Experimental and Theoretical Investigation of Effects of Ethanol and

Article pubs.acs.org/JPCA

Experimental and Theoretical Investigation of Effects of Ethanol and Acetic Acid on Carcinogenic NDMA Formation in Simulated Gastric Fluid Ou Zhang,† Xuan Zou,‡ Qi-Hong Li,‡ Zhi Sun,† Yong Dong Liu,*,† and Ru Gang Zhong† †

College of Life Science & Bioengineering, Beijing University of Technology, Beijing 100124, P. R. China Department of Stomatology, Chinese PLA 307 Hospital, Academy of Military Medical Sciences, Beijing, 100071, P. R. China

‡

S Supporting Information *

ABSTRACT: N-nitrosodimethylamine (NDMA), as a representative of endogenously formed N-nitroso compounds (NOCs), has become the focus of considerable research interest due to its unusually high carcinogenicity. In this study, effects of ethanol and acetic acid on the formation of NDMA from dimethylamine (DMA) and nitrite in simulated gastric fluid (SGF) were investigated. Experimental results showed that ethanol in the concentrations of 1−8% (v/v) and acetic acid in the concentrations of 0.01−8% (v/v) exhibit inhibitory and promotion effects on the formation of NDMA, respectively. Moreover, they are both in a dose-dependent manner with the largest inhibition/promotion rate reaching ∼70%. Further experimental investigations indicate that ethanol and acetic acid are both able to scavenge nitrite in SGF. It implies that there are interactions of ethanol and acetic acid with nitrite or nitrite-related nitrosating agents rather than DMA. Theoretical calculations confirm the above experimental results and demonstrate that ethanol and acetic acid can both react with nitrite-related nitrosating agents to produce ethyl nitrite (EtONO) and acetyl nitrite (AcONO), respectively. Furthermore, the reactivities of ethyl nitrite, acetyl nitrite, and dinitrogen trioxide reacting with DMA were found in the order of AcONO > N2O3 ≫ EtONO. This is probably the main reason why there are completely different effects of ethanol and acetic acid on NDMA formation. On the basis of the above results, two requirements for a potential inhibitor of NOCs formation in SGF were provided. The results obtained in this study will be helpful in better understanding the inhibition/promotion mechanisms of compounds on NDMA formation in SGF and searching for protective substances to prevent carcinogenic NOCs formation. is known that gastric fluid as a low pH environment is favorable for NOCs formation. Therefore, numerous researches about NOCs formation in the simulated gastric fluid (SGF) have been performed.12−22 Meanwhile, searching for protective substances to inhibit NOCs formation is a subject of intense research. Consumption of whole strawberries, kale juice, garlic juice, black chokeberry (Aronia melanocarpa), tomatoes, tomatobased products, black tea, and orange juice has been reported to be effective in inhibiting the formation of NDMA.12−18 Further research indicated that most of the above substances contain antioxidants such as ascorbic acid, polyphenols, lycopene, flavonoids, and allyl sulfur compounds. Thus, it can be concluded that antioxidants are effective inhibitors of NDMA formation. On the contrary, chemicals such as halide, 1,2benzendicarboxy acid, and thiocyanate have been reported to catalyze gastric NDMA formation.12,13 Why do different

1. INTRODUCTION N-Nitroso compounds (NOCs), composed of N-nitrosamines (NAs) and N-nitrosamides, have arisen widespread concern mainly because most of them are probable human carcinogens.1,2 NAs have been associated with an increased risk of gastric, esophageal, nasopharyngeal, and bladder cancers.3,4 Nnitrosodimethylamine (NDMA), as a representative of NOCs, has been demonstrated to be highly carcinogenic, mutagenic, and teratogenic.5 NDMA and three other NOCs, that is, Nnitrosodiethylamine, N-nitrosomethylurea, and N-nitrosoethylurea are classified by International Agency for Research on Cancer as carcinogens of the category 2A.6 In the U.S. EPA Integrated Risk Information Service database, NDMA has been identified to have an estimated 1 × 10−6 lifetime cancer risk level at a concentration in drinking water as low as 0.7 ng/L.7 Apart from exposure to exogenous NOCs through air, drinking water, food, and cosmetics,8,9 humans can also form NOCs endogenously in the stomach, intestine, and bladder.2,3,10,11 Moreover, endogenously formed NOCs have been estimated to account for 45−75% of total NOCs exposure.10 It © 2016 American Chemical Society

