NNK (100 pM; 0–60 minutes) directly causes c-Myc phosphorylation by stimulating PKCα and MAPK ERK1/2 activation [1]. NNK (100 pM; 96 hours) increases the proliferation of WT-expressing cells, but not of AAc-Myc mutant-expressing cells [1]. Western blot examination

Western blot analysis
Cell Types: NCI-H82 cells [1]
Tested Concentrations: 100 pM
Incubation Duration: 0-60 minutes
Experimental Results: Stimulates the activation of PKCα and MAPK ERK1/2 and directly induces c-Myc phosphorylation.

---

Apoptosis analysis
Cell Types: H1299 lung cancer cells [1]
Tested Concentrations: 100 pM
Incubation Duration: 96 hrs (hours)
Experimental Results: Cells expressing WT but not the T58A/S62A c-Myc mutant had enhanced proliferation.

Absorption, Distribution and Excretion
Numerous studies have investigated the metabolism of NNK and its metabolite 4-(methylnitroso)-1-(3-pyridyl)-1-butanol (NNAL) in humans and laboratory animals, as well as its adduct formation with DNA; the metabolic pathways and structures of DNA adducts have been comprehensively characterized. NNK and NNAL have been detected in the saliva of smokeless tobacco users, and NNAL and its other metabolite, NNAL-glucuronide, have been quantitatively detected in human urine. These metabolites are specific to tobacco product exposure (e.g., in smokers, smokeless tobacco users, and non-smokers exposed to secondhand smoke), and their presence indicates the absorption and metabolism of NNK in the human body. Quantification of these metabolites can estimate the absorbed dose of NNK. Dosage calculations indicate that the total amount of NNK ingested by individuals with long-term tobacco product use of 30 years or more is roughly equivalent to the total amount inducing tumors in rats.
The absorption of NNK by smokeless tobacco users was confirmed by the detection of the NNK metabolites 4-(methylnitrosamine)-1-(3-pyridyl)-1-butanol (NNAL) and NNAL-glucuronide (Gluc) in plasma and urine, and by the detection of NNAL-N-oxide in urine. Similar results were obtained in smokers, although NNAL was detected only in plasma. In the urine of smokers, NNAL-N-Gluc accounted for 50 ± 25% of total NNAL-Gluc, while in snuff users, the proportion was 24 ± 12%. The levels of NNAL and NNAL-Gluc excreted in the body of Tumbak pipe smokers were abnormally high (0.12 to 0.14 mg daily), indicating that the human body absorbs NNK at a higher rate than any other non-occupational carcinogen.
4-(methylnitrosamine)-1-(3-pyridyl)-1-butanol (NNAL) was detected in the amniotic fluid of mothers who smoked, but NNAL-Gluc was not detected. NNAL and NNAL-Gluc were detected in the urine of newborns from smoking mothers, but not in the urine of newborns from non-smoking mothers. These results suggest that NNK is converted to NNAL in the womb, and that NNAL can cross the placental barrier, be absorbed and metabolized to NNAL-Gluc during late fetal development. 4-(methylnitroso)-1-(3-pyridyl)-1-butanol (NNAL) and NNAL-Gluc were excreted in urine at a slower rate than expected based on their structures after smoking cessation or cessation of smoke-free tobacco use. One week after quitting smoking, 34.5% of baseline levels of NNAL and NNAL-Gluc were still detectable in urine, compared to 1.1% and 0.5% for the structure-related compounds cotinine and nicotine, respectively. Even six weeks after quitting smoking, 7.6% of NNAL and NNAL-Gluc remained in urine. The distribution half-lives of NNAL and NNAL-Gluc were 3 to 4 days, and the elimination half-lives were 40 to 45 days. The total clearance of NNAL was estimated at 61.4 ± 35.4 mL/min, and the β-phase distribution volume was estimated at 3800 ± 2100 L, indicating its extensive distribution in tissues. After cessation of smoke-free tobacco use, the distribution half-lives of both NNAL (1.32 ± 0.85 days vs. 3.35 ± 1.86 days) and NNAL-Gluc (1.53 ± 1.22 days vs. 3.89 ± 2.43 days) were significantly shorter in smokers. There was no significant difference in terminal half-lives. /NNAL/
For more complete data on absorption, distribution, and excretion of 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (out of 10), please visit the HSDB record page.

