Trace levels of mitomycin C disrupt genomic integrity and lead to DNA damage response defect in long-term-cultured human embryonic stem cells.

How to maintain the genetic integrity of cultured human embryonic stem (hES) cells is raising crucial concerns for future clinical use in regenerative medicine. Mitomycin C(MMC), a DNA damage agent, is widely used for preparation of feeder cells in many laboratories. However, to what extent MMC affects the karyotypic stability of hES cells is not clear. Here, we measured residual MMC using High Performance Liquid Chromatography-Mass Spectrometry/Mass Spectrometry following each step of feeder preparation and found that 2.26 ± 0.77 and 3.50 ± 0.92 ng/ml remained in mouse feeder cells and human feeder cells, respectively. In addition, different amounts of MMC caused different chromosomal aberrations in hES cells. In particular, one abnormality, dup(1)(p32p36), was the same identical to one we previously reported in another hES cell line. Using Affymetrix SNP 6.0 arrays, the copy number variation changes of the hES cells maintained on MMC-inactivated feeders (MMC-feeder) were significantly more than those cultured on γ-inactivated feeder (IR-feeder) cells. Furthermore, DNA damage response (DDR) genes were down-regulated during long-term culture in the MMC-containing system, leading to DDR defect and shortened telomeres of hES cells, a sign of genomic instability. Therefore, MMC-feeder and MMC-induced genomic variation present an important safety problem that would limit such hES from being applied for future clinic use and drug screening.

Analysis of the Systemic Exposure of Mitomycin C Used in Glaucoma Filtering Surgery Among Women in the Reproductive Age Group.

PURPOSE: Systemic absorbtion of topically applied mitomycin C (MMC) during trabeculectomy needs to be evaluated to look for any systemic toxicity, which might be a major concern in certain conditions like pregnancy. METHODS: After obtaining ethical committee clearance, female patients in the reproductive age group undergoing trabeculectomy with MMC were included. Pregnant/lactating patients, patients with any systemic illness were excluded. During trabeculectomy, 0.02% MMC was applied subconjunctivally for 2 min and then washed. Blood samples were withdrawn at 1, 2, 4, 8, 12, and 24 hrs after the surgery and analyzed of MMC levels using Liquid chromatography-tandem mass spectroscopy (LC-MS/MS). RESULTS: The mean age of the participants was 29 ± 12 years. MMC was not detected in any of the plasma samples analyzed as it was less than the detection limit (<1.56 ng/mL) of the employed LC-MS/MS assay. CONCLUSION: It can be deduced that the systemic absorption of MMC is negligible or the plasma concentration is less than 1.56 ng/ml (1000 times less than the concentration where systemic toxicity was not observed).

[Cyclic digene system as a control element of a bacterial biosensor].

The Escherichia coil JC1 58(pCIA12/pG FK5) strain carrying a cyclic digene system with a negative feedback on the pCIA12 plasmid reacting to the DNA damage by changing the synthesis level of reporter genes-GFP and beta-galactosidase-was tested. The acquired phenotype was inherited by the next generations after the removal of the genotoxic action when the concentration of the DNA-damaging compounds was above the threshold level. A potential has been shown for the application of bacterial biosensors to monitor the presence of genotoxicants in the environment and to test the consequences of short-term exposures to toxic compounds.

Smart activatable and traceable dual-prodrug for image-guided combination photodynamic and chemo-therapy.

Activatable photosensitizers (PSs) and chemo-prodrugs are highly desirable for anti-cancer therapy to reduce systemic toxicity. However, it is difficult to integrate both together into a molecular probe for combination therapy due to the complexity of introducing PS, singlet oxygen quencher, chemo-drug, chemo-drug inhibitor and active linker at the same time. To realize activatable PS and chemo-prodrug combination therapy, we develop a smart therapeutic platform in which the chemo-prodrug serves as the singlet oxygen quencher for the PS. Specifically, the photosensitizing activity and fluorescence of the PS (TPEPY-SH) are blocked by the chemo-prodrug (Mitomycin C, MMC) in the probe. Meanwhile, the cytotoxicity of MMC is also inhibited by the electron-withdrawing acyl at the nitrogen position next to the linker. Upon glutathione activation, TPEPY-S-MMC can simultaneously release active PS and MMC for combination therapy. The restored fluorescence of TPEPY-SH is also used to report the activation for both PS and MMC as well as to guide the photodynamic therapy.