Received: March 11, 2016 Revised: May 24, 2016 Published: June 14, 2016 4505

DOI: 10.1021/acs.jpca.6b02582 J. Phys. Chem. A 2016, 120, 4505−4513

Article

The Journal of Physical Chemistry A

2.1.4. Mechanisms Studies. Aqueous stock solution of ethanol (50% and 100% v/v) or equal concentrations of acetic acid and NDMA (800 μg/L), nitrite (50 mM), or DMA (50 mM) were mixed and then diluted 1:10 (v/v) with SGF without pepsin. Reaction mixtures were incubated for 2 h at 37 °C in a dark cabinet and then prepared for the analysis. 2.1.5. N-Nitrosodimethylamine Analysis. The samples were prepared directly after incubation with an addition of 200 μL of NDMA-d6 (1.0 mg/L) as internal standard. Then, samples were transferred to solid-phase extraction (SPE) columns filled with coconut charcoal. Prior to use, the SPE columns were activated by methanol. After hexane leaching, NDMA was eluted with dichloromethane. After the withdrawal of water, the concentrated extract was obtained. In each step, the SPE cartridge was drained to dry thoroughly, and the flow rate was maintained at ∼2−3 mL/min. After eluting, the extract was concentrated to 1 mL with dichloromethane. NDMA from the concentrated extract was measured using a Thermo Fisher Scientific gas chromatography mass (GC-MS, Trace DSQ) equipped with electron ionization (EI) source. The determination of NDMA was achieved by using an isotopic dilution method based on the mass detection of molecular ion (m/z = 74) and the characteristic molecular ion of NDMA-d6 (m/z = 80). NDMA was analyzed by both full scan (SM) and selected ion monitoring (SIM). The GC/MS operating conditions were set as follows: splitless injections of 1 μL at 200 °C for inlet, 230 °C for transfer line, and 250 °C for ion source, respectively. The chromatographic separation was performed on a DB-1701 column (30 m × 0.25 mm × 0.25 μm, Agilent Technology, USA) with the following temperature program: 45 °C was held for 5 min, and then the temperature was increased by 10 °C/ min until 200 °C, which was held for 5 min. One microliter of extract was injected into GC-MS. Helium carrier gas was maintained at 1.0 mL/min, and the retention time of NDMA was 6.85 min. The instrument and method detection limits for NDMA are 1 and 5 ng/L, respectively. 2.1.6. Nitrite and Dimethylamine Analysis. In the analysis of nitrite, 1 mL aliquots of the samples were withdrawn and diluted with deionized water to make a final volume of 10 mL after nitrite and ethanol or acetic acid incubated. Prior to determination, the samples were filtrated through 0.22 μm nylon films. Finally, the samples were subjected to ion chromatography (IC) analysis. The determination of nitrite was performed on a Dionex ICS-2100 IC with a suppressed conductivity detector equipped with a Dionex ICS series ASDV autosampling system. An IonPac AS11-HC Analytical column (4 × 250 mm) and an ASRS-4 mm suppressor with 75 mA current were applied to the IC in the determination of nitrite. The eluent was 30 mM NaOH with a flow rate of 1 mL/ min. In the determination of DMA, samples were also diluted 10:1 and filtrated through 0.22 μm nylon films. The determination of DMA was also performed on a Dionex ICS-2100 IC with a suppressed conductivity detector equipped with a Dionex ICS SERIES AS-DV autosampling system. An IonPac CS12A Analytical column (4 × 250 mm) and a CSRS-4 mm suppressor with 59 mA current were chosen. The eluent was 20 mM methanesulfonic acid with the flow rate of 1 mL/min. For both nitrite and DMA analysis, the column temperature was 30 °C, and the injection volume was 25 μL. The standard curves of nitrite and DMA were shown in Figures S1 and S2. The concentrations of nitrite and DMA were calculated using the standard curves. The instrument and method detection

compounds play completely different roles in NDMA formation? What kinds of underlying mechanisms lie in these reactions? To date, little knowledge of the above questions has been known. In our daily diet, it is easy to intake ethanol- and acetic acidrich foods, such as traditional typical ethanol drinks and vinegar, fermentation condiment, storage of fruit, and so on. So far ethanol has been found to have inhibitory effect on NDMA formation under acidic condition;23−25 however, there is little information about the effect of acetic acid. Although alkyl nitrite was proposed to be generated as an inactive nitrosating agent,23,24 the inhibition mechanisms for the ethanol are still not very clear. Thus, it is necessary to figure out how ethanol interacts with nitrite and why alkyl nitrite lacks nitrosating ability. In addition, in view of previous results, it seems that the substances that have nitrite scavenging abilities can generally inhibit the formation of N-nitrosamines. Is this assumption correct? Can an inhibitor of NOCs formation be identified by its ability to scavenge nitrite? To address the above questions, herein, we investigated the effects of ethanol and acetic acid on the formation of NDMA in SGF and further studied the reaction mechanisms from experimental and theoretical aspects. This research sought to fill the knowledge gaps of inhibitory/promotion effects and mechanisms of compounds on NDMA formation in SGF, provide requirements for potential inhibitors of NDMA formation, and help to search for more protective substances to prevent NOCs formation in the human body.

2. EXPERIMENTAL AND THEORETICAL METHODS 2.1. Laboratory Study. 2.1.1. Chemicals and Reagents. Dimethylamine hydrochloride, sodium nitrite, sodium chloride, Pepsin P7000, methanesulfonic acid, methanol, ethanol, hexane, and dichloromethane were provided by Sigma-Aldrich (Steinheim, Germany). Hydrochloric acid and acetic acid were purchased from Beijing Chemical Works (Beijing, China). NDMA and deuterated N-nitrosodimethylamine (NDMA-d6) were obtained from Dr. Ehrenstorfer GmbH (Augsburg, Germany). Standard solutions of nitrite and DMA were purchased from National Institute of Metrology (Beijing, China). Coconut charcoal was provided by Agela Technologies (Tianjing, China). All reagents used without further purification were of high-purity analytical grade or high-performance liquid chromatography grade. Deionized water was purified by a Millipore Milli-Q water system (Schwalbach, Germany) to a specific resistance of 18.2 MΩ·cm. 2.1.2. Preparation of Simulated Gastric Fluid. SGF was prepared based on the instructions of the United States Pharmacopoeia26 and contained 3.2 g of pepsin, 2.0 g of sodium chloride, and 7.0 mL of hydrochloric acid dissolved in sufficient water to make 1000 mL, adjusted to pH 2.0 using 4 M HCl. 2.1.3. N-Nitrosodimethylamine Formation. Aqueous stock solutions of nitrite (50 mM), DMA (50 mM), and ethanol (10%, 20%, 50%, 80% v/v) or acetic acid (0.1%, 0.5%, 1%, 3%, 10%, 20%, 50%, 80% v/v) were 1:10 (v/v) with SGF. Blank samples including aqueous stock solutions of nitrite (50 mM) and DMA (50 mM) were diluted 1:10 (v/v) with SGF in the absence of ethanol and acetic acid. Reaction mixtures were incubated for 2 h at 37 °C in a dark cabinet and then prepared for the analysis. Each incubation experiment was performed in triplicate. 4506


Article


Figure 1. Concentration of NDMA after reaction mixture (5 mM DMA, 5 mM NaNO2 and varied concentrations of ethanol or acetic acid) incubated for 2 h at 37 °C in SGF.