---

Metabolism/Metabolites
Many studies have reported assessing NNK absorption by measuring urinary metabolites 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanol and its glucuronide (total NNAL), but there is a lack of data in the literature regarding the percentage of NNK dose converted to NNAL in smoke-free tobacco users. …Fifteen male subjects had quit smoking for three weeks before placing 2 grams of smoke-free tobacco between their cheeks and gums 30 minutes prior. They continued to quit smoking and collected urine samples for three consecutive 24-hour periods. This study measured the NNK content in tobacco and saliva before and after smoking. The calculated NNK dose was compared with the total NNAL excreted over the next 72 hours. These data, combined with previous pharmacokinetic data, suggest that the percentage of NNK converted to total NNAL in smoke-free tobacco users is approximately 14% to 17%. This value can be used to calculate the daily NNK exposure (approximately 6 micrograms) in smoke-free tobacco users. The results of this study also indicate that the main metabolic pathway of NNK in smokeless tobacco users is metabolic activation into intermediates that can react with DNA. NNK can be converted into pyridine oxidation products 4-(methylnitrosoamino)-1-[3-(6-hydroxypyridinyl)]-1-butanone (6-HONNK) and NNK-N-oxide. After denitrosylation, NNK is oxidized to sarcosine. NNK can replace nicotinamide in NADP+ or NADPH to generate NNK adenosine dinucleotide phosphate ((NNK)ADP+) and (NNK)ADPH (reduced form). The carbonyl reduction of NNK yields NNAL, which can be further converted to four diastereomers of NNAL-Gluc via glucuronidation: two 4-(methylnitrosoamino)-1-(3-pyridyl)-1-(O-β-D-glucuronylpyranoside)butane isomers (NNAL-O-Gluc) and two 4-(methylnitrosoamino)-1-(3-pyridyl-N-β-D-glucuronylpyranoside)-1-butane inner salt isomers (NNAL-N-Gluc). NNAL can also be converted to NNAL-N-oxide and NNAL(ADP+). The α-hydroxylation of NNK with 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanol (NNAL) generates DNA and hemoglobin adducts. Hydroxylation of the NNK methyl group yields 4-(hydroxymethylnitrosoamino)-1-(3-pyridyl)-1-butanone (α-HOMeNNK), which can combine with glucuronic acid to form α-HOMeNNK-Gluc. α-HOMeNNK spontaneously decomposes to produce 4-oxo-4-(3-pyridyl)-1-butane diazonium hydroxide (POB-DZH) and formaldehyde. POB-DZH reacts with water to form 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB), which can combine with glucuronic acid to form HPB-Gluc. POB-DZH can also react with DNA and hemoglobin to form a series of adducts. α-Hydroxylation of the NNK methylene group yields 4-(methylnitrosoamino)-1-(3-pyridyl)-1-(4-hydroxy)butanone (α-HOMethyleneNNK). This metabolite spontaneously decomposes into 4-(3-pyridyl)-4-oxobutanal (ketaldehyde) and methyldiazo hydroxide (Me-DZH). The ketaldehyde is further metabolized to 4-(3-pyridyl)-4-oxobutyric acid (keto acid), which can then be converted into 4-hydroxy-4-(3-pyridyl)butyric acid (hydroxy acid). Me-DZH reacts with water to produce methanol and with DNA to form a methyl adduct, as shown in Figures 2 and 5. Similarly, NNAL undergoes α-hydroxylation on its methylene group to produce 4-(methylnitrosoamino)-1-(3-pyridyl)-1-(4-hydroxy)butanol (α-HOMethyleneNNAL), and α-hydroxylation on its methyl group to produce 4-(hydroxymethylnitrosoamino)-1-(3-pyridyl)-1-butanol (α-HOMeNNAL). α-HOMethyleneNNAL spontaneously decomposes into Me-DZH and 5-(3-pyridyl)-2-hydroxytetrahydrofuran (lactone alcohol), the latter