Investigations on safety of hyperthermic intraoperative intraperitoneal chemotherapy (HIPEC) with Mitomycin C.

AIMS: Previous safety monitoring of hyperthermic intraoperative intraperitoneal chemotherapy (HIPEC) with Mitomycin C (MMC) did not demonstrate any detectable safety hazard to the personnel. Nevertheless, those results have been discussed controversially because of the methodological problems employed in the evaluation of potential exposure. We re-evaluated possible safety hazards of HIPEC by applying different monitoring strategies. METHODS: We monitored air samples in the operation room during HIPEC. In addition, we measured MMC in plasma of the surgeon with a newly developed analytical method. All samples were analysed by HPLC-UV at 360nm. The permeability of the gloves was tested using two in vitro techniques: diffusion cells and a glass cell chamber. In-use and worst-case exposure scenarios were imitated for in vitro experiments. RESULTS: The analysis of the air samples (n=3) could not detect any MMC. We found no drug above the limit of detection (1microg MMC/L) in the plasma samples of the surgeons (n=5). A breakthrough of latex glove material was detected in only one (worst-case exposure scenario) of 40 diffusion cell experiments. CONCLUSIONS: Established methods of safety monitoring could not reveal any detectable risk on in-use exposure conditions. The wearing of doubled latex gloves should prevent the surgeon from dermal exposure to MMC during HIPEC.

LC-ESI-MSD fast determination of residual mitomycin C in hen aqueous humour after corneal refractive surgery.

A simple, fast and reliable method has been developed for the assay of traces of mitomycin C (MMC) in hen aqueous humour samples. The determination was carried out by high-performance liquid chromatography with electrospray ionization mass spectrometric detection. In isocratic elution analysis, the mobile phase was a mixture of water-acetonitrile (78:22, v/v) and the chromatographic column was C(18) at 35 degrees C. The method has been validated over a range from 0.1 to 250 microg L(-1) in hen aqueous humour with correlation coefficients higher than 0.999. Limit of detection and limit of quantification for MMC based in signal to noise ratio of 3 and 10, respectively, were 20 and 71 ng L(-1). The developed method allows the analysis of MMC in hen aqueous humour samples obtained at different times and conditions in order to evaluate and compare the efficacy of the drug administration.

Concentration Accuracy of Compounded Mitomycin C for Ophthalmic Surgery.

IMPORTANCE: Ophthalmologists rely on accurate concentrations of mitomycin C (MMC) to prevent scarring with trabeculectomy surgery. To our knowledge, the concentration accuracy and variability of compounded MMC are unknown. OBJECTIVE: To determine whether the measured concentration differs from the expected concentration of 0.4 mg/mL of MMC used in ophthalmic surgery. DESIGN, SETTING, AND PARTICIPANTS: Laboratory experimental investigation conducted in July 2013. We acquired 60 samples of 0.4 mg/mL of MMC from a spectrum of common compounding and storage techniques (refrigeration, freezing, and immediately compounded dry powder) and a variety of pharmacies (an academic hospital, a community hospital, and an independent Pharmacy Compounding Accreditation Board-accredited pharmacy). We used C18 reversed-phase high-performance liquid chromatography to measure the MMC concentration of all samples. We used pure MMC (Medisca Inc) to generate calibration curves and sulfanilamide as an internal standard. MAIN OUTCOMES AND MEASURES: We calculated MMC concentration using a calibration curve (range, 0.3-0.5 mg/mL) generated by dividing MMC peak area by internal standard peak area and plotting the area ratio against the calibrant concentrations. We compared the measured concentration against the expected 0.4 mg/mL concentration for all samples. RESULTS: Measurement of MMC using the high-performance liquid chromatography method demonstrated acceptable accuracy (92%-100%), precision (2%-6% coefficient of variation), and linearity (mean correlation coefficient of r2 = 0.99). The measured MMC concentration determined using the high-performance liquid chromatography method for all samples was 12.5% lower than the expected 0.4 mg/mL value (mean [SD], 0.35 [0.04] mg/mL; 95% CI, 0.34-0.36; P < .001) with a wide concentration range between 0.26 and 0.46 mg/mL. CONCLUSIONS AND RELEVANCE: Common compounding and storage techniques for MMC resulted in a lower accuracy and wider range of concentration than expected. These differences in concentration may result from compounding techniques and/or MMC degradation. Variability in MMC concentration could cause inconsistency in glaucoma surgical results, but the clinical relevance of such findings on glaucoma surgery outcomes remains unknown.