3. RESULTS AND DISCUSSION 3.1. Experimental Investigation of Effects of Ethanol and Acetic Acid on N-Nitrosodimethylamine Formation in Simulated Gastric Fluid. Taking the real situations of ethanol and acetic acid in the stomach into account and referring to some studies,23,25 ethanol concentrations of 1%, 2%, 5%, and 8% and acetic acid concentrations of 0.01%, 0.05%, 0.1%, 0.3%, and 1% were chosen to evaluate their effects on NDMA formation in SGF. Moreover, acetic acid concentrations of 2%, 5%, and 8% were also investigated for comparison with ethanol. Figure 1 illustrated the observed NDMA formation yields at different concentrations of ethanol and acetic acid, and the corresponding values were listed in Table S1. 3.1.1. Effects of Ethanol and Acetic Acid on N-Nitrosodimethylamine Formation in Simulated Gastric Fluid. As shown in Figure 1, NDMA yield obviously decreases with the increasing ethanol concentrations. Table S1 showed that the yields of NDMA were 78.7, 48.2, 36.6, 29.0, and 18.7 μg/L at ethanol concentrations of 0, 1%, 2%, 5%, and 8%, respectively. It indicates that ethanol plays an inhibitory effect on NDMA formation from DMA and nitrite in SGF, which is consistent with previous studies.24,25 Moreover, it can be found that NDMA formation yield initially decreases rapidly in the ethanol concentrations from 0 to 2%, and it decreases slowly when concentrations are in the range from 2% to 8%; however, the inhibition rate during these concentrations increases largely from ∼53% to 76%. Thus, it can be concluded that ethanol significantly inhibits the formation of NDMA in a dosedependent manner from DMA and nitrite in SGF with the largest inhibition rate reaching ∼80%. Figure 1 shows that the formation of NDMA obviously increases with the increasing acetic acid concentrations, indicating that acetic acid plays a promotion effect on NDMA formation from DMA and nitrite in SGF. Table S1 showed that the yields of NDMA were 78.7, 82.1, 91.0, 94.1, 96.6, 107.8, 114.5, 119.3, and 132.4 μg/L at acetic acid concentrations of 0, 0.01%, 0.05%, 0.1%, 0.3%, 1%, 2%, 5%, and 8%, respectively. Moreover, it can be found that NDMA formation initially increases slowly with the promotion rate under 30% in acetic acid concentration of 0 to 0.3%, whereas it increased rapidly when ethanol concentrations increase from 1% to 8% with the promotion rate from ∼37% to 68%. Thus, it can be concluded that contrary to ethanol, acetic acid can

limits for nitrite are 0.02 and 0.08 mg/L as well as DMA, respectively. 2.2. Theoretical Methods. All quantum chemical computations were performed with the Gaussian-09 program package.27 The geometry optimizations and vibrational frequencies of all complexes involved in this study were first calculated using the B3LYP28,29 with the 6-311G(d)30 basis set. To obtain more accurate energies, the composite Gaussian-4 (G4) method was used.31 All structures of the reactants, transition states, and products were fully optimized. Vibrational frequencies were calculated at the same level of theory to characterize the nature of the stationary points. The minimumenergy path (MEP) was obtained using intrinsic reaction coordinate (IRC) calculations32 to confirm the connection of each transition state with the designated isomers. The activation free energies (ΔG‡) and energy barriers are calculated relative to the isolated species.33−35 The solvation free energy corrections were performed following Trogolo’s very recent work33 with the solute electron density (SMD) implicit solvation model36 at the M05/aug-cc-pvtz levels.37−41 Because of the importance of solvent effect, the treatment including explicit water molecules was also investigated for all the reactions involved in ethanol and acetic acid. However, the results indicate that the explicit water does not make the reactions more favorable than those without explicit water. Therefore, only data without explicit water were listed and discussed in the text, and the results including one explicit water molecule were listed in Supporting Information. According to transition state theory (TST)42,43 approximate reaction rate constants for some elementary reactions in which the reactants directly generate products through a concerted pathway were estimated based on the Eyring−Polanyi equation: k TST = (kBT /h)exp(ΔG‡sol /RT )

where kB is the Boltzmann constant, T is temperature, h is Planck’s constant, R is the molar gas constant, and ΔG‡sol is the corrected solvation activation free energy. Since the uncertainty of ±3 kcal/mol in the ΔG‡sol, although the calculated rate constants k for the elementary reactions are generally comparable to the experimentally measured kapparent values, three is an uncertainty of 1 × 10±2.5 for the computed rate constant k.33 4507


Article


Figure 2. Concentrations of NDMA, DMA, and nitrite after reaction mixture (5 mM DMA or 5 mM NaNO2 with three varied ethanol or acetic acid concentrations of 0, 5%, and 10%) incubated for 2 h at 37 °C in SGF, respectively.