of which can be converted into a hydroxy acid. α-HOMethyleneNNAL spontaneously decomposes into 4-hydroxy-4-(3-pyridyl)-1-butane diazonium hydroxide (PHB-DZH) and formaldehyde. PHB-DZH reacts with water to form 4-(3-pyridyl)butane-1,4-diol (diol), which cyclizes to form 2-(3-pyridyl)tetrahydrofuran (pyridyltetrahydrofuran), and reacts with DNA and hemoglobin to form adducts. Cytochrome P450 (CYP) is the main catalyst for the α-hydroxylation of NNK in humans and rodents. The relative efficiency of human CYPs in NNK metabolism (from highest to lowest catalytic activity) is: 2A13 > 2B6 > 2A6 > 1A2 ~ 1A1 > 2D6 ~ 2E1 ~ 3A4. The actual participation of these enzymes in NNK metabolism in vivo depends on various factors, including relative expression levels, expression levels of CYP oxidoreductases in specific tissues, tissue localization and inducibility of each CYP, and the concentration of NNK in human tissues. Among liver CYPs, 2B6 has the highest affinity for NNK. However, the content of this enzyme is low in most liver samples. The content of CYP2A6 is also relatively low, accounting for only 1% to 4% of the total CYP. The content of CYP1A2 is 4 to 20 times higher than that of CYP2A6. Therefore, although CYP1A2 has a slightly higher Km value and a slightly lower Vmax/Km value, it is likely, like CYP2A6, an important catalyst for NNK α-hydroxylation in human liver. CYP3A4 may also be involved in hepatic NNK α-hydroxylation, as its concentration is typically 10 to 50 times higher than that of CYP2A6. The presence of CYP2A13 (the most important known catalyst for NNK metabolism) in the liver cannot be completely ruled out. However, the mRNA level of CYP2A13 in the liver is much lower than that of CYP2A6, suggesting that even if this enzyme is present, its level is extremely low. To date, no single CYP enzyme in the liver has been identified as a key player in NNK activation. Multiple enzymes, including CYP1A2, CYP2A6, CYP2B6, and CYP3A4, clearly play a role. The relative contributions of these CYP enzymes vary from person to person, and their relative abundance and catalytic efficiency suggest that few (if any) of them are major catalysts. For more complete metabolite/metabolite data on 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (19 metabolites in total), please visit the HSDB record page.
4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK) Known third-hand smoke metabolites include 4-hydroxy-4-pyridin-3-ylbutanal, 4-oxo-4-pyridin-3-ylbutane-1-diazonium salt, 5-(3-pyridyl)-tetrahydrofuran-2-one (lactone), and 5-(3-pyridyl)-2-hydroxytetrahydrofuran (lactone alcohol). NNAL-O-glucuronide, NNAL-N-glucuronide, 4-[(hydroxymethyl)nitrosoamino]-1-(3-pyridyl)-1-butanone, 1-(methylnitrosoamino)-4-(3-pyridyl)-1,4-butanediol, NNAL-N-oxide, 1-(3-pyridyl)-1,4-butanediol (1,4-diol), 3-(oxacyclopentan-2-yl)pyridine, 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB), 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK), 4-hydroxy-4-(3-pyridyl)-butyric acid (HPBA) 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanol (NNAL), isoNNAL, α-[3-[(hydroxymethyl)nitrosoamino]propyl]-3-pyridinemethanol, 4-oxo-4-(pyridin-3-yl)butanal, 2-hydroxy-1-pyridin-3-ylprop-1-one, NNK-N-oxide, 4-oxo-4-(3-pyridyl)butyric acid (OPBA), and 4-hydroxy-4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone. 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK) is a known third-hand smoke metabolite.
The known human metabolites of NNK include 4-[(hydroxymethyl)nitrosoamino]-1-(3-pyridyl)-1-butanone.