Trypan blue identifies antimetabolite treatment area in trabeculectomy.

AIM: Colourless solutions of mitomycin C (MMC) and 5-fluorouracil (5-FU) are widely used during trabeculectomy to inhibit postoperative scarring. The poor visibility of these agents on the eye has several drawbacks including the inability to accurately assess the area of treatment. This study examined the utility of using trypan blue dye to colour antimetabolites used during trabeculectomy and the effect of trypan blue on antimetabolite cytotoxicity in vitro. METHODS: For in vitro experiments, MMC (0.4 mg/ml) and 5-FU (25 mg/ml) were reconstituted with or without trypan blue. A lactate dehydrogenase release assay was used to measure drug induced cell death and viable cell number 7 days after treatment. For clinical assessment, trypan blue 0.1% was added to MMC and 5-FU to final concentrations of between 0.01% and 0.05%. The mixture was applied to Tenon's capsule and sclera via pre-wet or into dry 5x8 mm sponges (MMC and 5-FU) for 3 minutes or by direct subconjunctival injection after completion of surgery (5-FU). Twenty two consecutive patients undergoing trabeculectomy either with or without trypan blue were followed for 2 years postoperatively. RESULTS: The addition of 0.05% trypan blue to MMC or 5-FU did not alter MMC induced cell death or the number of viable fibroblast in vitro. In vivo, trypan blue clearly delineated the antimetabolite treatment area and facilitated control of excess antimetabolite at the wound margins as well as sponge removal. With direct subconjunctival injection, total staining area varied for a given volume with location of the needle tip. Any leakage from the injection site could be easily seen. No adverse effects attributable to trypan blue were found in 2 years of follow up. CONCLUSIONS: Trypan blue permits delineation of antimetabolite/tissue interactions without affecting cytoxicity for the assays investigated. Trypan blue can be used to visualise antimetabolite soaked sponges, estimate treatment area, and show areas of unintended tissue contact during trabeculectomy. The addition of trypan blue to antimetabolites has potential benefits in clinical, research, and teaching aspects of ocular surgery and therapy.

Detection of mitomycin C-DNA adducts in human breast cancer cells grown in culture, as xenografted tumors in nude mice, and in biopsies of human breast cancer patient tumors as determined by (32)P-postlabeling.

Mitomycin C (MMC) is a DNA cross-linking agent that has been used in cancer chemotherapy for >20 years. However, little is known either qualitatively or quantitatively about the relationship between formation and repair of specific MMC-DNA adducts and specific biological outcomes. The goal of this study was to examine formation and removal of specific MMC-DNA adducts in breast cancer cells using a (32)P-postlabeling assay in relation to cytotoxicity and other biological end points. MMC-DNA adducts were measured in cultured human metastatic MDA-MB-435 cells, in the same cells xenografted as a mammary tumor in nude mice, and in metastatic tumor biopsies obtained from human breast cancer patients undergoing MMC-based therapy. MMC adducts corresponding to the CpG interstrand cross-link, the MMC-G bifunctional monoadduct, and two isomers of the MMC-G monofunctional monoadduct were detected in most samples. Despite similarities in the overall patterns of adduct formation, there were substantial differences between the cultured cells and the in vivo tumors in their adduct distribution profile, kinetics of adduct formation and removal, and relationship of specific adduct levels to cytotoxicity, suggesting that the in vivo microenvironment (e.g., degree of oxygenation, pH, activity of oxidoreductases, and other factors) of breast cancer cells may significantly modulate these parameters.

Validation of an HPLC-MS/MS and wipe procedure for mitomycin C contamination.