highly reactive dinitrogen trioxide (N2O3),44,45 which is the substantial nitrosating agent to produce nitrosamines by nitrosating of amines. Thus, it implies that ethanol and acetic acid are probable to react with HONO or N2O3, the mostly existing nitrite-related nitrosating agents in SGF, leading to the decrease of nitrite concentration. In summary, experimental results illustrated that ethanol and acetic acid both affect NDMA formation from DMA and nitrite in SGF through reacting with nitrite-related species HONO or N2O3, rather than DMA. However, it is interesting that ethanol and acetic acid have completely different influences on NDMA formation, in which the former plays an inhibitory effect, whereas the latter plays a promotion effect. Thus, we infer that the products from reactions of ethanol and acetic acid with nitrite-related species probably have completely different properties in the following nitrosating reactions. 3.2. Theoretical Investigation of Effects of Ethanol and Acetic Acid on N-Nitrosodimethylamine Formation in Simulated Gastric Fluid. To further understand the inhibitory and promotion effects of the respective ethanol and acetic acid on NDMA formation, the reactions of ethanol and acetic acid with nitrite-related species HONO and N2O3 in SGF, were investigated by using G4. Activation free energies, reaction energies, and reaction rate constants (kTST) of these reactions were listed in Table 1, and optimized structures and some important geometric parameters of reactants, transition states, and products involved in these reactions were shown in Figure 3. 3.2.1. Interactions of HONO with Ethanol and Acetic Acid. HONO has trans and cis configurations, and trans-HONO is energically favored over cis-HONO.46−48 Thus, interactions of ethanol and acetic acid with trans-HONO were investigated, and reaction pathways were depicted in Scheme 1. As shown in Scheme 1, the reactions of ethanol and acetic acid with HONO proceed through four- and six-membered ring transition states, respectively. Figure 3 demonstrated that the O1−H1 and N−O2 bond lengths are 1.19 and 1.99 Å in TS-EtHONO, respectively. With the O1 and H1 atoms of ethanol further shifting to respective N and O2 atoms of HONO, ethyl nitrite (EtONO) and water are finally formed. Similar to the reaction processes of ethanol with HONO, acetic acid reacts

substantially promote the generation of NDMA in a dosedependent manner from DMA and nitrite in SGF with the largest inhibition rate reaching ∼68%. 3.1.2. Mechanisms of Ethanol and Acetic Acid Affecting NNitrosodimethylamine Formation in Simulated Gastric Fluid. To elucidate the inhibition/promotion mechanisms of ethanol and acetic acid on the formation of NDMA from DMA and nitrite in SGF, interactions of ethanol and acetic acid with NDMA, DMA, and nitrite were investigated. The concentrations of NDMA, DMA, and nitrite after addition of three varied ethanol and acetic acid concentrations of 0, 5%, and 10% were measured; the results were demonstrated in Figure 2, and the corresponding values were listed in Table S2. Figure 2 (left) showed that the concentrations of NDMA after interaction with ethanol and acetic acid concentrations of 0, 5%, and 10% are all ∼78−79 μg/L. It indicates that the concentrations of NDMA are nearly the same in the absence and presence of ethanol and acetic acid. Thus, it infers that there are no interactions of NDMA with ethanol or acetic acid. Similar to the interactions of ethanol and acetic acid with NDMA, as shown in Figure 2 and Table S2, DMA concentrations after interacting with 5% and 10% ethanol and acetic acid are nearly the same, with the values of 4.8 and 4.9 mM for ethanol as well as 4.9 and 5.0 mM for acetic acid, which are almost the same as 4.8 and 4.9 mM for DMA concentrations in the absence of ethanol and acetic acid, respectively. Again, it can be concluded that there are no interactions between DMA with ethanol or acetic acid. Figure 2 clearly demonstrates that the concentrations of nitrite after addition of varied concentrations of ethanol and acetic acid obviously decrease. As listed in Table S2, the content of nitrite is ∼2.3 mM in the absence of ethanol, whereas that decreases to 1.3 and 1.0 mM in the presence of 5% and 10% ethanol, respectively. Similarly, the content of nitrite is ∼2.1 mM in the absence of acetic acid, whereas that decreases to 1.2 and 1.1 mM in the presence of 5% and 10% acetic acid, respectively. For both ethanol and acetic acid, the nitrite decreasing rate after addition of them reaches ∼45−60%. It is known that nitrous acid (HONO) form predominates over the form of nitrite ion under acid conditions like SGF, and two nitrous acid molecules can react with each other to form a 4508


