms. The telomeres of the 1p, 3p, 4q, 5p, 12q, and 13q chromosome arms throughout all experiments were among the longest; and those of 13p, 15p, 21p, and 22p were among the shortest telomeres of the karyotype. From these data, it can be concluded that MMC affects the telomeric repeat area of chromosomes more than BLM, which mostly had no significant effect on telomere length in the performed experiments.