A high-performance liquid chromatography-tandem mass spectrometric (HPLC-MS/MS) method was developed for the determination of mitomycin C, an anticancer drug, from contamination on various surfaces. Mitomycin C is often used in various forms of intraperitoneal chemotherapy, and operating room healthcare worker exposure to this drug is possible. The surface testing method consisted of a wiping procedure utilizing a solution of 20/45/35 (v/v/v) of acetonitrile-isopropanol-water made 0.01 M in ammonium citrate (apparent pH 7.0). The wipe solutions were analyzed by means of HPLC-MS/MS using a reversed-phase gradient system and electrospray ionization in positive ion mode with a triple-quadrupole MS detector. Accuracy and precision of this method were demonstrated by a series of recovery studies of both spiked solutions and extracted wipes from various surfaces (stainless steel, vinyl and Formica(®)) spiked with known levels of mitomycin C. Recoveries of spiked solutions containing the analyte demonstrate mean recoveries (accuracy) ranged from 93 to 105%. Precision as measured by the relative standard deviation (% RSD) of multiple samples (n= 10) at each concentration level demonstrated values of 7.5% or less. The recoveries from spiked surfaces varied from 30 to 99%. The limit of detection for this methodology is ∼2 ng/100 cm(2) equivalent surface area, and the limit of quantitation is ∼6 ng/100 cm(2).

A two-stage minibioreactor system for continuous toxicity monitoring.

A two-stage minibioreactor system was successfully developed for continuous toxicity monitoring. This system consists of two minibioreactors in series. Recombinant Escherichia coli DPD2794 containing a RecA::luxCDABE fusion as a model strain was utilized to monitor environmental insults to DNA, with mitomycin C as a model toxicant. Pulse type exposures were used to evaluate the system's reproducibility and reliability. Step inputs of mitomycin C have been adopted to show the system's stability. The system's ability to monitor the possible upsets or accidental discharges of toxic chemicals was also evaluated with these step insults. All the data demonstrated that this two-stage minibioreactor system using recombinant bacteria containing stress promoters fused with lux genes is quite appropriate for continuous toxicity monitoring. Long-term operation and minimized media-usage have been investigated. Thus application to many different areas, including an early warning system of wastewater biotreatment plant upsets and the monitoring and tracking of accidental spills, discharges or failures in plant operation are plausible.

Construction and characterization of novel dual stress-responsive bacterial biosensors.

Using the genes for the green fluorescence protein and Xenorhabdus luminescens luciferase operon and the promoters for the recA and katG genes, two stress-responsive Escherichia coli biosensor strains have been constructed that can individually or concurrently respond to oxidative and genotoxic conditions. Strain DUO-1 carries the pRGDK1 plasmid, which has the recA::GFPuv4 and katG::luxCDABE fusion genes oriented divergently with each other, while in DUO-2, i.e., pRGDK2, they are in a tandem orientation, with the recA promoter showing run-though transcription of the katG::luxCDABE fusion. These two strains and their responses were characterized using several known hydroxyl radical-forming chemicals, e.g., hydrogen peroxide and cadmium chloride, along with some genotoxins, e.g., mitomycin C and methyl-N-nitro-N-nitrosoguanidine, and some general toxicants. Both strains showed an induction of green fluorescent protein (GFP) and bioluminescence when they experienced DNA and oxidative damage, respectively, while the tandem orientation of the two fusion genes within DUO-2 allowed it to also sensitively respond to genotoxins via the production of bioluminescence. However, the characteristics of DUO-2's bioluminescent response to each stress were easily distinguishable, making it useful for the detection of both stresses. Furthermore, tests with mixtures of chemicals showed that both DUO-1 and DUO-2 were responsive when chemicals causing oxidative or genotoxic stress were present as a single chemical or within complex chemical mixtures.

A bioluminescent sensor for high throughput toxicity classification.

A high throughput toxicity monitoring and classification biosensor system has been successfully developed using four immobilized bioluminescent Escherichia coli strains, DPD2511, DPD2540, DPD2794 and TV1061, which have plasmids bearing a fusion of a specific promoter to the luxCDABE operon. The bioluminescence of DPD2511 increases in the presence of oxidative damage, DPD2540 by membrane damage, DPD2794 by DNA damage and TV1061 by protein damage. In the developed biosensor these strains are immobilized in a single 96 well plate using an LB-agar matrix, and are able to detect the toxicities of hydrogen peroxide, phenol and mitomycin C in water samples. As the concentration of each chemical was increased, the bioluminescence levels from the corresponding wells, containing either DPD2511, DPD2540, DPD2794 or TV1061, increased. This increase in bioluminescence followed a dose dependent response to the toxic chemicals within a specific concentration range. In particular, each test requires only 4 h to give clear bioluminescent response signature. Storage of the biosensor at 4 degrees C for 2 weeks caused no change in its dose-dependent response. The fast and easy detection of oxidative, membrane, protein and DNA damaging agents in aqueous environments is possible due to the high throughput capability of this biosensor.