Article


similar process, and finally acetyl nitrite (AcONO) and nitrous acid are formed through the transition state TS−Ac-N2O3. Table 1 shows that the enthalpy changes (ΔH) of ethanol and acetic acid reacting with N2O3 are ca. −6 and 5 kcal/mol, respectively, indicating that they are exothermic and endothermic processes, respectively. The activation free energies in the aqueous phase (ΔG‡sol) of ethanol and acetic acid reacting with N2O3 to produce EtONO and AcONO are 22.4 and 29.0 kcal/mol, respectively. Obviously the energy barrier of ethanol reacting with N2O3 is significantly lower than that of acetic acid reacting with N2O3 by ∼7 kcal/mol. It implies that both ethanol and acetic acid are kinetically feasible to react with N2O3 and that the reaction of ethanol with N2O3 is much easier than acetic acid. Moreover, these energy barriers are lower and higher than that of two HONO molecules reacting to produce N2O3 with the value of 27.2 kcal/mol by ∼5 and 2 kcal/mol, respectively. Compared to HONO, ethanol prefers reacting with N2O3 due to its ΔG‡sol being considerably lower relative to that of ethanol reacting with HONO. However, acetic acid is feasible to react with both N2O3 and HONO with relatively low energy barriers. These theoretical results confirm our proposed mechanisms that ethanol and acetic acid can react with the nitrite-related nitrosating agents in SGF and also explain the experimental observations that nitrite concentrations decreased in SGF when ethanol or acetic acid were added. 3.2.3. N-Nitrosodimethylamine Formation from Dimethylamine Reacting with EtONO and AcONO. EtONO and AcONO produced upon the reactions of ethanol and acetic acid with HONO/N2O3 are also nitrosating agents as ON+ carriers.52 Therefore, the formation of NDMA from DMA reacting with EtONO and AcONO were investigated, and reactions of DMA with N2O3 were also studied for comparison. The reaction pathways of DMA with EtONO and AcONO were shown in Scheme 3. Scheme 3 illustrated that EtONO and AcONO can react with DMA through a transition sate of four- and six-membered ring, respectively. As shown in Figure 3, with the shifting of atom H1 from atom N2 to atom O1 and atom N1 from atom O1 to atom N2, the bond distances of H1−N2, O1−N1, and N1−N2 in the transition state TS-EtONO−DMA became 1.19, 2.09, and 1.90 Å, respectively. With the further shifting of atom H1 to atom O1 and atom N1 to atom N2, the products NDMA and ethanol formed. Regarding reaction of AcONO and DMA, with the shifting of atom H1 from atom N2 to atom O1 and atom N1 from atom O2 to atom N2, the bond distances of H1−N2, O2−N1, and N1−N2 in the transition state TSAcONO−DMA became 1.20, 2.25, and 1.77 Å, respectively. Finally, with the further shifting of atom H1 to atom O1 and atom N1 to atom N2, the product NDMA and acetic acid are formed. As listed in Table 1, the ΔH of reactions of DMA with EtONO and AcONO are −14.7 and −25.8 kcal/mol, respectively, indicating that these reactions are exothermic processes. Thus, these reactions are thermodynamically favorable. The values of ΔG‡sol for EtONO and AcONO reacting with DMA were calculated to be 33.3 and 2.0 kcal/ mol, respectively, whereas that for N2O3 reacting with DMA is 7.4 kcal/mol. Such low energy barrier of AcONO reacting with DMA indicates that AcONO is definitely a reactive nitrosating agent to generate NDMA, and it is even more reactive than N2O3. This is probably the main reason why acetic acid promotes the formation of NDMA in SGF. Meanwhile,

Table 1. Activation Free Energies, Reaction Energies (in kcal/mol), and Reaction Rate Constants (kTST, in M−1 s−1) for all Reactions of Ethanol and Acetic Acid with HONO and N2O3 as well as Reactions of Products EtONO and AcONO with DMA Calculated at G4 Level reactions C2H5OH + HONO → C2H5ONO + H2O CH3COOH + HONO → CH3COONO + H2O C2H5OH + N2O3 → C2H5ONO + HNO2 CH3COOH + N2O3 → CH3COONO + HNO2 HONO + HONO → N2O3 + H2O DMA + C2H5ONO → NDMA + C2H5OH DMA + CH3COONO → NDMA + CH3COOH DMA + N2O3 → NDMA + HNO2

ΔG‡gas ΔG‡sol

ΔH

ΔGsol

kTST

34.5

35.9

−6.1

−5.8

23.2

25.6

5.0

4.5

3.3 × 10−14±2.5 1.2 × 10−6±2.5

20.6

22.4

−5.8

−4.0

30.3

29.0

5.3

6.2

27.1

27.2

−0.3

−1.7

31.8

33.3

−14.7

−20.5

2.8

2.0

−25.8

−30.8

10.1

7.4

−20.5

−24.5

2.5 × 10−4±2.5 3.7 × 10−9±2.5 7.8 × 10−8±2.5 2.6 × 10−12±2.5 2.3 × 1011±2.5 2.5 × 107±2.5

with HONO through the transition state TS-Ac-HONO and generates acetyl nitrite (AcONO) and water. Table 1 shows that the reaction enthalpies (ΔH) in the reactions of ethanol and acetic acid with HONO were calculated to be −6.1 and 5.0 kcal/mol, respectively. It indicates that the reaction of ethanol with HONO is a slightly exothermic process, whereas that of acetic acid with HONO is an endothermic reaction. The activation free energies in the aqueous phase (ΔG‡sol) of ethanol and acetic acid reacting with HONO to produce EtONO and AcONO are 35.9 and 25.6 kcal/mol, respectively. Obviously the energy barrier of acetic acid reacting with HONO is significantly lower than that of ethanol reacting with HONO by ∼10 kcal/mol, which is likely due to the relatively stable six-membered ring transition state of acetic acid with HONO. It implies that although acetic acid reacting with HONO to form AcONO is thermodynamically unfavorable, it is kinetically much more feasible than ethanol reacting with HONO to produce EtONO. It is notable that ΔG‡sol of acetic acid reacting with HONO to generate AcONO (25.6 kcal/mol) is even lower than that of two HONO molecules reacting to produce N2O3 (27.2 kcal/mol). 3.2.2. Interactions of N2O3 with Ethanol and Acetic Acid. It is known that asym-N2O3 is the most stable isomer of N2O3.49 Thus, interactions of ethanol and acetic acid with asymN2O3 were investigated, and the reaction pathways were shown in Scheme 2. Scheme 2 demonstrates that the reactions of ethanol and acetic acid with N2O3 both proceed through five-membered ring transition states. As shown in Figure 3, with the shifting of atom H1 to atom O2 and atom N1 to atom O1, the bond distances of O1−H1 and N1−N2 in the transition state TS-EtN2O3 became 1.21 and 2.33 Å, respectively, and the bond length of N1−O1 shortened to 1.86 Å. Finally with O1−N1 and H1−O2 bonds forming and O1−H1 and N1−N2 bonds breaking, ethyl nitrite (EtONO) and nitrous acid are generated (see Scheme 2), which agrees well with previous results that ethyl nitrite is observed in vivo upon the consumption of nitrite- and ethanol-rich food.50,51 Acetic acid goes through a 4509


