Metabolism / Metabolites
This study investigated the peroxynitrite-induced metabolism of indo[1,2,3-cd]pyrene (IND) using a porphyrin/peroxynitrite biomimetic system. High-performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) was used to identify metabolites, including hydroxypyrene (OH-IND), pyrenequinone (IND-quinone), and nitrite (2NO2-IND). By terminating the reaction at different stages, we found that IND was initially converted to IND-quinone and 2NO2-IND, the latter subsequently to OH-IND. Mutagenicity assays (including Ames assay and cell transformation assay) showed that activation of the peroxynitrite/Fe(III)porphyrin system enhanced the mutagenicity of IND. The results also indicated that 2NO2-IND and IND-quinone play key roles in the mutagenicity following activation of polycyclic aromatic hydrocarbons (PAHs). …The major metabolites of pyrene formed in mouse skin have been identified. 8-Hydroxyindo[1,2,3-cd]pyrene was the most abundant metabolite identified. 9-Hydroxyindo[1,2,3-cd]pyrene and trans-1,2-dihydro-1,2-dihydroxyindo[1,2,3-cd]pyrene were also major in vivo metabolites in mouse skin. In addition, several minor metabolites were identified, including trans-1,2-dihydro-1,2,8-trihydroxyindo[1,2,3-cd]pyrene, trans-1,2-dihydro-1,2,9-trihydroxyindo[1,2,3-cd]pyrene, indo[1,2,3-cd]pyrene-1,2-dione, and 10-hydroxyindo[1,2,3-cd]pyrene. This study investigated the tumorigenic activity of several major in vivo metabolites of indo[1,2,3-cd]pyrene on mouse skin. Both trans-1,2-dihydro-1,2-dihydroxyindo[1,2,3-cd]pyrene and 1,2-dihydro-1,2-epoxyindo[1,2,3-cd]pyrene induced tumors in 80% of mice at a total starting dose of 1.0 mg. However, the activity of these K-region dihydrodiols and K-region oxides was lower than that of their parent hydrocarbons. These data suggest that 1,2-dihydro-1,2-epoxyindo[1,2,3-cd]pyrene, the final mutagenic metabolite of indo[1,2,3-cd]pyrene, is not the final tumorigenic metabolite observed on mouse skin. 8-Hydroxyindo[1,2,3-cd]pyrene exhibits mutagenicity when detected in the presence of a microsomal activation system, but only weak tumor initiation activity. These results indicate that the major metabolic activation pathway associated with the mutagenic activity of indo[1,2,3-cd]pyrene is independent of its tumor initiation activity in mouse skin. Indo[1,2,3-cd]pyranone (ip)…is carcinogenic to mouse skin and rat lung. Unlike benzo[a]pyrene, IP is a non-alternating polycyclic aromatic hydrocarbon that does not contain benzo[a]pyrene. IP is mutagenic to Salmonella Typhimurium TA100 in the presence of 9000 x g supernatant of Aroclor-pretreated rat liver. Using a similar activation system, the major metabolites of IP were identified… trans-1,2-dihydro-1,2-dihydroxy-IP, 8-, 9-, and 10-hydroxy-IP, 8- and 9-hydroxy-trans-1,2-dihydro-1,2-dihydroxy-IP, and IP-1,2-quinone are in vitro metabolites. The 1,2-epoxide of indo[1,2,3-cd]pyrene is a potent direct mutagen. 8- and 9-hydroxy-IP and trans-1,2-dihydrodiol did not exhibit significant mutagenic activity in metabolically activated Salmonella typhimurium TA100. These data suggest that the K-zone oxides of IP, as well as 8- and 9-hydroxy-IP, are ultimately the root cause of its mutagenic activity. The metabolism of polycyclic aromatic hydrocarbons occurs in all tissues and is generally catalyzed by cytochrome P-450 and its associated enzymes. Polycyclic aromatic hydrocarbons (PAHs) metabolize to produce 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 bind to glutathione. (L10)