Mitomycin C dissolved in a reversible thermosetting gel: target tissue concentrations in the rabbit eye.

AIMS: To determine whether a new, reversible thermosetting gel enhances mitomycin C transfer to target ocular tissues in the rabbit eye. METHODS: A 0.1 ml solution of mitomycin C containing 0.22 microgram, 2.9 micrograms, or 28 micrograms of the agent dissolved in a reversible thermosetting gel consisting of methylcellulose, citric acid, and polyethylene glycol was injected subconjunctivally in 30 New Zealand albino rabbits. Scleral and conjunctival tissues were excised at 0.5, 1, 2, 4, or 24 hours after the injection and mitomycin C concentrations in these tissues were determined by high performance liquid chromatography. The concentration over time was approximated to a single exponential curve, and initial mitomycin C concentrations, time constants, and half life values were determined. Finally, the areas under the curves (AUCs) between 0.5 and 24 hours were calculated. RESULTS: The mitomycin C concentrations in the target tissues were dose dependent and decreased rapidly over 24 hours. Both the initial mitomycin C concentrations as well as AUCs in these eyes treated with mitomycin C, dissolved in a reversible thermosetting gel, were higher than those in eyes treated similarly in a previous study in which the gel was not used. CONCLUSION: Applied subconjunctivally in the rabbit eye, mitomycin C dissolved in the reversible thermosetting gel enhanced transfer of the agent to the sclera and the conjunctiva.

Variability of mitomycin C adsorption by activated charcoal.

A saline suspension of mitomycin C adsorbed on activated charcoal and administered intraperitoneally has been reported to be safe and effective in the treatment of gastric carcinoma. Activated charcoal specifically targets tumour and lymph-node tissues and the sustained higher local drug concentration is thought to be beneficial. The charcoal particles used in these suspensions have varied in size from > 147 microm to < 20 nm in diameter, but no data have been published to show how this might affect drug adsorption and delivery. Any variability in drug adsorption could pose a serious clinical risk for drugs with a narrow therapeutic index. We have, therefore, investigated the adsorption of mitomycin C on activated charcoal in-vitro. Activated charcoal was ground and sieved to yield four size-fractions between 180 and 53 microm. Adsorption isotherms (n > or = 3) were constructed and applied to the Freundlich model with 0-l00 microg mL(-1) mitomycin C measured by HPLC with detection at 365 nm. Adsorption of mitomycin C by activated charcoal varied by a factor of three under identical conditions at room temperature (21 degrees C) and at 37 degrees C. The specific adsorption (microg mitomycin C (mg activated charcoal)(-1)) was generally higher at 37 degrees C than at room temperature. The variability of mitomycin C adsorption was greatly reduced by addition of the surface-active agent polyvinylpyrollidone, used to determine that adsorption of mitomycin C was independent of activated charcoal particle size. The characteristics of adsorption of mitomycin C by activated charcoal are complex and should be thoroughly investigated to discover the critical controlling factors before submitting the suspensions for further clinical evaluation.

Serum complement enhances the responses of genotoxin- and oxidative stress-sensitive Escherichia coli bioreporters.

Bacterial bioreporters are limited in their abilities to detect large polar molecules due to their membrane selectivity. In this study, the activity of serum complement was used to bypass this undesired selectivity. Initially, the serum complement activity was assessed using the responses of a bacterial bioreporter harboring a recA::luxCDABE transcriptional fusion when exposed to the chemotherapy drug, mitomycin C (MMC). Using 50 °C-treated serum, the limit of detection for this bacterial sensor was lowered by nearly 450-fold, from 31 µg/L to 0.07 µg/L MMC. Real-time quantitative PCR demonstrated that serum-treated cultures responded more strongly to 100 µg/L MMC, with 3.1-fold higher recA expression levels. Subsequent experiments with other bioreporter strains also found enhanced sensitivities and responses. Finally, combining each of the above findings, tests were performed to demonstrate the potential application of the recA::luxCDABE bioreporter within a lab-on-a-CD platform as a point-of-care diagnostic to measure chemotherapeutic drug concentrations within blood.