Article


Figure 3. Fully optimized structures and some important geometric parameters of complexes involved in all reactions of ethanol and acetic acid with HONO and N2O3 as well as reactions of EtONO and AcONO with DMA (distances in angstroms, atom in red color represents oxygen atom; atom in blue color represents nitrogen atom; atom in white represents hydrogen atom; atom in gray color represents carbon atom).

explain well the experimental results about the inhibitory effect of ethanol and promotion effect of acetic acid on NDMA formation in SGF. 3.3. Proposed Other Inhibitors of N-Nitrosodimethylamine in Simulated Gastric Fluid. On the basis of the above investigations, it can be found that ethanol and acetic acid can both scavenge nitrite; however, only ethanol has inhibitory

EtONO is a relatively weak nitrosating agent and substantially weaker than N2O3. Thus, ethanol restrains NDMA formation through EtONO reacting with DMA. Moreover, as shown in Table 2, the reaction rate constants kTST for EtONO, AcONO, and N2O3 reacting with DMA at 298 K and 1 atm calculated following the TST are 2.6 × 10−12±2.5, 2.3 × 1011±2.5, and 2.5 × 107±2.5 M−1 s−1, respectively. Clearly, these theoretical results 4510


Article


ability. According to the first requirement, the compounds HY, that is, ethanethiol and phenol, as shown in Scheme 4, are two

Scheme 1. Reaction Pathways of Ethanol and Acetic Acid with HONO

Scheme 4. Schematic Transition States for the Reaction of N2O3 with Proposed Inhibitor HY

Scheme 2. Reaction Pathways of Ethanol and Acetic Acid with N2O3

possible inhibitors of NDMA formation, since they can both first react with N2O3 to produce Y-NO (EtSNO and PhONO for ethanethiol and phenol, respectively) and HONO through a five-membered rings. However, the more important thing, which is needed by the second requirement, is that the products Y-NO should have low nitrosating abilities. To further examine whether these two compounds can inhibit NDMA formation or not, the calculations of HY reacting with N2O3 and the products YON reacting with DMA were performed, and the results were listed in Table 2. Similar to the results of ethanol and acetic acid, ethanethiol and phenol are both favorable to react with N2O3 with ΔG‡sol being ∼23 kcal/mol. However, only product EtSNO is a weak nitrosating agent with ΔG‡sol in the reaction of EtSNO with DMA being ∼36 kcal/mol, whereas PhONO is still a moderate nitrosating agent with ΔG‡sol in the reaction of PhONO with DMA being ∼17 kcal/mol. This result is consistent with previous findings that sulfur-containing compounds are likely to play the inhibitory effects on NOCs formation.13,14

Scheme 3. Reaction Pathways of EtONO and AcONO with DMA

4. CONCLUSIONS The effects of ethanol and acetic acid on the formation of NDMA in SGF were investigated from experimental and theoretical aspects. The experimental results demonstrate that ethanol can inhibit, whereas acetic acid can promote, the formation of NDMA, and they are both dose-dependent to NDMA formation. Further studies demonstrated that ethanol and acetic acid both have interaction with nitrite or its related species in SGF rather than DMA. The theoretical results indicate that ethanol is feasible to react with dinitrogen trioxide (N2O3) rather than nitrous acid (HONO) to generate ethyl nitrite (EtONO), whereas acetic acid is favorable to react with both N2O3 and HONO to produce acetyl nitrite (AcONO). Moreover, the generated EtONO and AcONO have, respectively, weaker and stronger nitrosating abilities than N2O3, so that ethanol and acetic acid play inhibitory and promotion roles in NDMA formation from DMA and nitrite in SGF, respectively. In view of the above results, it can be concluded that, as a potential inhibitor of NOCs formation in SGF, two requirements, namely, feasible to react with nitriterelated species in SGF and the product of the former reaction unfavorable to nitrosating reactions, should be met. Results obtained in this study will be helpful in better understanding the inhibitory effect and searching for a series of inhibitors of NOCs formation.

Table 2. Activation Free Energies, Reaction Energies (in kcal/mol), and Reaction Rate Constants (kTST, in M−1 s−1) for all Reactions of Ethanethiol and Phenol with N2O3 as well as Reactions of Products EtSNO and PhONO with DMA Calculated at G4 Level reactions C2H5SH + N2O3 → C2H5SNO + HNO2 C6H6OH + N2O3 → C6H6ONO + HNO2 DMA + C2H5SNO → NDMA + C2H5SH DMA + C6H6ONO → NDMA + C6H6OH

ΔG‡gas ΔG‡sol

ΔH

ΔGsol

kTST

25.4

22.6

−13.9

−15.5

21.0

23.3

−1.5

1.3

34.1

35.9

−6.4

−9.1

20.0

17.0

−19.0

−25.9

1.8 × 10−4±2.5 5.6 × 10−5±2.5 3.3 × 10−14±2.5 2.3 × 100±2.5

effect on the formation of NDMA. Thus, the assumption that a compound as an inhibitor of NDMA formation can be identified by checking if it has the activity to scavenge nitrite is not correct. Subsequently, it can be concluded that a protective compound of NOCs formation in SGF should satisfy two requirements. One is that it should be feasible to react with nitrite-related species in SGF, and the other is that the product of the above reaction should have a relatively low nitrosating 4511


