Metabolism / Metabolites
4,5-Dihydrodiol is the major metabolite of benzo[e]pyrene, but 9,10-dihydrodiol was also detected as a minor product after incubation of benzo[e]pyrene with rat liver preparations. A glucuronic acid conjugate of 3-hydroxybenzo[e]pyrene and 4,5-dihydrodiol has been reported in hamster embryonic cells. 4,5,9,10-tetraols and 9,10,11,12-tetraols, as well as unidentified phenolic compounds, have been reported to be generated from the precursor 9,10-dihydrodiol using liver microsomal preparations from various species, including humans. The potential biological activity of benzo[e]pyrene was investigated by determining the mutagenic activity of polycyclic aromatic hydrocarbons and six of their derivatives in the presence and absence of a cytochrome P-450-dependent monooxygenase system. In rat liver microsomes pretreated with Aroclor 1254 or in the presence of a near-homogeneous cytochrome P-450-dependent monooxygenase system purified from these microsomes, benzo[e]pyrene, trans-9,10-dihydroxy-9,10-dihydrobenzo[e]pyrene (K-block dihydrodiol), and trans-9,10-dihydroxy-9,10,11,12-tetrahydrobenzo[e]pyrene were metabolized to products with little or no mutagenic activity against Salmonella Typhimurium strains TA 98 and TA 100. Under the same assay conditions, the product derived from trans-4,5-dihydroxy-4,5-dihydrobenzo[e]pyrene (K-block dihydrodiol) exhibited moderate mutagenic activity. Contrary to the above results, 9,10-dihydrobenzo[e]pyrene, upon metabolic activation, yields a potent mutagenic product with activity 10–25 times higher than that of the benzo[e]pyrene metabolites. The tetrahydrobenzene ring epoxide of chemically synthesized 9,10-dihydrobenzo[e]pyrene exhibited high intrinsic mutagenic activity in two bacterial strains and cultured Chinese hamster V79 cells, indicating that the high mutagenicity of 9,10-dihydrobenzo[e]pyrene metabolism is mediated by this tetrahydrobenzene ring epoxide. High-performance liquid chromatography (HPLC) was used to analyze the liver microsomal metabolites of several benzo[e]pyrene derivatives. Incubation of 9,10-dihydrobenzo[e]pyrene with untreated or Aroclor 1254-pretreated rat liver microsomes confirmed the metabolism of 9,10-dihydrobenzo[e]pyrene to form a benzene ring epoxide and identified the formation of several other metabolites. Contrary to these results, trans-9,10-dihydroxy-9,10-dihydrobenzo[e]pyrene was not metabolized to phenylcyclic diol epoxides, but instead converted to 4,5,9,10-tetrahydroxy-4,5,9,10-tetrahydrobenzo[e]pyrene and three phenolic derivatives of this dihydrodiol. Therefore, the lack of benzo[e]oxides could explain why 9,10-dihydroxy-9,10-dihydrobenzo[e]pyrene cannot be activated by rat liver enzymes to become a mutagenic product, and may contribute to the lower carcinogenic activity of benzo[e]pyrene in rodents. After 24 hours of culture with B[e]P (hamster embryonic cells), the main organic solvent sol metabolites formed in the extracellular culture medium were K-block dihydrodiol 4,5-dihydro-4,5-dihydroxybenzo[e]pyrene and a small amount of monohydroxybenzo[e]pyrene.
The genotoxicity of 15 polycyclic aromatic hydrocarbons (PAHs) was determined using the alkaline comet assay with Chinese hamster V79 lung fibroblasts as target cells. These cells lacked the enzymes required to convert PAHs into DNA-binding metabolites. Eleven PAHs—benzo[a]pyrene (BaP), benzo[a]anthracene, 7,12-dimethylbenzo[a]anthracene, 3-methylcholanthracene, fluoranthracene, anthracene anthracene, 11H-benzo[b]fluorene, dibenzo[a,h]anthracene, pyrene, benzo[ghi]perylene, and benzo[e]pyrene—caused DNA strand breaks even without external metabolic activation, while naphthalene, anthracene, phenanthrene, and benzonaphthalene did not exhibit this activity. The DNA-damaging effects of these 11 PAHs disappeared when the comet assay was performed in the dark or under yellow fluorescent illumination. White fluorescent lamps exhibit emission peaks at 334.1, 365.0, 404.7, and 435.8 nm, corresponding to the spectral lines of mercury. These emission phenomena are absent with yellow fluorescent lamps. Clearly, under standard laboratory lighting conditions, many polycyclic aromatic hydrocarbons (PAHs) are photoactivated, producing DNA-damaging substances. This property of PAHs should be considered when using these compounds to induce skin cancer. ...
PAH metabolism occurs in all tissues, typically catalyzed by cytochrome P-450 and its associated enzymes. PAHs are metabolized into reactive intermediates, including epoxide intermediates, dihydrodiols, phenols, quinones, and various combinations thereof. Phenols, quinones, and dihydrodiols can all bind to glucuronides and sulfates; quinones can also form glutathione conjugates. (L10)

