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.

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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
Extensive studies have examined the metabolism of NNK and the formation of DNA adducts by NNK and its metabolite 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) in humans and laboratory animals; the metabolic pathways and structures of DNA adducts have been characterized comprehensively. NNK and NNAL have been detected in the saliva of smokeless tobacco users, and NNAL and another metabolite of NNK, NNAL-glucuronide, have been quantified in human urine. The presence of these metabolites, which are specific to exposure to tobacco products (e.g. in smokers, users of smokeless tobacco and nonsmokers exposed to secondhand tobacco smoke), signals human uptake and metabolism of NNK, and their quantification allows an estimation of the dose of NNK absorbed. Dose calculations show that the total amounts of NNK taken up by people who used tobacco products for a period of 30 years or more approximate the total amounts that induce tumors in rats.
Absorption of NNK in smokeless tobacco users was demonstrated by the detection of its metabolites, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and NNAL-glucuronide (Gluc), in plasma and urine and of NNAL-N-oxide in urine. Similar results have been obtained in smokers, although only NNAL has been reported in plasma. NNAL-N-Gluc comprised 50 +/- 25% of total NNAL-Gluc in the urine of smokers and 24 +/- 12% in snuff-dippers. Toombak users excreted exceptionally high levels of NNAL and NNAL-Gluc (0.12 to 0.14 mg per day), which demonstrated a higher uptake of NNK by humans than of any other non-occupational carcinogen.
4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), but not NNAL-Gluc, was detected in the amniotic fluid of mothers who smoked. Both NNAL and NNAL-Gluc were detected in the urine of neonates born to mothers who smoked, but not in the urine of newborns of nonsmoking mothers. These results indicate that NNK is converted to NNAL in the mother, and that NNAL crosses the placental barrier and is absorbed and metabolized to NNAL-Gluc in the late stages of fetal development. /NNAL/
4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and NNAL-Gluc are excreted in the urine more slowly than would be expected, based on their structures, after cessation of smoking or smokeless tobacco use. One week after smoking cessation, 34.5% of baseline NNAL plus NNALGluc was detected in urine, whereas the corresponding values for the structurally related compounds cotinine and nicotine were 1.1 and 0.5%, respectively. Even 6 weeks after cessation, 7.6% of the original levels of NNAL plus NNAL-Gluc remained. The distribution half-life of NNAL and NNAL-Gluc was 3 to 4 days, while the elimination half-life was 40 to 45 days. Total body clearance of NNAL was estimated to be 61.4 +/- 35.4 mL/min, and the volume of distribution in the beta-phase was estimated to be 3800 +/- 2100 L, which indicates substantial distribution into tissues. After cessation of smokeless tobacco use, the distribution half-lives of NNAL (1.32 +/- 0.85 versus 3.35 +/- 1.86 days) and NNAL-Gluc (1.53 +/- 1.22 versus 3.89 +/- 2.43 days) were significantly shorter than those in smokers. There were no significant differences in the terminal half-lives. /NNAL/
For more Absorption, Distribution and Excretion (Complete) data for 4-(N-Nitrosomethylamino)-1-(3-pyridyl)-1-butanone (10 total), please visit the HSDB record page.

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Metabolism / Metabolites
NNK uptake by measurement of the urinary metabolites 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol and its glucuronides (total NNAL) has been reported in many studies, but there are no data in the literature on the percentage of the NNK dose that is converted to NNAL in smokeless tobacco users. ... 15 male subjects abstained from tobacco use for 3 weeks before placing 2 g smokeless tobacco between their cheeks and gums for 30 min. They then continued abstinence and collected three consecutive 24-hr urine samples. The amount of NNK in the tobacco before and after use was determined along with the amount in expectorated saliva. The NNK dose thus calculated was compared with the amount of total NNAL excreted in the next 72 hr. These data, taken together with previous pharmacokinetic data, show that the percent conversion of NNK to total NNAL in smokeless tobacco users is approximately 14% to 17%. This figure can be used to calculate daily exposure to NNK in smokeless tobacco users ( approximately 6 ug). The results of this study also indicate that metabolic activation of NNK to intermediates that can react with DNA is its major pathway of metabolism in smokeless tobacco users.