Toxicity Summary
Routes of Exposure: Tobacco-specific N-nitrosamines, including 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK), N'-nitrosonornicotinic acid (NNN), N'-nitrosoanabasine (NAB), and N'-nitrosoanatabine (NAT), are widely present in tobacco and tobacco smoke. They are formed by the nitrosation of nicotine and other tobacco alkaloids and have been detected in the green leaves of common tobacco (Nicotiana tabacum) and rust tobacco (N. rustica); however, the main formation of tobacco-specific N-nitrosamines occurs during tobacco curing and processing, with smaller amounts also generated during smoking. All commercially available and non-commercially available tobacco products, including cigarettes, cigars, bisques, pipe tobacco, and smokeless tobacco products, contain tobacco-specific N-nitrosamines. N-nitrosamines are widely present in various foods and non-foods, but the content of tobacco-specific N-nitrosamines in all tobacco products is several orders of magnitude higher than in other commercial products. Smokeless tobacco products have the highest content of tobacco-specific N-nitrosamines. …The degree of exposure to tobacco-specific N-nitrosamines depends not only on the amount of these compounds in the tobacco product or smoke, but also on the method of product use. Effects on humans: No relevant data available. Effects on animals: In multiple mouse studies, NNK induced lung adenomas, regardless of the route of administration. In subcutaneous injection studies, benign and malignant tumors of the lung, nasal cavity, and liver were induced in rats. In two of four hamster studies, lung adenomas, adenocarcinomas, or adenosquamous carcinomas were induced in both male and female hamsters. Adenomas were observed in two other studies. A limited study in mink observed nasal tumors involving the forebrain. In one study administered via drinking water and another via oral swab, benign and malignant lung tumors (adenomas, adenosquamous carcinomas, and carcinomas) were induced in male rats. In the drinking water administration study, NNK induced benign and malignant pancreatic tumors. In the oral swab administration study, benign and malignant tumors of the liver and nasal cavity were observed. The incidence of liver and lung tumors was significantly increased when NNK was instilled into the bladder of female rats. In two studies, NNK was delivered to mouse offspring via intraperitoneal injection. Liver tumors were observed in male offspring in both studies, and in one study, liver tumors were also observed in female offspring. Lung tumors were also observed in male offspring in one study. In studies of hamster offspring treated with NNK during pregnancy, intratracheal instillation in mothers led to nasal adenocarcinoma in male offspring, and one study also found adrenal pheochromocytoma in both male and female offspring. In another study, subcutaneous injection of NNK into female mice induced respiratory tract (nasal cavity, larynx, and trachea) tumors in both male and female offspring. In a third study, subcutaneous injection or intratracheal instillation of NNK into female mice resulted in nasal and adrenal tumors in both male and female offspring. Intraperitoneal injection of NNK-N-oxide induced pulmonary adenomas in female mice. In an oral swab study, the combined use of NNK and NNN increased the incidence of oral tumors in rats. Interactions… Male ICR mice were administered NNK (0.5 mg/mouse) and sodium arsenite (0, 10, or 20 mg/kg) daily by gavage for 10 days, with urine collected on day 10 for NNK metabolite analysis. Liver samples were also collected for CYP2A enzyme and DNA adduct assessment. Arsenic-treated mice showed significantly increased levels of cyp2a4/5 mRNA and CYP2A enzyme activity in their livers. Furthermore, compared with mice treated with NNK alone, mice treated with a combination of NNK and arsenic showed increased levels of NNK metabolites in their urine. Simultaneously, DNA adducts (N(7)-methylguanine and O(6)-methylguanine) were significantly increased in the livers of mice simultaneously treated with NNK and arsenic. Our results clearly demonstrate that arsenic increases NNK metabolism by upregulating CYP2A expression and activity, leading to increased NNK metabolism and DNA adducts (N(7)-methylguanine and O(6)-methylguanine). These findings suggest that NNK may induce the formation of more DNA adducts in liver tissue in the presence of arsenic, resulting in a higher carcinogenic potential. The NNK/mouse lung model has been widely used by numerous researchers to identify factors, conditions, drugs, or chemopreventive compounds that can regulate lung tumor formation in mice. One such study is summarized below. This study aimed to determine the ability of cigarette smoke to induce lung tumors and promote NNK-induced lung tumor development. Twenty 7-week-old female A/J mice were randomly divided into four groups and exposed to filtered air (FA), cigarette smoke (CS; diluted mainstream smoke with a target concentration of 250 mg/m³ total particulate matter, from cigarettes used in the IR3 study), NNK, or NNK+CS for 26 weeks, 5 days a week. Before full exposure, mice were first exposed to 50% of the target concentration of CS for 3 days, followed by 75% of the target concentration of CS for 4 days. Three days before CS exposure, mice were intraperitoneally injected with 100 mg/kg body weight of NNK (dissolved in 0.1 mL of physiological saline). Mice were sacrificed 5 weeks after the end of exposure. The total number of tumors was counted visually and characterized microscopically. Kaplan-Meier survival analysis was performed, and the Breslow statistic was used to analyze survival differences. Bonferroni's multiple comparisons corrected Student's t-test was used to test for differences in lung weight, tumor number in all animals, and tumor number in tumor-bearing animals among the groups, with a significance level set at p < 0.05. The incidence rates of lung tumors in the four groups were as follows: FA group, 5/19 (26%); CS group, 0/19 (0%); FA + NNK group, 19/20 (95%); CS + NNK group, 13/16 (81%). The number of lung tumors (total number of tumors per animal) in each group were as follows: FA group, 0.32 ± 0.58 tumors per animal; FA + NNK group, 2.50 ± 1.67 tumors per animal; CS + NNK group, 2.50 ± 1.97 tumors per animal. The number of lung tumors in tumor-bearing animals was as follows: FA group, 1.20 ± 0.44 tumors per animal; FA + NNK group, 2.63 ± 1.61 tumors per animal; CS + NNK group, 3.08 ± 1.71 tumors per animal. CS exposure reduced the animals' body weight and lung weight, but NNK treatment had no additional effect. In all animals, the number of tumors in the FA+NNK and CS+NNK groups was higher than that in the FA and CS groups (p<0.05), but there was no significant difference in the number of tumors among the FA, FA+NNK, and CS+NNK groups in tumor-bearing animals.

