Metabolism / Metabolites
This study employed high-performance liquid chromatography (HPLC) to analyze in vitro metabolites and detect their mutagenic activity against *Salmonella typhimurium*, thereby investigating the metabolic activation process of the environmental carcinogen 5-methylchrysene. The metabolites were generated by incubating 5-methylchrysene with Aroclor-treated liver tissue from 9000 rats. Using a reversed-phase column, the metabolites were separated into nine peaks, labeled A through I. Each peak was collected, and its mutagenic activity was detected. The results showed that the major mutagenic activity was present in peak E, while peak D exhibited lower mutagenic activity. Other metabolites did not show significant mutagenic activity. The major mutagenic metabolite (peak E) was identified as 1,2-dihydro-1,2-dihydroxy-5-methylchrysene (7.0% of 5-methylchrysene). Peak D was identified as 7,8-dihydro-7,8-dihydroxy-5-methylchrysene (2.6% of 5-methylchrysene). Other metabolites included 9,10-dihydro-9,10-dihydroxy-5-methylpyrrol, 9-hydroxy-5-methylpyrrol, 7-hydroxy-5-methylpyrrol, 1-hydroxy-5-methylpyrrol, and 5-hydroxymethylpyrrol. These results indicate that 1,2-dihydro-1,2-dihydroxy-5-methylpyrrol is the major proximal mutagen of 5-methylpyrrol. 1,2- and 7,8-dihydrodiols were the major metabolites of 5-methylpyrrol, and small amounts of 9,10-dihydrodiol were also generated upon incubation of the compound with a rat liver formulation. Other metabolites detected included 1-, 7-, and 9-hydroxy-5-methylpyrrol, as well as 5-hydroxymethylpyrrol. The metabolic activation of the potent carcinogen 5-methylpyrrol (5-MeC) in mouse skin was compared with the metabolic activation of the inactive compound 6-nitro-5-methylpyrrol (6-NO₂-5-MeC). 6-NO₂-5-MeC metabolites generated from rat liver homogenate were identified based on their spectral characteristics and used as markers for in vivo studies. In mouse epidermal tissue, the metabolites of 6-NO₂-5-MeC include trans-1,2-dihydro-1,2-dihydroxy-6-nitro-5-methyltriol (6-NO₂-5-MeC-1,2-diol), a precursor of bay dihydrodiol epoxides; trans-9,10-dihydro-9,10-dihydroxy-6-nitro-5-methyltriol; and 6-nitro-5-hydroxymethyltriol. The content of 6-NO₂-5-MeC-1,2-diol generated from 6-NO₂-5-MeC in mouse epidermal tissue was higher than that of the proximal carcinogen trans-1,2-dihydro-1,2-dihydroxy-5-methyltriol (5-MeC-1,2-diol) generated from 5-MeC. Further metabolism of 6-NO2-5-MeC-1,2-diol in mouse epidermis was investigated under conditions similar to those previously described for 5-MeC-1,2-diol. The extent to which both dihydrodiols formed 1,2,3,4-tetraols was similar. The chromatograms of DNA adducts formed by 6-NO2-5-MeC and 5-MeC in mouse epidermis were qualitatively similar; however, the extent of DNA adduct formation by 5-MeC was 15 times greater than that by 6-NO2-5-MeC. The reaction between calf thymus DNA and the bay area 1,2-diol-3,4-epoxides of 5-MeC and 6-NO2-5-MeC was compared; the adduct level formed by the benzene ring diol epoxide of 5-methylcyclohexane (5-MeC) was approximately four times that of the adduct formed by the benzene ring diol epoxide of 6-nitro-5-methylcyclohexane (6-NO2-5-MeC). The results suggest that the relatively low DNA-binding capacity of 6-NO2-5-MeC in vivo may explain its lack of tumorigenicity compared to 5-MeC. The nitro substitution at the 6-position of 5-MeC may interfere with the structural requirements of 1,2-diol-3,4-epoxides, which are essential for specific DNA interactions. We investigated the metabolism of cyclohexenoic acid (CHR) and 5-methylcyclohexenoic acid (5-MeCHR) in liver microsomes of Shasta rainbow trout (Oncorhynchus mykiss) and Long Evans rats to assess the effect of non-benzene ring methyl substituents on the metabolism of polycyclic aromatic hydrocarbons (PAHs). The metabolic rates of cyclohexenoic acid (CHR) and 5-methylcyclohexenoic acid (5-MeCHR) in trout and rat liver microsomes were substantially similar, indicating that methyl substituents do not alter the substrate specificity of the cytochrome P450 enzymes involved in the metabolism of these two PAHs. Dihydrodiols are the main CHR metabolites produced by trout and mouse liver microsomes. However, trout liver microsomes produce a significantly higher proportion of 5-MeCHR phenolic compounds than diols, indicating that 5-methyl substitution alters the substrate specificity of trout microsomal epoxide hydrolases for 5-MeCHR epoxides. Unlike trout liver microsomes, mouse liver microsomes produce a significantly higher proportion of 5-MeCHR diols than 5-MeCHR phenolic compounds, suggesting that 5-MeCHR epoxides are a better substrate for mouse liver microsomal epoxide hydrolases than for trout liver microsomal epoxide hydrolases. Trout and mouse liver microsomes exhibit higher attack efficiency on the benzene ring double bond in benzo[a]pyrene than on the non-benzene ring double bond. Conversely, the opposite is true for 5-methylbenzo[a]pyrene, indicating that non-benzene ring methyl substituents alter the regioselectivity of enzymes involved in the oxidation metabolism of polycyclic aromatic hydrocarbons. For more complete data on the metabolism/metabolites of 5-methylbenzo[a]pyrene (10 metabolites in total), please visit the HSDB record page. Known human metabolites of 5-methylbenzo[a]pyrene include 5-methyl-1,2-dihydrobenzo[a]pyrene-1,2-diol.