NNK can be converted to the pyridine oxidation products 4-(methylnitrosamino)-1-[3- (6-hydroxypyridyl)]-1-butanone (6-HONNK) and NNK-N-oxide. Denitrosation of NNK followed by oxidation produces myosmine. NNK can replace nicotinamide in NADP+ or NADPH, to yield NNK adenosine dinucleotide phosphate ((NNK)ADP+) and (NNK)ADPH (reduced form). Carbonyl reduction of NNK produces NNAL which can be conjugated by glucuronidation giving four diastereomers of NNAL-Gluc: two isomers of 4- (methylnitrosamino)-1-(3-pyridyl)-1-(O-beta-D-glucopyranuronosyl)butane (NNAL-O-Gluc) and two isomers of 4-(methylnitrosamino)-1-(3-pyridyl-N-beta-D-glucopyranuronosyl)-1- butanolonium inner salt (NNAL-N-Gluc). NNAL is also converted to NNAL-N-oxide and NNAL(ADP+).
Alpha-Hydroxylation of NNK and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) leads to DNA and hemoglobin adducts. Hydroxylation of the NNK methyl group gives 4-(hydroxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (alpha-HOMeNNK) which can be conjugated as a glucuronide, alpha-HOMeNNK-Gluc. -HOMeNNK spontaneously decomposes to 4-oxo-4-(3-pyridyl)-1-butanediazohydroxide (POB-DZH) and formaldehyde. POB-DZH reacts with water to give 4-hydroxy-1-(3-pyridyl)- 1-butanone (HPB) which can be conjugated as its glucuronide, HPB-Gluc. POB-DZH also reacts with DNA and haemoglobin to produce a series of adducts. Alpha-Hydroxylation of the NNK methylene group produces 4-(methylnitrosamino)-1-(3-pyridyl)- 1-(4-hydroxy)butanone (alpha-HOMethyleneNNK). This metabolite spontaneously decomposes to 4-(3-pyridyl)-4-oxobutanal (keto aldehyde) and methanediazohydroxide (Me- DZH). Keto aldehyde is further metabolized to 4-(3-pyridyl)-4-oxobutanoic acid (keto acid) which in turn can be converted to 4-hydroxy-4-(3-pyridyl)butanoic acid (hydroxy acid). Me-DZH reacts with water to yield methanol and with DNA to produce methyl adducts as shown in Figures 2 and 5. NNAL similarly undergoes alpha-hydroxylation at its methylene group to yield 4-(methylnitrosamino)-1-(3-pyridyl)-1-(4-hydroxy)butanol (alpha-HOMethyleneNNAL) and at its methyl group to yield 4-(hydroxymethylnitrosamino)-1-(3-pyridyl)- 1-butanol (alpha-HOMeNNAL). Alpha-HOMethyleneNNAL spontaneously decomposes to Me- DZH and 5-(3-pyridyl)-2-hydroxytetrahydrofuran (lactol), which can be converted to hydroxy acid. Alpha-HOMeNNAL spontaneously decomposes to 4-hydroxy-4-(3-pyridyl)-1- butanediazohydroxide (PHB-DZH) and formaldehyde. PHB-DZH reacts with water to yield 4-(3-pyridyl)butane-1,4-diol (diol), cyclizes to 2-(3-pyridyl)tetrahydrofuran (pyridylTHF) and reacts with DNA and hemoglobin to produce adducts.
CYPs are the major catalysts of NNK alpha-hydroxylation in humans and rodents. ... The relative efficiencies in NNK metabolism by human CYP are (from greatest catalyst to least): 2A13 > 2B6 > 2A6 > 1A2 ~ 1A1 > 2D6 ~ 2E1 ~ 3A4. ... The actual involvement of these enzymes in NNK metabolism in vivo depends on many factors that include relative expression levels, the amount of CYP oxido-reductase expressed in a given tissue, tissue localization and inducibility of individual CYPs, and the concentration of NNK in human tissues. Among hepatic CYPs, 2B6 has the highest affinity for NNK. However, low levels of this enzyme are present in most liver samples. CYP2A6 is also present at relatively low levels, and accounts for < 1% to 4% of the total CYP content. Levels of CYP1A2 are four- to 20-fold higher than those of CYP2A6. Therefore, despite its somewhat higher Km and lower Vmax/Km, CYP1A2 is most probably as important a catalyst of NNK alpha-hydroxylation in human liver as CYP2A6. CYP3A4 may also play a role in hepatic NNK alpha-hydroxylation, since it is often present at concentrations that are 10 to 50 times greater than those of CYP2A6. It is not possible to rule out completely the presence of CYP2A13, the best known human catalyst of NNK metabolism, in the liver. However, the very low hepatic mRNA levels of CYP2A13 relative to CYP2A6 suggest that, if this enzyme is present, it is so at very low levels. Results to date do not identify that any single CYP in the liver is a key player in NNK activation. Several enzymes, including CYP1A2, CYP2A6, CYP2B6 and CYP3A4, clearly play a role. The relative contribution of any one of these CYPs varies among individuals, and their relative abundance and catalytic efficiencies suggest that rarely, if ever, is one of them the dominant catalyst.