Toxicity Summary
Identification and Uses: Indo[1,2,3-cd]pyrene (IP) forms yellow, flaky or needle-like crystals with a yellow-green fluorescence. It has no known uses other than for research purposes. Polycyclic aromatic hydrocarbons (PAHs) are a class of chemicals formed during the incomplete combustion of coal, petroleum, natural gas, wood, waste, or other organic matter (e.g., tobacco and barbecued meat). Human Exposure and Toxicity: This substance may be carcinogenic to humans. The more IP a mother is exposed to, the higher the risk of her child developing neuroblastoma. Animal Studies: In rats, intrapulmonary implantation of 0, 0.16, 0.83, or 4.15 mg doses of IP resulted in a dose-dependent increase in the incidence of squamous cell carcinoma and sarcoma of the lung. In studies of newborn male and female mice, intraperitoneal injection of IP on days 1, 8, and 15 after birth induced lung adenoma in 1 of 11 treated male mice. Topical application of IP to the skin of mice did not show carcinogenic effects. In the presence of an exogenous metabolic system, IP at a concentration of 20 μg/plate induced mutations in *Salmonella Typhimurium* TA100 (his-/his+) strains, and at a concentration of 2 μg/plate induced mutations in *Salmonella Typhimurium* TA98 strains. A strong photomutagenic response was observed in *Salmonella Typhimurium* TA102 strain after simultaneous exposure to IP and light. Ecotoxicity studies: IP induced the activity of 7-ethoxyhalothrin-O-deethylase (EROD) in rainbow trout hepatocyte cell lines. 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, especially CYP1A1, CYP1A2, and CYP1B1, by binding to aryl hydrocarbon 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
Pyrene, benzo[a]pyrene (BaP), and indo[1,2,3-cd]pyrene (IND) are polycyclic aromatic hydrocarbons (PAHs) with four to six fused benzene rings. Dexamethasone (Dex) is a synthetic glucocorticoid agonist. The aryl hydrocarbon receptor (AhR) ligands BaP and IND do not directly activate the glucocorticoid receptor (GR), nor does Dex activate AhR. When BaP and IND are added to Dex-treated cultures, they coexist with Dex for a longer period, and Dex-induced GR transcriptional activation is significantly enhanced, suggesting that newly activated AhR is crucial for synergistic effects with activated GR. The enhancement of dexamethasone (Dex)-induced glucocorticoid receptor (GR) transcriptional activation by polycyclic aromatic hydrocarbons (PAHs) (benzo[a]pyrene (BaP) / approximately equal to / indole (IND) > pyrene) was positively correlated with the efficacy of PAHs in activating the aryl hydrocarbon receptor (AhR). This synergistic effect was more pronounced in ovarian granulosa cells (HO23) than in HepG2, 293T, or HeLa cells. Conversely, Dex inhibited AhR-mediated expression, including AhR and cytochrome P450 (CYP) 1A1 expression. Dex also antagonized BaP-induced cell viability decline. Crosstalk between AhR and GR was independent of their expression levels. We conclude that there is a functional cross-reaction between AhR and GR, through which GR transcriptional activation activity is further enhanced while AhR transcriptional activation activity is inhibited. In this study, we evaluated how particulate polycyclic aromatic hydrocarbons (PAHs) modulate the formation of DNA damage induced by the carcinogen benzo[a]pyrene (B[a]P). We used a comet assay and high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) to detect single-strand breaks, base-labile sites, and BPDE-N2-dGuo DNA adducts in HepG2 competent cells. B[a]P, alone or in combination with other PAHs (1 μM each), resulted in only a small number of single-strand breaks. In contrast, the formation of BPDE-N2-dGuo adducts was significant, and in HepG2 cells, the formation of BPDE-N2-dGuo adducts was significantly enhanced after 14 hours of co-treatment with B[a]P in combination with benzo[b]fluoranthene (B[b]F), dibenzo[a,h]anthracene (DB[a,h]A), or indo[1,2,3-cd]pyrene (IP). The opposite results were obtained using benzo[k]fluoranthene (B[k]F). The same phenomenon was observed when cells were pre-incubated with polycyclic aromatic hydrocarbons (PAHs) before being incubated with benzo[a]pyrene (B[a]P). These results indicate that the interactions between PAHs are not directly competitive. Subsequently, the focus shifted to the regulatory roles of B[b]F and B[k]F in B[a]P-induced DNA damage. No difference was observed in the time course of DNA damage formation. However, the dose-response relationship between the two PAHs differed: B[k]F exhibited a concentration-dependent inhibitory effect on BPDE-N2-dGuo DNA, while B[b]F showed a constant enhancing effect at concentrations above 1 μM. In summary, these results suggest that the genotoxicity of B[a]P in binary mixtures with other carcinogenic PAHs may be modulated. In this case, enhanced BPDE-N2-dGuo adduct formation was generally observed, in addition to B[k]F. Several biological mechanisms may explain these phenomena, including the binding of polycyclic aromatic hydrocarbons (PAHs) to aryl hydrocarbon acceptors (AhRs), their affinity for CYP450, and metabolic competition. This article reports the in vitro effects of single heavy metals (Cd²⁺, Cu²⁺, and Hg²⁺) and polycyclic aromatic hydrocarbons (including benzo[a]pyrene (BaP), indo[1,2,3-cd]pyrene (IP), and fluoranthene (FL)) and mixtures thereof on the activity of ethoxyhalothrin-O-deethylase (EROD), as determined by microplate reader assay. The results showed that all three metals inhibited EROD activity, while BaP/IP significantly induced the enzyme's activity. However, FL alone reduced EROD activity. Furthermore, combined treatment with heavy metals and BaP/IP inhibited PAH-induced EROD activity, while combined exposure to heavy metals and FL induced FL-inhibited EROD activity. This study investigated the photomutability of 16 polycyclic aromatic hydrocarbons (PAHs), all of which are on the US Environmental Protection Agency (EPA) Priority Pollutant List. Without the use of activator S9, *Salmonella typhimurium* strain TA102 was simultaneously exposed to one of the PAHs and light (1.1 J/sq cm UVA + 2.1 J/aq cm visible light). Anthracene, benzo[a]anthracene, benzo[ghi]perylene, benzo[a]pyrene, indo[1,2,3-cd]pyrene, and pyrene all exhibited strong photomutagenic responses. Under the same conditions, acenaphthene, acenaphthene, benzo[k]fluoranthene, chlorophenanthrene, and fluorene showed weak photomutability. Benzo[b]fluoranthene, fluoranthene, naphthalene, phenanthrene, and dibenzo[a,h]anthracene did not exhibit photomutability. These results indicate that polycyclic aromatic hydrocarbons (PAHs) can be photoactivated and exhibit mutagenicity in Salmonella TA102. Simultaneously, a standard mutagenicity assay using 10% S9 as the activation system was employed to detect the mutagenicity of all 16 PAHs. The results showed that benzo[b]fluoranthene, benzo[k]fluoranthene, chrysene, acenaphthene, and fluorene exhibited weak mutagenicity, while the remaining PAHs were not mutagenic. Generally, the photomutability of PAHs in TA102 is not correlated with their mutagenicity after S9 activation in TA102 or TA98/TA100, as they involve different activation mechanisms. This study investigated the metabolism of indo[1,2,3-cd]pyrene (IND) induced by peroxynitrite using a porphyrin/peroxynitrite biomimetic system. Metabolites, including OH-IND, IND-quinone, and 2NO2-IND, were identified using high-performance liquid chromatography-electrospray ionization tandem mass spectrometry. By terminating the reaction at different stages, we found that IND is first converted to IND-quinone and 2NO2-IND, and then further converted to OH-IND. Mutagenicity analysis, including Ames assay and cell transformation experiments, showed that activation by the oxynitrite/Fe(III)porphyrin system enhanced mutagenicity. The results also indicated that 2NO2-IND and IND-quinone play a key role in the mutagenicity following activation by polycyclic aromatic hydrocarbon metabolism.

