Metabolism / Metabolites
Alkyl-substituted polycyclic aromatic hydrocarbons can be metabolized to highly reactive benzyl sulfates via benzylic hydroxylation and subsequent sulfonation. We investigated the benzylic hydroxylation of 1-methylpyrene (MP, a rodent liver carcinogen) and 1-ethylpyrene (EP). Unlike the primary alcohol (α-HMP) generated by MP, the benzylic hydroxylation of EP yields a secondary alcohol (α-HEP). We incubated these hydrocarbons with human and mouse liver microsomes, as well as V79-derived cell lines genetically engineered to express specific human (1A1, 1A2, 1B1, 2A6, 2E1, 3A4) and mouse (1A1, 1A2, 2B1) cytochrome P450 (CYP) forms. All microsomal systems and CYP-expressing cell lines (except for CYP-deficient V79 cells) showed biotransformation of both hydrocarbons. Benzyl alcohol formation was detected in all cases. In the presence of human liver microsomes (38-64%) and cells expressing human 3A4, 2E1, or 1B1 (80-85%), α-HMP and its oxidation product 1-pyrene carboxylic acid (COOH-P) account for a large proportion of the total MP metabolites. Similarly, in cells expressing human 1A1, the contribution of α-HMP and COOH-P to total metabolites (45%) is higher than in cells expressing rat homologous enzymes (3%). Compared to MP, EP exhibits a higher metabolic rate and altered regioselectivity. Although ω-hydroxylation of the side chain was not detected in cell lines, and this reaction accounts for only a small portion of biotransformation in microsomal formulations, both the metabolic rate and regioselectivity of EP are altered. In cells expressing human 1A1, 1B1, and 3A4, α-HEP contributes the highest percentage (38-51%) to the total EP metabolites. In the presence of human liver microsomes, α-HEP accounts for 16% of the generated metabolites. Therefore, benzylic hydroxylation is the major initial step in the metabolism of both MP and EP. This pathway appears to be more significant in humans than in rats. Previously, we demonstrated that the second step of activation, namely the sulfonation of α-HMP and α-HEP, can also be efficiently catalyzed by a variety of human sulfonyltransferases. 1-Methylpyrene is an alkylated polycyclic aromatic hydrocarbon and an environmental carcinogen. It is activated to 1-hydroxymethylpyrene (1-HMP) via side-chain hydroxylation, which then undergoes sulfonation with the DNA reactant 1-sulfonyloxymethylpyrene. In addition to bioactivation, metabolic detoxification and transport processes also greatly influence the genotoxicity of 1-methylpyrene. To better understand the detoxification process of 1-HMP in vivo, we investigated the metabolites in the urine and feces of rats after intraperitoneal injection of 19.3 mg/kg body weight of 1-HMP (5 rats) or the same dose containing 200 μCi [(14)C]1-HMP/kg body weight of the same dose (2 rats). Forty-eight hours later, 48.0% (rat 1) and 29.1% (rat 2) of the radioactive material were recovered from the feces as 1-HMP. Six major metabolites were detected in urine and fecal samples by high-performance liquid chromatography (HPLC) followed by UV detection and online radioactivity detection. These compounds were characterized by mass spectrometry, proton NMR spectroscopy, and proton-COSY NMR spectroscopy to determine their preliminary molecular structures. Two major metabolites, 1-pyrenecarboxylic acid (M-6) and acylglucuronide of 1-pyrenecarboxylic acid (M-5), accounted for 17.7% (rat 1) and 25.2% (rat 2) of the total radioactive dose, respectively. In addition, acylglucuronide of 6-hydroxy-1-pyrenecarboxylic acid (M-1) and 8-sulfonoxy-1-pyrenecarboxylic acid (M-3), as well as two regioisomers of M-3 (M-2 and M-4), differing in the position of the sulfate group on the pyrene ring, were also detected. In urine samples, the radioactivity of 1-pyrene carboxylic acid and its five derivatives accounted for 32.4% (rat 1) or 45.5% (rat 2) of the total [(14)C]1-HMP dose. This study compared the transformation of unsubstituted and alkyl-substituted polycyclic aromatic hydrocarbons (PAHs) by the benthic invertebrate Nereis diversicolor. Pyrene and 1-methylpyrene were used as model compounds for unsubstituted and alkyl-substituted PAHs, respectively. Qualitative and quantitative analyses were performed on metabolites and parent compounds in worm tissues, water, and sediments. The transformation of 1-methylpyrene yielded a benzyl hydroxylated phase I product, 1-pyrene carboxylic acid, which accounted for 90% of the total 1-methylpyrene metabolites and was mainly found in the water extract. We preliminarily identified 1-methylpyrene glucuronide and 1-carbonylpyrene glycine as previously unreported phase II metabolites. Pyrene can be bioconverted into 1-hydroxypyrene, pyrene-1-sulfate, pyrene-1-glucuronide, and pyrene-glucuronide sulfate, with pyrene-1-glucuronide being the most significant metabolite. The conversion efficiency of 1-methylpyrene (21%) is more than three times that of pyrene (5.6%). Since the content of CC-substituted polycyclic aromatic hydrocarbons (PAHs) in crude oil is higher than that of unsubstituted PAHs, the rapid and efficient conversion of 1-methylpyrene in sediments may lead to significant exposure of aquatic organisms to the metabolites of alkyl-substituted PAHs, the toxicity of which remains unclear. The common PAH 1-methylpyrene is carcinogenic in a neonatal mouse liver cancer model. In vitro studies have shown that 1-methylpyrene is metabolized and activated via benzylic hydroxylation and sulfation to form an active ester, which can form benzylic DNA adducts, such as N(2)-(1-methylpyrene)-2'-deoxyguanosine (MPdG) and N(6)-(1-methylpyrene)-2'-deoxyadenosine (MPdA). The formation of these adducts has also been observed in animals treated with the metabolites 1-hydroxymethylpyrene and 1-sulfonyloxymethylpyrene (1-SMP), but data on 1-methylpyrene are lacking. In this study, we treated mice with 1-methylpyrene and subsequently analyzed the presence of the active metabolite 1-SMP in serum and the presence of MPdG and MPdA adducts in tissue DNA. We used wild-type mice and human sulfotransferase (SULT) 1A1 and 1A2 transgenic mouse strains, including both males and females. All analyses were performed using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) with isotopically labeled internal standards for adduct analysis. 1-SMP was detected in all treated animals. Serum 1-SMP levels in transgenic mice were higher than in wild-type mice (p < 0.001). Similarly, both adducts were detected in liver, kidney, and lung DNA from all exposed animals. Transgenic studies significantly increased the levels of each adduct in all tissues of both male and female mice (p < 0.01–0.001). The highest adduct levels were observed in the liver (the carcinogenic target tissue) among all animal models used. MPdG and MPdA adducts were also observed in rats treated with 1-methylpyrene. Our results confirm that 1-SMP is indeed the ultimate carcinogen of 1-methylpyrene, and that human SULT can mediate its ultimate activation in vivo.
For more complete data on the metabolism/metabolites of 1-methylpyrene (6 in total), please visit the HSDB record page.

