(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)

KB cells were treated with MTT at final concentrations up to 0.2 mg/ml alone or in combination with methotrexate (10 and 32 uM). When used alone, as little as 0.025 mg/ml increased population doubling time from 26 hr (control) to 96 hr. At 0.2 mg/ml or higher, total cell number declined. MTT, 0.1 mg/ml, reduced the number of cells in S and G2/M phases after 8 hr treatment. However, no significant effect was seen at 2 and 4 hr. The effect of 0.2 mg/ml MTT was more immediate and more pronounced, resulting in accumulation of cells in G0/G1 and S phases. MTT in combination with methotrexate produced a more significant perturbation in the KB cell cycle.

...Phenazine methosulfate (PMS) and MTT are often used in combination: however, PMS did not potentiate the mutagenicity of MTT.

/GENOTOXICITY/ Tetrazolium salts, in combination with phenazine methosulfate (PMS) are widely used laboratory reagents. PMS, and 3 tetrazolium salts (MTT, [3-(4,5-dimethylthiazolyl-2)-2,5-diphenyl tetrazolium bromide]; TTC,[2,3,5-triphenyl tetrazolium chloride]; and NBT, [2,2'-di-p-nitrophenyl-5,5'-diphenyl-3,3'-(3,3'-dimethoxy-4,4' were tested for mutagenicity in a range of Escherichia coli WP2 (trp-) and Salmonella typhimurium ... . Without S-9, PMS was mutagenic to E. coli uvrApKM101 /and/ S. typhimurium TA 100 and TA 98 giving (in the dose range 0.5-10 ug/plate) linear dose response curves with slopes of 2.3, 1.3 and 0.5 revertants/nmol respectively. Addition of /rat liver/ S-9 drastically reduced the mutagenicity of PMS in these bacteria. MTT, in the absence of S-9, was mutagenic to E. coli uvrApKM101, S.typhimurium TA 100 and TA 98, giving linear dose-response curves with slopes of 3.8, 12.1 and 1.0 respectively, at doses ranging from 0.5 to 10 or 50 ug/plate. Addition of S-9 markedly reduced the mutagenicity of MTT. MTT was very weakly mutagenic in E. coli WP2 (0.22 revertants/nmol), significantly more mutagenic in WP2 uvrA (1.29 revertants/nmol), and non-mutagenic in WP2 lexA (negative slope due to toxicity), suggesting that MTT causes excisable misrepair DNA damage. PMS and MTT are often used in combination: however, PMS did not potentiate the mutagenicity of MTT.

/ALTERNATIVE IN VITRO TESTS/ ... /The authors/... investigated the effects of MTT on cell growth and cell cycle distribution using DNA flow cytometry. KB cells were treated with MTT at final concentrations up to 0.2 mg/ml alone or in combination with methotrexate (10 and 32 uM). When used alone, as little as 0.025 mg/ml increased population doubling time from 26 hr (control) to 96 hr. At 0.2 mg/ml or higher, total cell number declined. MTT, 0.1 mg/ml, reduced the number of cells in S and G2/M phases after 8 hr treatment. However, no significant effect was seen at 2 and 4 hr. The effect of 0.2 mg/ml MTT was more immediate and more pronounced, resulting in accumulation of cells in G0/G1 and S phases. MTT in combination with methotrexate produced a more significant perturbation in the KB cell cycle. Although the MTT assay measures cell viability and proliferation via mitochondrial dehydrogenases, it showed definite effects on cellular DNA content and cell proliferation. These observations question the credibility of such an assay, indicating it may be measuring combined effects of MTT and drug and not drug alone.

/OTHER TOXICITY INFORMATION/ Despite widespread use of various tetrazolium assays, the mechanisms of bioreduction of these compounds have not been fully elucidated. /The authors/ investigated the capacity of tetrazolium salts to penetrate through intact cell plasma membranes. 5-Cyano-2,3-ditolyl tetrazolium chloride (CTC) and 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) tetrazolium salts appear to represent examples of species that are reduced by different mechanisms. /The authors/ provide evidence suggesting that MTT readily crosses intact plasma membranes and is reduced intracellularly. MTT appears to be reduced by both plasma membrane and intracellular reductases; reducing cells are not damaged and remain metabolically active for at least 45 min. In contrast, CTC remains extracellular with respect to viable cells and thus requires plasma membrane permeable electron carrier to be reduced efficiently. However, reduction of CTC in the presence of an electron carrier inflicts damage on plasma membranes. The intracellular vs extracellular sites of reduction of tetrazolium salts were established on the basis of deposition of formazans. Crystals of formazan were detected using fluorescence or backscattered light confocal laser microscopy. /The authors/ postulate that the capacity of a tetrazolium salt to cross intact plasma membranes constitutes an important experimental variable which needs to be controlled in order to correctly interpret the outcome of tetrazolium assays designed to measure cellular production of oxygen radicals, activity of mitochondrial, cytosolic, or outer membrane reductases, etc.

Basic treatment: Establish a patent airway. Suction if necessary. Watch for signs of respiratory insufficiency and assist ventilations if needed. Administer oxygen by nonrebreather mask at 10 to 15 L/min. Monitor for pulmonary edema and treat if necessary ... . Monitor for shock and treat if necessary ... . Anticipate seizures and treat if necessary ... . For eye contamination, flush eyes immediately with water. Irrigate each eye continuously with normal saline during transport ... . Do not use emetics. For ingestion, rinse mouth and administer 5 ml/kg up to 200 ml of water for dilution if the patient can swallow, has a strong gag reflex, and does not drool ... . Cover skin burns with dry sterile dressings after decontamination ... . /Poison A and B/

Advanced treatment: Consider orotracheal or nasotracheal intubation for airway control in the patient who is unconscious, has severe pulmonary edema, or is in respiratory arrest. Positive pressure ventilation techniques with a bag valve mask device may be beneficial. Monitor cardiac rhythm and treat arrhythmias as necessary ... . Start an IV with D5W /SRP: "To keep open", minimal flow rate/. Use lactated Ringer's if signs of hypovolemia are present. Watch for signs of fluid overload. Consider drug therapy for pulmonary edema ... . For hypotension with signs of hypovolemia, administer fluid cautiously. Watch for signs of fluid overload ... . Treat seizures with diazepam (Valium) ... . Use proparacaine hydrochloride to assist eye irrigation ... . /Poison A and B/