For more Metabolism/Metabolites (Complete) data for 4-(N-Nitrosomethylamino)-1-(3-pyridyl)-1-butanone (19 total), please visit the HSDB record page.
4-(Methylnitrosoamino)-1-(3-pyridinyl)-1-butanone (NNK) has known thirdhand smoke metabolites that include 4-Hydroxy-4-pyridin-3-ylbutanal, 4-Oxo-4-pyridin-3-ylbutane-1-diazonium, 5-(3-Pyridyl)-tetrahydro-furan-2-one(lactone), 5-(3-Pyridyl))-2-hydroxytetrahydrofuran (lactol), NNAL-O-glucoronide, NNAL-N-glucoronide, 4-[(Hydroxymethyl)nitrosoamino]-1-(3-pyridinyl)-1-butanone, 1-(Methylnitrosoamino)-4-(3-pyridinyl)-1,4-butanediol, NNAL-N-Oxide, 1-(3-Pyridinyl)-1,4-butanediol (1,4-Diol), 3-(Oxolan-2-yl)pyridine, 4-Hydroxy-1-(3-pyridyl)-1-butanone (HPB), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), 4-hydroxy-4-(3-pyridyl)-butanoic acid (HPBA), 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), iso-NNAL, alpha-[3-[(Hydroxymethyl)nitrosoamino]propyl]-3-pyridinemethanol, 4-Oxo-4-(pyridin-3-yl)butanal, 2-Hydroxy-1-pyridin-3-ylpropan-1-one, NNK-N-Oxide, 4-Oxo-4-(3-pyridyl)-butanoic acid (OPBA), and 4-Hydroxy-4-(methylnitrosoamino)-1-(3-pyridinyl)-1-butanone.
4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a known thirdhand smoke metabolite of 4-(Methylnitrosoamino)-1-(3-pyridinyl)-1-butanone (NNK).
NNK has known human metabolites that include 4-[(Hydroxymethyl)nitrosoamino]-1-(3-pyridinyl)-1-butanone.

Toxicity Summary
Exposure: Tobacco-specific N-nitrosamines, including 4-(methylnitrosamino)-1-(3-pyridyl)-1- butanone (NNK), N'-nitrosonornicotine (NNN), N'-nitrosoanabasine (NAB) and N'-nitrosoanatabine (NAT), occur widely in tobacco and tobacco smoke. They are formed by the nitrosation of nicotine and other tobacco alkaloids and have been detected in green tobacco leaves from Nicotiana tabacum and N. rustica species; however, the largest quantities of tobacco-specific N-nitrosamines are formed during tobacco curing and processing and additional amounts are formed during smoking. Tobacco-specific N-nitrosamines occur in all commercially and non-commercially prepared tobacco products including cigarettes, cigars, bidis, pipe tobacco and smokeless tobacco products. N-Nitrosamines occur in a wide variety of both food and non-food products, but the amounts of tobacco-specific N-nitrosamines in all tobacco products exceed the levels of other N-nitrosamines in other commercial products by several orders of magnitude. The highest levels of tobacco-specific N-nitrosamines are measured in smokeless tobacco products. ... The degree of exposure to tobacco-specific N-nitrosamines depends not only on the levels of these compounds in tobacco products or smoke, but also on the manner in which the products are used. Effects in Humans: No data were available. Effects in Animals: In numerous studies in mice, NNK induced lung adenomas independent of the route of administration. In studies by subcutaneous injection, benign and malignant tumors of the lung, nasal cavity and liver were induced in rats. In two of four experiments in hamsters, lung adenomas and adenocarcinomas or adenosquamous carcinomas were induced in males and females. In the two other experiments, adenomas were observed. Nasal cavity tumours involving the forebrain were observed in a limited study in mink. In a study by administration in the drinking-water and another by oral swabbing, combined benign and malignant lung tumours (adenoma, adenosquamous carcinoma and carcinoma) were induced in male rats. In the drinking-water study, NNK produced benign and malignant pancreatic tumours. In the oral swabbing study, combined benign and malignant tumours of the liver and nasal cavity were observed. Asignificant increase in the incidence of liver and lung tumours was reported in female rats when NNK was instilled into the urinary bladder. In two studies, the offspring of mice were exposed transplacentally by intraperitoneal injection of the dams. Liver tumors were observed in male offspring in both studies and in female offspring in one study. In one of these studies, lung tumors were also observed in male offspring. In studies of the offspring