[1]. An in vitro cytotoxicities comparison of 16 priority polycyclic aromatic hydrocarbons in human pulmonary alveolar epithelial cells HPAEpiC. Toxicol Lett. 2018 Jun 15;290:10-18.

[2]. A prominent air pollutant, Indeno1,2,3-cdpyrene, enhances allergic lung inflammation via aryl hydrocarbon receptor. Sci Rep. 2018 Mar 26;8(1):5198.

According to an independent committee of scientific and health experts, indo[1,2,3-cd]pyrene is likely carcinogenic. Indo[1,2,3-cd]pyrene is a yellow crystalline substance. It belongs to the pyrene family of compounds. Indo[1,2,3-cd]pyrene is a pale yellow aromatic hydrocarbon composed of six fused rings, a product of the incomplete combustion of organic matter. It is primarily found in certain foods, gasoline and diesel exhaust, cigarette smoke, coal tar and coal tar pitch, soot, and petroleum asphalt. This substance is for research purposes only. Indo[1,2,3-cd]pyrene is likely a human carcinogen. (NCI05)
Indo-(1,2,3-cd)pyrene is one of more than 100 polycyclic aromatic hydrocarbons (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) It is one of the components of cigarettes.
Structure see first source.

C22H12

276.33

276.094

193-39-5

Indeno[1,2,3-cd]pyrene-d12;203578-33-0;Indeno[1,2,3-cd]pyrene (Standard);193-39-5

9131

Colorless to light yellow liquid

1.4±0.1 g/cm3

497.1±12.0 °C at 760 mmHg

320 to 325 °F (NTP, 1992) ; 164 °C ; 164 °C

247.2±13.7 °C

0.0±0.6 mmHg at 25°C

2.009

6.89

0

0

22

453

0

C1=CC=C2C(=C1)C3=C4C2=CC5=CC=CC6=C5C4=C(C=C6)C=C3

SXQBHARYMNFBPS-UHFFFAOYSA-N

InChI=1S/C22H12/c1-2-7-17-16(6-1)18-11-10-14-9-8-13-4-3-5-15-12-19(17)22(18)21(14)20(13)15/h1-12H

hexacyclo[16.3.1.02,7.08,21.011,20.014,19]docosa-1(22),2,4,6,8(21),9,11(20),12,14(19),15,17-undecaene

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)

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.)