Toxicity Summary
Identification and Uses: Benzo[e]pyrene (B(e)P) is a component of coal tar and is commonly used in experimental research. Polycyclic aromatic hydrocarbons (PAHs) are a class of chemical substances formed during the incomplete combustion of coal, petroleum, natural gas, wood, waste, or other organic matter (such as tobacco and barbecued meat). Human Exposure and Toxicity: B(e)P is a toxic component of cigarette smoke. In vitro experiments have shown that it is toxic to human retinal pigment epithelial cells. It induces cell death and apoptosis through multiple caspase pathways. Exposure to B(e)P reduces the viability of human microvascular endothelial cells. In the presence of an exogenous metabolic system, it can induce unplanned DNA synthesis in HeLa cells. The carcinogenicity of this substance in humans is currently unclassified. Animal Experiments: Observations have shown that intraocular injection of benzo[e]pyrene into the eyes of animals can cause severe uveitis. One study showed that benzo[e]pyrene is a very weak tumor initiator, a weak complete carcinogen, and a moderate tumor promoter. When used in combination with benzo[a]pyrene, it may act as a weak co-tumor initiator, while when combined with 7,12-dimethylbenzo[a]anthracene, it becomes a potent antitumor initiator. In another study, 20 female mice received dermal application of a 0.1% benzo[e]pyrene solution for life. Of the five mice that survived 13 months after treatment initiation, two developed cutaneous papillomas, and three developed cancer. In another experiment, 30 female mice received dermal application of 100 micrograms of benzo[e]pyrene for 30 weeks. After 30 weeks, 68% of the mice developed papillomas (an average of 2.1 papillomas per mouse). At 40 weeks, 24% of the mice developed cancer. Benzo[e]pyrene is mutagenic to Salmonella Typhimurium in the presence of an exogenous metabolic system. It does not induce mitotic recombination in yeast. It does not induce mutations or sister chromatid exchange in cultured mammalian cells, and morphological transformation assays are negative. In the only reported case to date, it does not induce chromosomal aberrations in vitro. In the only in vivo study to date, it induced sister chromatid exchange in hamster bone marrow, but did not induce chromosomal aberrations. Polycyclic aromatic hydrocarbons (PAHs) can bind to blood proteins such as albumin, enabling their transport in vivo. Many PAHs induce the expression of cytochrome P450 enzymes, particularly CYP1A1, CYP1A2, and CYP1B1, by binding to aryl receptors or glycine N-methyltransferases. These enzymes metabolize PAHs into their toxic intermediates. The active metabolites of PAHs (epoxide intermediates, dihydrodiols, phenols, quinones, and various combinations thereof) covalently bind to DNA and other cellular macromolecules, inducing mutagenic and carcinogenic effects. (L10, L23, A27, A32)

