Metabolism / Metabolites
Gut bacteria and hepatic azo reductases can reduce azo dyes to the aromatic amines that compose them. Azo dyes based on benzidine and its homologues have attracted considerable attention due to their widespread use and the known carcinogenicity of benzidine in humans. Azo dyes based on β-diketone coupling components exist primarily as tautomer hydrazones. We prepared a series of hydrazone dyes based on benzidine and its homologues and characterized them by NMR and UV-Vis spectroscopy. The mutagenicity of these dyes was tested using a modified Ames assay, and the results showed that, unlike true azo dyes, these dyes did not exhibit significant mutagenic activity. These hydrazone dyes were resistant to enzymatic reduction in the postmitochondrial supernatant (S-9) of hamster liver supplemented with FMN; under the same conditions, azo dyes (e.g., trypan blue) were rapidly reduced. In an H₂O₂-dependent Ames assay system, benzidine and several of its derivatives were activated into mutagens. Optical and electron paramagnetic resonance (EPR) spectroscopy was used to investigate the H₂O₂-dependent oxidation of benzidine and 3,5,3',5'-tetramethylbenzidine (TMB) catalyzed by intact bacteria, providing direct evidence for peroxidase activity in Salmonella typhimurium. The Ames test strain TA98, possessing acetyltransferase activity, and its acetyltransferase-deficient derivative TA98/1,8-DNP6 exhibited identical H₂O₂-dependent mutagenic responses; in this system, enzymatic acetylation appeared not to be involved in benzidine activation. H₂O₂-dependent mutagenicity of benzidine and oxidation of TMB were observed when assayed in acetate buffer (pH 5.5), but not in 2-[N-morpholino]ethanesulfonic acid (MES) buffer at the same pH. This difference can be explained by the effect of these buffers on the intracellular pH of the bacteria. The H₂O₂-dependent mutagenicity of several benzidine homologues is also described in this paper.
Dichlorobenzidine can be activated by peroxidation in Salmonella typhimurium Ames test strains. Mutagenicity was observed when Salmonella typhimurium strains sensitive to frameshift mutagen were incubated with dichlorobenzidine and hydrogen peroxide. This study demonstrates that the bacterial enzyme hydroperoxidase I is the primary responsible party for this activation. We constructed homologous test strains lacking hydroperoxidase I or II due to the insertion of a Tn10 gene encoding these proteins. Hydrogen peroxide-dependent mutagenicity of dichlorobenzidine was determined in each strain. Test strains lacking hydroperoxidase I activity were significantly less sensitive than the parental strains. When hydroperoxidase I activity was restored in these strains via a copy of the E. coli protein-encoding gene carried by a plasmid, their sensitivity to hydrogen peroxide-dependent dichlorobenzidine mutagenicity was enhanced.
Acquired using specialized “dry fracture” techniques and scanning electron microscopy, an accumulation of insoluble fine particulate matter was observed beneath the pigment surface of Xenopus laevis eggs. These techniques allowed for the identification of cortical and pigment granules, which were observed to be embedded within this material. Ultrathin sections revealed that this region also contained mitochondria and membranous vesicles or reticular structures. Yolk platelets were not significantly included in the largest accumulation of this substance. This substance was densest beneath the cortex, gradually transitioning to the more diffuse, yolk-containing endoplasmic reticulum. In the animal hemisphere of the oocyte, this substance was much thicker than in the plant hemisphere, with embedded pigments defining the pigmented region of the animal hemisphere. In the pigmented region, this substance excluded the yolk, reaching a thickness of 3–7 micrometers or more from the surface. In the plant hemisphere, this accumulation was absent, with yolk flakes almost touching the plasma membrane. Experiments induced cortical contraction in the oocyte. Its relative intensity correlated with the relative thickness of the subcortical fine granular material. During contraction, this substance accumulated to a greater thickness, excluding the yolk at a thickness of 15–30 micrometers or more from the surface. The entity undergoing contraction was either this fine granular material or present within it. Injection of cytochalasin into the oocyte inhibited cleavage formation but did not inhibit induced cortical contraction. Therefore, cortical contraction does not appear to depend on the formation of actin microfilaments as much as the cleavage contraction ring. The difference in sensitivity to cytochalasin between the contraction ring and the system inducing cortical contraction suggests that oocyte cortical contraction is a two-component system. This paper presents a model that can explain the existing data. For more complete metabolite/metabolite data on 3,3',5,5'-tetramethylbenzidine (6 metabolites in total), please visit the HSDB record page.