Article


■

Nitroso Compounds and Risk of Childhood Brain Tumors. Cancer Causes Control 2005, 16, 619−635. (2) Engemann, A.; Focke, C.; Humpf, H. U. Intestinal Formation of N-Nitroso Compounds in the Pig Cecum Model. J. Agric. Food Chem. 2013, 61, 998−1005. (3) Mirvish, S. S. Role of N-Nitroso Compounds (NOC) and NNitrosation in Etiology of Gastric, Esophageal, Nasopharyngeal and Bladder-Cancer and Contribution to Cancer of Known Exposures to NOC. Cancer Lett. 1995, 93, 17−48. (4) Bartsch, H.; Ohshima, H.; Shuker, D. E. G.; Pignatelli, B.; Calmels, S. Exposure of Humans to Endogenous N-Nitroso Compounds-Implications in Cancer Etiology. Mutat. Res., Rev. Genet. Toxicol. 1990, 238, 255−267. (5) Dennehy, M. K.; Loeppky, R. N. Mass Spectrometric Methodology for the Determination of Glyoxaldeoxyguanosine and O-6-Hydroxyethyldeoxyguanosine DNA Adducts Produced by Nitrosamine Bident Carcinogens. Chem. Res. Toxicol. 2005, 18, 556−565. (6) IARC. Some N-Nitroso Compounds. IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans; IARC Publications: Lyon, France, 1978. (7) U.S. EPA. Integrated Risk Information System (IRIS Summaries): N-Nitrosodimethylamine (CASRN 62-79-9). Available online: http://www.epa.gov./iris/subst/0045.htm. Accessed 5/20/2016. (8) Gloria, M. B. A.; Barbour, J. F.; Scanlan, R. A. Volatile Nitrosamines in Fried Bacon. J. Agric. Food Chem. 1997, 45, 1816− 1818. (9) Haruta, S.; Chen, W. P.; Gan, J.; Simunek, J.; Chang, A. C.; Wu, L. S. Leaching Risk of N-Nitrosodimethylamine (NDMA) in Soil Receiving Reclaimed Wastewater. Ecotoxicol. Environ. Saf. 2008, 69, 374−380. (10) Tricker, A. R. N-Nitroso Compounds and Man: Sources of Exposure, Endogenous Formation and Occurrence in Body Fluids. Eur. J. Cancer Prev. 1997, 6, 226−268. (11) Holtrop, G.; Johnstone, A. M.; Fyfe, C.; Gratz, S. W. Diet Composition is Associated with Endogenous Formation of N-Nitroso Compounds in Obese Men. J. Nutr. 2012, 142, 1652−1658. (12) Krul, C. A. M.; Zeilmaker, M. J.; Schothorst, R. C.; Havenaar, R. Intragastric Formation and Modulation of N-Nitrosodimethylamine in a Dynamic in Vitro Gastrointestinal Model under Human Physiological Conditions. Food Chem. Toxicol. 2004, 42, 51−63. (13) Choi, S. Y.; Chung, M. J.; Lee, S. J.; Shin, J. H.; Sung, N. J. NNitrosamine Inhibition by Strawberry, Garlic, Kale, and the Effects of Nitrite-Scavenging and N-Nitrosamine Formation by Functional Compounds in Strawberry and Garlic. Food Control 2007, 18, 485− 491. (14) Rahman, M. S. Allicin and Other Functional Active Components in Garlic: Health Benefits and Bioavailability. Int. J. Food Prop. 2007, 10, 245−268. (15) Denev, P. N.; Kratchanov, C. G.; Ciz, M.; Lojek, A.; Kratchanova, M. G. Bioavailability and Antioxidant Activity of Black Chokeberry (Aronia Melanocarpa) Polyphenols: In Vitro and in Vivo Evidences and Possible Mechanisms of Action: A Review. Compr. Rev. Food Sci. Food Saf. 2012, 11, 471−489. (16) Giovannucci, E. Tomatoes, Tomato-Based Products, Lycopene, and Cancer: Review of the Epidemiologic Literature. J. Natl. Cancer I. 1999, 91, 317−331. (17) Abraham, S. K.; Khandelwal, N. Ascorbic Acid and Dietary Polyphenol Combinations Protect against Genotoxic Damage Induced in Mice by Endogenous Nitrosation. Mutat. Res., Genet. Toxicol. Environ. Mutagen. 2013, 757, 167−172. (18) Mirvish, S. S.; Grandjean, A. C.; Chen, S. C.; Gallagher, J.; Maynard, T. Time of Dosing with Ascorbic Acid and Nitrate, Chewing Gum and Tobacco, and Other Factors Affecting N-nitrosoproline Formation in Healthy Adults Taking Proline with a Standard Meal. Cancer Epidem Biomar. 1995, 4, 775−782. (19) Lane, R. P.; Bailey, M. E. The Effect of pH on Dimethylnitrosamine Formation in Human Gastric Juice. Food Cosmet. Toxicol. 1973, 11, 851−854.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpca.6b02582. The details of calculations, standard curves of NO2− and DMA, yield data of NDMA after incubation, concentrations of NDMA, DMA, and nitrite after incubation, activation free energies, Cartesian coordinates and the lowest harmonic vibrational frequency (LHVF) of transition states for each reaction. (PDF)

■

AUTHOR INFORMATION

Corresponding Author

*Fax: +86-10-6739-2001. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We thank the High Performance Computing (HPC) Center in Beijing Computing Center for providing the high-performance computing clusters. This work was supported by Beijing Natural Science Foundation (No. 8132015) and the Project of Construction of Innovative Teams and Teacher Career Development for Universities and Colleges under Beijing Municipality (CIT & TCD201304057). The first two authors contributed equally to this work.