---

Non-human toxicity values
Mouse intraperitoneal injection LD50 1 g/kg

[1]. Tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone promotes functional cooperation of Bcl2 and c-Myc through phosphorylation in regulating cell survival and proliferation. J Biol Chem. 2004;279(38):40209-40219.

[2]. Lung tumorigenicity of NNK given orally to A/J mice: its application to chemopreventive efficacy studies. Exp Lung Res. 1991;17(2):485-499.

According to the International Agency for Research on Cancer (IARC) of the World Health Organization, 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone is potentially carcinogenic. 4-(N-nitroso-N-methylamino)-1-(3-pyridyl)-1-butanone (nnk) is a pale yellow crystalline solid. (NTP, 1992) 4-(N-nitrosomethylamino)-1-(3-pyridyl)butanone is a nitrosamine, belonging to the pyridine class of compounds. There have been reports of the presence of 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone in tobacco (Nicotiana tabacum), and relevant data exist. 4-Methylnitrosoamino-1,3-pyridyl-1-butanone is a pale yellow crystalline solid nitrosamine, naturally occurring in tobacco products, and is a product of nicotine oxidation and nitrosation during tobacco production and consumption. 4-Methylnitrosoamino-1,3-pyridyl-1-butanone is currently used only as a research chemical to induce tumorigenesis. This substance is considered potentially carcinogenic to humans. (NCI05)
See also: Tobacco (partial).