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Interactions
……In this study, we investigated the reactions of the isomers methylbenzo[a]pyrene (MBaP) and methylbenzo[e]pyrene (MBeP) under UVA light irradiation in the presence of lipid methyl linoleate and evaluated the potential of these compounds to induce lipid peroxidation. The compounds used in this study included benzo[a]pyrene (BaP), 3-methylbenzo[a]pyrene (3-MBaP), 4-methylbenzo[a]pyrene (4-MBaP), 6-methylbenzo[a]pyrene (6-MBaP), 7-methylbenzo[a]pyrene (7-MBaP), 10-methylbenzo[a]pyrene (10-MBaP), benzo[b]pyrene (BeP), 4-methylbenzo[b]pyrene (4-MBeP), and 9-methylbenzo[a]pyrene (9-MBeP). The results showed that these compounds induced lipid peroxidation under UVA irradiation at 7 and 21 J/cm². The levels of lipid peroxidation induced by BaP and its isomer MBaP, and by BeP and MBeP, were similar, but the lipid peroxidation level in the BaP group was higher than that in the BeP group. Furthermore, a correlation was found between UVA light dose and the induced lipid peroxidation level. Sodium azide (NaN₃) (a singlet oxygen and free radical scavenger) inhibits the formation of lipid peroxides, while heavy water (D₂O) (which prolongs the singlet oxygen lifetime) promotes lipid peroxide formation. These results indicate that UVA irradiation of MBaPs and MBePs generates reactive oxygen species (ROS), thereby inducing lipid peroxidation. Benzo[e]pyrene (B[e]P) inhibited the occurrence of 7,12-dimethylbenzo[a]anthracene (DMBA) skin tumors in mice by 84%, while pyrene and fluoranthene inhibited the occurrence of DMBA tumors by 50% and 34%, respectively. ...
When benzo[e]pyrene was administered to mice via skin application in combination with 7,12-dimethylbenzo[a]anthracene or benzo[a]pyrene, it resulted in fewer skin tumors compared to 7,12-dimethylbenzo[a]anthracene alone, and more skin tumors compared to benzo[a]pyrene alone. Twenty female Fischer 344 rats (age unspecified) were divided into several groups. In each group, syngeneic donor tracheae were subcutaneously transplanted into the retroscapular region (two tracheae per animal). Beeswax particles containing the following substances were implanted into the tracheae: 1 mg benzo[a]pyrene, 0.5 mg benzo[a]pyrene, 1 mg benzo[e]pyrene (purity unspecified), 0.5 mg benzo[a]pyrene + 1 mg benzo[e]pyrene, or 1 mg benzo[a]pyrene + 1 mg benzo[e]pyrene. All surviving animals were sacrificed 28 months after the start of exposure. Benzo[e]pyrene did not induce tumors in the tracheal explants, while 1 mg benzo[a]pyrene induced carcinogenesis in 65% of the transplanted tissues. Benzo[e]pyrene appears to reduce the incidence of carcinogenesis from 65% (benzo[a]pyrene alone) to 40% (benzo[a]pyrene in combination with benzo[e]pyrene). However, the incidence of sarcomas in the trachea and peritracheal explants increased 2 to 3 times compared to benzo[a]pyrene alone. More complete data on interactions with benzo[e]pyrene (6 items in total) can be found on the HSDB record page.

[1]. Fluorescence measurements of benzene, naphthalene, anthracene, pyrene, fluoranthene, and benzo(e)pyrene in water. Anal Chem. 1976 Mar;48(3):524-8.

Benzo[e]pyrene is a colorless crystal or a white crystalline solid. (NTP, 1992)
Benzo[e]pyrene is a fused polycyclic aromatic hydrocarbon (PAH) consisting of five fused benzene rings in both ortho and periphery. The International Agency for Research on Cancer (IARC) classifies it as a Group 3 carcinogen. It is mutagenic and carcinogenic.
Benzo[e]pyrene has been reported in tobacco (Nicotiana tabacum), and relevant data exist.
Benzo[e]pyrene is one of more than 100 different PAHs. PAHs are chemical substances formed during the incomplete combustion of organic matter (such as fossil fuels). They usually exist as mixtures of two or more compounds. (L10)