Non-Human Toxicity Values
Mouse intraperitoneal injection LD50: 135 mg/kg

3,3',5,5'-Tetramethylbenzidine is a pale yellow crystal or grayish-white powder. (NTP, 1992)

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Mechanism of Action
Histological analysis of surgically removed adrenal masses often makes it difficult to distinguish between benign and malignant tumors. In normal cells, the telomeres at the ends of chromosomes shorten with each cell division, leading to chromosomal instability and cellular senescence after a certain number of cell cycles. In tumor cells, a specific DNA polymerase called telomerase can prevent telomere shortening. To elucidate the role of telomerase in the pathogenesis of adrenal tumors and to examine whether its activity can serve as a marker of malignancy, we measured telomerase activity in 41 human adrenal tissue samples, categorized according to clinical course and histological findings. Telomerase activity was measured using the TRAP ELISA method and categorized as high (>50%), moderate (31-50%), low (11-30%), very low (≤10%), or none (0%), representing 50% or more of the positive control telomerase activity. Telomerase activity was extremely low in all 8 normal adrenal tissue samples. The mean telomerase activity was also extremely low in 3 adrenal incidental tumors, 6 Cushing's adenomas, 6 Conn's adenomas, 7 adrenocortical carcinomas, 8 benign pheochromocytomas, and 2 malignant pheochromocytomas. Conversely, 1 malignant pheochromocytoma showed relatively high telomerase activity. These data suggest that telomerase activity may not be a suitable biomarker for adrenal malignancies. Our results also challenge the current conventional wisdom regarding the close relationship between cell dedifferentiation and telomerase activity. Previous studies on the oxidation of 3,5,3',5'-tetramethylbenzidine (TMB) using horseradish peroxidase and prostaglandin H synthase showed that TMB forms cationic radicals and reaches equilibrium with charge-transfer complexes, consistent with the two-electron or one-electron transfer of the initial oxidation process. In this study, we utilized the unique spectroscopic properties of myeloperoxidase and its oxidation intermediates, compounds I and II, to confirm a two-stage, consecutive one-electron oxidation process of TMB. We also determined the rate constants of the fundamental steps of myeloperoxidase-catalyzed oxidation of TMB at pH 5.4 and 20 °C using a stop-flow technique under transient and steady-state conditions. The second-order rate constant for the reaction of the native enzyme with H₂O₂ to form compound I is 2.6 × 10⁷ M⁻¹ s⁻¹. Compound I undergoes one-electron reduction in the presence of TMB to form compound II, with a rate constant of (3.6 ± 0.1) × 10⁶ M⁻¹ s⁻¹. Spectroscopic scanning revealed that compound II accumulates under steady-state conditions. Under steady-state conditions, the rate constant for the reduction of compound II to the native enzyme by TMB is (9.4 ± 0.6) × 10⁵ M⁻¹ s⁻¹. These findings were applied to a novel and more accurate method for myeloperoxidase assay based on the formation of a charge-transfer complex between TMB and its diimine final product.

C16H20N2

240.3434

240.162

54827-17-7

TMB dihydrochloride;64285-73-0;TMB monosulfate;54827-18-8;TMB dihydrochloride hydrate;312693-82-6

41206

White to light yellow solid powder

1.1±0.1 g/cm3

368.6±37.0 °C at 760 mmHg

168-171 °C(lit.)

210.8±26.0 °C

0.0±0.8 mmHg at 25°C

1.618

3.4

2

2

1

18

226

0

UAIUNKRWKOVEES-UHFFFAOYSA-N

InChI=1S/C16H20N2/c1-9-5-13(6-10(2)15(9)17)14-7-11(3)16(18)12(4)8-14/h5-8H,17-18H2,1-4H3

4-(4-amino-3,5-dimethylphenyl)-2,6-dimethylaniline

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~25 mg/mL (~104.02 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)