of hamsters given NNK during pregnancy, intratracheal instillation of the dams resulted in adenocarcinomas of the nasal cavity in male offspring and adrenal pheochromocytomas in male and female offspring in one study. In a second study, subcutaneous injection of NNK into dams induced respiratory tract (nasal cavity, larynx and trachea) tumors in male and female offspring. When dams were injected subcutanously or treated by intratracheal instillation, nasal cavity and adrenal gland tumors developed in male and female offspring in a third study. Intraperitoneal administration of NNK-N-oxide induced lung adenomas in female mice. In an oral swabbing study, NNK in combination with NNN increased the incidence of oral tumors in rats.

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Interactions
... Male ICR mice were exposed to NNK (0.5 mg/mouse) and sodium arsenite (0, 10, or 20 mg/kg) daily via gavaging for 10 days and their urine was collected at day 10 for NNK metabolite analysis. Liver samples were also obtained for CYP2A enzyme and DNA adducts evaluations. Both the cyp2a4/5 mRNA levels and the CYP2A enzyme activity were significantly elevated in arsenic-treated mice liver. Furthermore, urinary NNK metabolites in NNK/arsenic co-treated mice also increased compared to those treated with NNK alone. Concomitantly, DNA adducts (N(7)-methylguanine and O(6)-methylguanine) were significantly elevated in the livers of mice co-treated with NNK and arsenic. Our findings provide clear evidence that arsenic increased NNK metabolism by up-regulation of CYP2A expression and activity leading to an increased NNK metabolism and DNA adducts (N(7)-methylguanine and O(6)-methylguanine). These findings suggest that in the presence of arsenic, NNK could induce greater DNA adducts formation in hepatic tissues resulting in higher carcinogenic potential.
The NNK/mouse lung model has been used extensively by numerous investigators to determine factors, conditions, drugs or chemopreventive compounds that can modulate the formation of lung tumors in mice. One of these studies is summarized below. A study was conducted to determine the capacity of cigarette smoke to induce lung tumors and promote lung tumorigenesis induced by NNK. Groups of 20 female A/J mice, 7 weeks of age, were exposed for 6 hr per day on 5 days per week for 26 weeks to filtered air (FA), cigarette smoke (CS; diluted mainstream smoke (target concentration, 250 mg total particulate matter/cu m) from IR3 research cigarettes), NNK or NNK plus CS. Mice were exposed for 3 days to 50% of the target concentration of CS and for 4 days to 75% of the target concentration of CS before full exposure. Three days before CS exposure, mice received an intraperitoneal injection of 100 mg/kg bw NNK in 0.1 mL saline. Mice were killed 5 weeks after the exposures were terminated. Total tumors were enumerated macroscopically and characterized microscopically. Differences in survival were analysed by Breslow statistics in a Kaplan-Meier survival analysis. Student's t-test with a Bonferroni multiple comparisons correction was used to examine group differences in lung weight, tumor multiplicity for all animals and tumor multiplicity in tumor-bearing animals, with significance set at the p < 0.05 level. The lung tumor incidence among the four groups was: FA, 5/19 (26%); CS, 0/19 (0%); FA + NNK, 19/20 (95%); and CS + NNK, 13/16 (81%). The lung tumor multiplicities (total tumors/animal at risk) were: FA, 0.32 +/- 0.58 tumors per animal; FA+ NNK, 2.50 +/- 1.67 tumors per animal; and CS + NNK, 2.50 +/- 1.97. Those among tumour-bearing animals were: FA, 1.20 +/- 0.44 tumors per animal; FA+ NNK, 2.63 1.61 tumors per animal; and CS + NNK, 3.08 +/- 1.71. CS exposure decreased both body weights and lung weights, but treatment with NNK had no additional effect. Tumor multiplicity was greater in the FA + NNK- and the CS + NNK-treated groups compared with the FA- and CS-treated groups (p < 0.05) among all animals, but tumor multiplicity in the tumor-bearing animals did not differ between the FA-, FA + NNK- or CS + NNK-treated groups.