Signal enhancement by a multi-layered substrate for mutagen detection using an SOS response-induced green fluorescent protein in genetically modified Escherichia coli.

In this paper, we describe a method to enhance the fluorescence signal of mutagen detection using SOS response-induced green fluorescence protein (GFP) in genetically modified Escherichia coli using a multi-layered substrate. To generate E. coli that express SOS response-induced GFP, we constructed a plasmid carrying the RecA promoter located upstream of the GFP gene and used it to transform E. coli BL21. The transformed strain was incubated with mitomycin C (MMC), a typical mutagen, and then immobilized on a multi-layered substrate with Ag and a thin Al(2)O(3) layer on a glass slide. Since the multi-layered substrate technique is an optical technique with potential to enhance the fluorescence of fluorophore placed on top of the substrate, the multi-layered substrate was expected to improve the fluorescence signal of mutagen detection. We obtained an average 14-fold fluorescence enhancement of MMC-induced GFP in the concentration range 1 to 1000 ng/ml. In addition, the lower detection limit of MMC was improved using this technique, and was estimated to be 1 ng/ml because of an enlargement of the difference between the blank and the signal of 1 ng/ml of MMC.

Optimization of bacterial whole cell bioreporters for toxicity assay of environmental samples.

In a study to optimize bacterial whole cell biosensors (bioreporters) for the detection of environmental contaminants, we constructed a toxicity sensing strain Acinetobacter baylyi ADP1_recA_lux. The ADP1_recA_lux is a chromosomally based bioreporter which makes the sensing system stable and negates the need for antibiotics to maintain the trait. The AOP1_recA_lux is activated to express bioluminescence when it is exposed to DNA damaging toxicants. Since the ADP1_recA_lux constantly expresses a baseline level of bioluminescence, false negative results are avoided. The host strain, A. baylyi ADP1, is an ideal model strain typical of water and soil bacteria occurring in the natural environment, and it is more robust than E. coli in terms of viability, maintenance, and storage. The expression of reporter genes - luxCDABE cloned from Photorhabdus luminescens - is robust in a broad range of temperature (10-40 degrees C). The ADP1_recA_lux was used to detect a variety of toxic or potentially toxic compounds including mitomycin C (MMC), methyl methanesulfonate, ethidium bromide, H2O2, toluene, single-wall nanocarbon tubes (SWNCT), nano Au colloids (20 nm), pyrene, beno[a]pyrene, and UV light. These exposures revealed that the ADP1_recA_lux was able to detect both genotoxicity and cytoxicity, qualitatively and quantitatively. The optimal induction time of the ADP1_recA_lux bioreporter was 3 h, and the detection limits for MMC and benezo[a]pyrene were 1.5 nM and 0.4 nM, respectively. The ADP1_recA_lux was also used to detect toxicity of groundwater contaminated by a mixture of phenolic compounds, and the bioreporter toxicity detection was in a good agreement with chemical analysis. The optimized whole cell bioreporter ADP1_recA_lux could be valuable in providing a simple, rapid, stable, quantitative, robust, and costly efficient approach for the detection of toxicity in environmental samples.

[Construction of the pUCD-recA recombinate luminescence bacterium to evaluate the genetoxicty of environment pollutants].

Recombinate luminescence bacteria have the important role in evaluating water toxicity. A recombinate luminescence bacteria vector pUCD-recA was constructed to investigate the general toxicity of pollutants. The gene of recA amplified by PCR from W3110 was cloned into pGEM-T easy vector and sequencing. The correct PCR product and pUCD615 vector were digested with BamH I, EcoR I, then be fused and imported into JM109 with electrotransformation. Several clones were selected and identificated by PCR and sequencing. The result revealed that the length of the recA fragment was 293 bp. When it was sequenced and blasted with the recA in GenBank, the homology of the sequences reached 99% indicating the amplified result correct. The sequencing result of the fragment fused with pUCD615 revealed that the gene of recA had been inserted into the multiple clone site correctly, and the insert direction and reading frame were also exactly. Transforming the pUCD-recA vector to E. coli RFM443, then added the genetoxic pollutants and observed the responding effect. The effect of MMC inducing the pUCD-recA strain was best with the dose only of 0.01 mg/L, while need the dosage of MNNG reached 50-100 mg/L.