Toxicity Summary
Identification and Uses: 5-Methylcytosine (5-MeC) is a solid polycyclic aromatic hydrocarbon. There is currently no commercial production or known use of this compound. Certified high-purity reference samples are available. Human Exposure and Toxicity: Based on sufficient evidence of carcinogenicity from animal studies, it is reasonable to expect 5-MeC to be carcinogenic to humans. Animal Studies: A group of 25 male mice were subcutaneously injected with 0.05 mg of high-purity 5-MeC every 2 weeks for 20 weeks (total dose 0.5 mg), and observed for 12 weeks. 22 of the 25 mice developed 24 fibrosarcomas, with a mean latency of 25 weeks. The study found that 5-MeC is a potent lung carcinogen in A/J strain mice, inducing more than 100 tumors per mouse at a concentration of 200 mg/kg. 5-Methylcytosine (5-MeC) induces the formation of lung adenomas and DNA adducts in mice in a dose-dependent manner. The induction of lung adenomas was significantly correlated with changes in DNA adduct levels over time. 5-MeC is a potent tumor inducer in mouse skin. The environmental carcinogen 5-MeC can be activated by various cytochrome P-450 isoenzymes into mutagenic metabolites. The resulting reactive diol epoxides can be detoxified by binding to glutathione S-transferase.

[1]. Cytotoxicity and mutagenicity of 5-methylchrysene and its 1,2-dihydrodiol in V79MZ cells modified to express human CYP1A1 or CYP1B1, in the presence or absence of human GSTP1 coexpression. Toxicol Lett. 2008 Dec 15;183(1-3):99-104.

According to an independent committee of scientific and health experts, 5-methylpyrrolidone is potentially carcinogenic. 5-Methylpyrrolidone is a purple crystalline compound, insoluble in water. It is a polycyclic aromatic hydrocarbon. 5-Methylpyrrolidone is a crystalline, carcinogenic aromatic hydrocarbon composed of four fused rings, produced by the incomplete combustion of organic matter. It is primarily found in gasoline exhaust and tobacco smoke. 5-Methylpyrrolidone is reasonably expected to be a human carcinogen. (NCI05) Methylpyrrolidone in tobacco smoke is suspected to be one of the causes of this inhalant carcinogenicity; RN refers to an unlabeled compound; structure

C19H14

242.31

242.11

3697-24-3

19427

Needles (recyrstallized from benzene/ethanol) with a brilliant bluish-violet fluorescence in ultraviolet light

1.2±0.1 g/cm3

449.4±12.0 °C at 760 mmHg

117.5 °C

217.8±13.7 °C

0.0±0.5 mmHg at 25°C

1.748

6.37

0

0

19

320

0

CC1=CC2=CC=CC=C2C3=C1C4=CC=CC=C4C=C3

GOHBXWHNJHENRX-UHFFFAOYSA-N

InChI=1S/C19H14/c1-13-12-15-7-3-4-8-16(15)18-11-10-14-6-2-5-9-17(14)19(13)18/h2-12H,1H3

5-methylchrysene

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.)