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Non-Human Toxicity Values
LD50 Mouse ip 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.

4-(N-Nitrosomethylamino)-1-(3-pyridyl)1-butanone can cause cancer according to The World Health Organization's International Agency for Research on Cancer (IARC).
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)butan-1-one is a nitrosamine and a member of pyridines.
4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone has been reported in Nicotiana tabacum with data available.
4-Methylnitrosamino-1,3-pyridyl-1-butanone is a yellowish crystalline solid nitrosamine that naturally occurs in tobacco products by oxidation and nitrosation of nicotine during the making and smoking of tobacco. 4-Methylnitrosamino-1,3-pyridyl-1-butanone is only used as a research chemical to induce tumorigenesis. This substance is reasonably anticipated to be a human carcinogen. (NCI05)
See also: Tobacco Leaf (part of).

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Mechanism of Action
The metabolic activation of NNK and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) to DNA adducts is critical for the expression of their carcinogenic activities. The metabolic activation process has been documented extensively in laboratory animals. Cytochrome P450 enzymes are the principal catalysts of this process, and those in the 2A family appear to be the most efficient in both humans and laboratory animals. NNK is a genotoxic compound. It was shown to be mutagenic in bacteria, in rodent fibroblasts and in human lymphoblastoid cells in vitro. It caused cytogenic effects in a variety of mammalian cells in vitro and induced transformation of the pancreatic duct cells of hamsters. In vivo, NNK induced micronucleus formation in the bone marrow of mice and DNA strand breaks in the hepatocytes of rats and hamsters. NNAL was reported to be mutagenic in Salmonella in a single study. In addition to the classical mechanisms of carcinogenesis that proceed through the formation of DNA adducts, NNK also binds to nicotinic and other receptors, which leads to downstream effects that contribute to the development of cancer. These effects have been observed in experimental systems including pancreatic and lung cells from humans and laboratory animals.
4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone and N'-nitrosonornicotine are the most abundant strong carcinogens in smokeless tobacco; uptake and metabolic activation in smokeless tobacco users have been clearly observed. In rats, combined application of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and N'-nitrosonornicotine induced oral tumors consistent with their induction by smokeless tobacco. One of the mechanisms of carcinogenicity is cytochrome P450-mediated alpha-hydroxylation, which leads to the formation of DNA and hemoglobin adducts that are commonly detected in users of tobacco.
Structure-activity studies suggest that both DNA methylation and pyridyloxobutylation are important in NNK-induced lung tumorigenesis in rats.
Persistent O6-MeGua is the critical determinant of lung tumor induction in A/J mice, but does not account for differences in sensitivity to NNK induced lung tumorigenesis between A/J and C57BL/6 mice. ... Levels of O6-methylguanine (O6-MedGuo) measured 96 hr after treatment of A/J mice correlate strongly with tumor multiplicity, independent of the source of the methylating agent, e.g. NNK ... . In addition, GC to AT transitions in codon 12 of the K-ras gene are observed in a high percentage of lung tumors induced by NNK in A/J mice, consistent with the importance of O6-MeGua. ... Pyridyloxobutyl adducts inhibit O6-alkylguanine-DNA alkyltransferase (AGT), the enzyme responsible for the repair of O6-MedGuo. Since O6-MedGuo is also formed by metabolic activation of NNK, this phenomenon is probably important in the persistence of O6-MedGuo in NNK-exposed tissues.
For more Mechanism of Action (Complete) data for 4-(N-Nitrosomethylamino)-1-(3-pyridyl)-1-butanone (13 total), 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.

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