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Mechanism of Action
...Previous studies in the authors' laboratory have shown that benzo[a]pyrene (Bap) affects the efflux transport of Rhodamine 123 (Rho-123) by inducing P-glycoprotein (P-gp) in Caco-2 cells. This study investigated whether benzo[e]pyrene (Bep) can also induce P-gp expression and enhance Rho-123 efflux transport. Bep has a similar structure to Bap, but is not a carcinogenic compound. In Caco-2 monolayer cells, after treatment with 50 μM Bep for 72 hours, the apparent permeability ratio (P(app)) of Rho-123 efflux was significantly higher than that in the control group. Similarly, compared with the control group, Caco-2 cells exposed to Bep showed significantly increased expression levels of MDR1 mRNA and P-gp protein, as detected by RT-PCR and Western blot analysis, respectively. Caco-2 cells exposed to Bep exhibited oxidative stress, detectable by aminophenyl fluorescein fluorescence microscopy. However, this oxidative stress was weaker compared to that of Bap. In Caco-2 cells exposed to Bep or Bap, intracellular GSH levels decreased to 80% and 59% of the control group, respectively. Our results further suggest that Bep- or Bap-induced P-gp in Caco-2 cells may be a result of oxidative stress rather than DNA damage. We investigated the co-carcinogenic mechanism of benzo[e]pyrene (BeP) by measuring the effect of benzo[a]pyrene (BeP) on the binding of the carcinogen benzo[a]pyrene (BaP) to epidermal DNA in Sencar mice. The benzo[a]pyrene dose used was 20 nmol/mouse, a dose that was not carcinogenic after a single administration but became carcinogenic after multiple administrations, similar to the dose used in the benzo[a]pyrene-benzo[e]pyrene co-carcinogenicity experiment described by Van Duuren and Goldschmidt. Three hours after exposure to [(3)H]benzo[a]pyrene and benzo[e]pyrene at dose ratios of 1:3 and 1:10, the levels of [(3)H]benzo[a]pyrene-DNA adduct in both benzo[e]pyrene treatment groups were lower than those in the acetone-benzo[a]pyrene control group. At 12 and 24 hours of exposure, the levels of [(3)H]benzo[a]pyrene-DNA adduct in the benzo[a]pyrene-benzo[e]pyrene (1:10) group were 19% and 33% higher than those in the control group, respectively. In the benzo[a]pyrene-benzo[e]pyrene (1:3) group, the levels of [(3)H]benzo[a]pyrene-DNA adduct were higher than those in the control group after 12 hours of exposure. Co-treatment with benzo[e]pyrene and [(3)H]benzo[a]pyrene-7,8-dihydrodiol or trans[(3)H]benzo[a]pyrene had no effect on the content of benzo[a]pyrene DE-DNA adduct. These results indicate that the co-carcinogen benzo[e]pyrene can increase the binding of low-dose benzo[a]pyrene to mouse epidermal DNA, suggesting that the increase in benzo[a]pyrene-DNA adduct is due to the metabolism of benzo[a]pyrene to the proximal carcinogen benzo[a]pyrene-7,8-dihydrodiol.

C20H12

252.30900

252.093

192-97-2

9128

Prisms or plates from benzene
Pale yellow needles (from benzene or methanol)
Colorless crystals or white crystalline solid

1.3±0.1 g/cm3

467.5±12.0 °C at 760 mmHg

177-180ºC(lit.)

228.6±13.7 °C

0.0±0.6 mmHg at 25°C

1.887

6.4

0

0

0

20

336

0

C1=CC=C2C(=C1)C3=CC=CC4=C3C5=C(C=CC=C25)C=C4

TXVHTIQJNYSSKO-UHFFFAOYSA-N

InChI=1S/C20H12/c1-2-8-16-15(7-1)17-9-3-5-13-11-12-14-6-4-10-18(16)20(14)19(13)17/h1-12H

benzo[e]pyrene

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.)