---

Mechanism of Action
The metabolic activation of NNK and 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanol (NNAL) into DNA adducts is key to its carcinogenic activity. The metabolic activation process has been extensively demonstrated in laboratory animals. Cytochrome P450 enzymes are the main catalysts in this process, with enzymes from the 2A family appearing to be the most efficient in humans and laboratory animals. NNK is a genotoxic compound. In vitro experiments have shown that it is mutagenic to bacteria, rodent fibroblasts, and human lymphoblasts. It has cytogenetic effects on various mammalian cells and can induce transformation of hamster pancreatic duct cells. In vivo, NNK induces micronucleus formation in mouse bone marrow and causes DNA strand breaks in rat and hamster hepatocytes. A study reported that NNAL is mutagenic to Salmonella. In addition to the classic carcinogenic mechanism through DNA adduct formation, NNK can also bind to nicotine receptors and other receptors, leading to downstream effects that ultimately promote cancer development. These effects have been observed in experimental systems including pancreatic and lung cells in humans and laboratory animals. 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone and N'-nitrosonicotinamide are the most abundant and potent carcinogens in smokeless tobacco; absorption and metabolic activation of these substances have been clearly observed in smokeless tobacco users. In rats, the combined use of 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone and N'-nitrosonicotinamide induced oral tumors, consistent with the tumor-inducing effects of smokeless tobacco. One carcinogenic mechanism is cytochrome P450-mediated α-hydroxylation, a process that leads to the formation of DNA and hemoglobin adducts commonly found in tobacco users. Structure-activity studies revealed that both DNA methylation and pyridoxybutylation play crucial roles in NNK-induced lung tumorigenesis in rats. Persistent O6-MeGua is a key determinant of lung tumorigenesis in A/J mice, but it does not explain the difference in sensitivity to NNK-induced lung tumorigenesis between A/J and C57BL/6 mice. O6-methylguanine (O6-MedGuo) levels, measured 96 hours after treatment in A/J mice, were strongly correlated with tumor number and independent of the source of the methylating agent (e.g., NNK). Furthermore, in A/J mice, a high proportion of GC-to-AT conversions occurred at codon 12 of the K-ras gene in NNK-induced lung tumors, consistent with the importance of O6-MeGuo. The pyridoxybutyl adduct inhibits O6-alkylguanine-DNA alkyltransferase (AGT), an enzyme responsible for repairing O6-MedGuo. Since O6-MedGuo is also a product of NNK metabolic activation, this phenomenon may be significant for the persistent presence of O6-MedGuo in NNK-exposed tissues. For more complete data on the mechanisms of action of 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanones (out of 13), please visit the HSDB record page.

C10H13N3O2

207.23

207.1

64091-91-4

NNK-d4;764661-24-7;NNK-d3;86270-92-0

47289

White to light yellow solid powder

1.2±0.1 g/cm3

423.9±25.0 °C at 760 mmHg

63-65ºC

210.2±23.2 °C

0.0±1.0 mmHg at 25°C

1.557

0.09

0

5

5

15

221

0

FLAQQSHRLBFIEZ-UHFFFAOYSA-N

InChI=1S/C10H13N3O2/c1-13(12-15)7-3-5-10(14)9-4-2-6-11-8-9/h2,4,6,8H,3,5,7H2,1H3

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone

Nicotine-derived nitrosamine ketoneNNK

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)

DMSO : ≥ 62.5 mg/mL (~301.60 mM)

Solubility in Formulation 1: 3.33 mg/mL (16.07 mM) in 1% CMC-Na/saline water (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication (<50°C).
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

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