Toxicity Summary
Identification and Uses: 1-Methylpyrene (1-MP) is a polycyclic aromatic hydrocarbon. Human Exposure and Toxicity: This study investigated the ability of 1-MP to induce cytotoxicity, micronuclei, and Hprt gene mutations in V79-hCYP2E1-hSULT1A1 cells using Chinese hamster V79-derived cell lines expressing human CYP2E1 and human SULT1A1. 1-MP induced micronuclei in V79-hCYP2E1-hSULT1A1 cells in a concentration-dependent manner; however, it had no effect on V79 cells. 1-MP led to an increased frequency of Hprt mutations in V79-hCYP2E1-hSULT1A1 cells, but had no effect on V79 cells. These results indicate that human CYP2E1 and SULT1A1 synergistically activate 1-MP, thereby leading to genotoxicity in mammalian cells. 1-Methylpyrene or perylene, alone or in combination, significantly upregulated the secretion of IL-1α and IL-6 in human keratinocytes. 1-Methylpyrene also exhibits cytotoxic effects on human keratinocytes. Animal experiments: Unactivated 1-methylpyrene showed no activity in the Chinese hamster V79-derived cell line. Modification of adenine residues with 1-methylpyrene caused E. coli DNA polymerase I (Klenow fragment) to terminate DNA replication in vitro, with the termination site located opposite the base of the 1-methylpyrene adduct and the base preceding it.

[1]. Time course of hepatic 1-methylpyrene DNA adducts in rats determined by isotope dilution LC-MS/MS and 32P-postlabeling. Chem Res Toxicol. 2008 Oct;21(10):2017-25.

[2]. 1-Methylpyrene induces chromosome loss and mitotic apparatus damage in a Chinese hamster V79-derived cell line expressing both human CYP1A2 and sulfotransferase 1A1. Chem Biol Interact. 2020 Dec 1;332:109283.

Plate-like substance (soluble in ethanol) or brownish-green powder. (NTP, 1992)
1-Methylpyrene is a member of the pyrene class of compounds.

C17H12

216.2772

216.093

2381-21-7

16932

Light yellow to yellow solid powder

1.2±0.1 g/cm3

387.4±9.0 °C at 760 mmHg

72-74 °C(lit.)

178.9±12.8 °C

0.0±0.4 mmHg at 25°C

1.816

5.63

0

0

0

17

294

0

KBSPJIWZDWBDGM-UHFFFAOYSA-N

InChI=1S/C17H12/c1-11-5-6-14-8-7-12-3-2-4-13-9-10-15(11)17(14)16(12)13/h2-10H,1H3

1-methylpyrene

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 : ~4.35 mg/mL (~20.11 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.)