■

ABBREVIATIONS NDMA; N-nitrosodimethylamine NOCs; N-nitroso compounds DMA; dimethylamine SGF; simulated gastric fluid EtONO; ethyl nitrite AcONO; acetyl nitrite N2O3; dinitrogen trioxide HONO; nitrous acid NAs; N-nitrosamines NDEA; N-nitrosodiethylamine NMU; N-nitrosomethylurea NEU; N-nitrosoethylurea IARC; International Agency for Research on Cancer U.S. EPA; The U.S. Environment Protection Agency IRIS; Integrated Risk Information Service DMA·HCl; dimethylamine hydrochloride NaNO2; sodium nitrite NaCl; sodium chloride HCl; hydrochloric acid NDMA-d6; deuterated N-nitrosodimethylamine GC-MS; gas chromatography mass spectrometry SM; full scan SIM; selected ion monitoring EI; electron ionization IC; ion chromatography MEP; minimum-energy path IRC; intrinsic reaction coordinate SED; Solute Electron Density TST; transition state theory

■

REFERENCES

(1) Dietrich, M.; Block, G.; Pogoda, J. M.; Buffler, P.; Hecht, S.; Preston-Martin, S. A Review: Dietary and Endogenously Formed N4512


Article


(42) Hill, T. L. An Introduction to Statistical Thermodynamics; Dover Publications, Inc: New York, 1986, 508. (43) Steinfeld, J. I.; Francisco, J. S.; Hase, W. L. Chemical Kinetics and Dynamics, 2nd ed.; Pearson, 1999; p 518. (44) Sun, Z.; Liu, Y. D.; Zhong, R. G. Theoretical Investigation of NNitrosation Mechanism of Amino Acids Mediated by N2O3. iCBBE 2009, 4150−4153. (45) Lv, C. L.; Liu, Y. D.; Wang, Y. H.; Zhong, R. G. Theoretical Studies on the N-Nitrosodimethylamine Formation from Dimethylamine and Nitrous Acid. Acta Chim. Sinica. 2007, 65, 1568−1572. (46) Jones, L. H.; Badger, R. M.; Moore, G. E. The Infrared Spectrum and the Structure of Gaseous Nitrous Acid. J. Chem. Phys. 1951, 19, 1599. (47) Mcgraw, G. E. Infrared Spectra of Isotopic Nitrous Acids. J. Chem. Phys. 1966, 45, 1392. (48) Varma, R.; Curl, R. F. Study of the N2O3-H2O-HNO2 Equilibrium by Intensity Measurements in Microwave Spectroscopy. J. Phys. Chem. 1976, 80, 402−409. (49) Sun, Z.; Liu, Y. D.; Lv, C. L.; Zhong, R. G. Theoretical Investigation of the Isomerization of N2O3 and the N-Nitrosation of Dimethylamine by Asym-N2O3, Sym-N2O3, and Trans-Cis N2O3 Isomers. J. Mol. Struct.: THEOCHEM 2009, 908, 107−113. (50) Rocha, B. S.; Gago, B.; Barbosa, R. M.; Cavaleiro, C.; Laranjinha, J. Ethyl Nitrite Is Produced in the Human Stomach from Dietary Nitrate and Ethanol, Releasing Nitric Oxide at Physiological pH: Potential Impact on Gastric Motility. Free Radical Biol. Med. 2015, 82, 160−166. (51) Gago, B.; Nystrom, T.; Cavaleiro, C.; Rocha, B. S.; Barbosa, R. M.; Laranjinha, J.; Lundberg, J. O. The Potent Vasodilator Ethyl Nitrite Is Formed Upon Reaction of Nitrite and Ethanol under Gastric Conditions. Free Radical Biol. Med. 2008, 45, 404−412. (52) Nicolescu, A. C.; Reynolds, J. N.; Barclay, L. R.; Thatcher, G. R. Organic Nitrites and No: Inhibition of Lipid Peroxidation and Radical Reactions. Chem. Res. Toxicol. 2004, 17, 185−196.

(20) Yermilov, V. B.; Shendrikova, I. A.; Volkov, D. P.; Stefanenko, Y. F.; Chernomordikov, V. G.; Pavlov, K. A.; Dikun, P. P. Anomalous Action of Nitrosation Inhibitors in Human Gastric-Juice. Vop Onkol. 1986, 32, 58−64. (21) Yermilov, V. B.; Shendrikova, I. A.; Dikun, P. P. On the Effect of Inhibitors of Amine Nitrosation in Human Gastric-Juice and Animal Body. Vop Onkol. 1989, 35, 38−44. (22) Mirvish, S. S. Erratum to Role of N-Nitroso Compounds (NOC) and N-Nitrosation in Etiology of Gastric, Esophageal, Nasopharyngeal and Bladder-Cancer and Contribution to Cancer of Known Exposures to NOC. Cancer Lett. 1995, 97, 271−271. (23) Kurechi, T.; Kikugawa, K.; Kato, T. Effect of Alcohols on Nitrosamine Formation. Food Cosmet. Toxicol. 1980, 18, 591−595. (24) Williams, D. L. H.; Aldred, S. E. Inhibition of Nitrosation of Amines by Thiols, Alcohols and Carbohydrates. Food Chem. Toxicol. 1982, 20, 79−81. (25) Shendrikova, I. A.; Ermilov, V. B.; Dikun, P. P. The Influence of Ethanol on Synthesis of N-Nitrosodimethylamine in Vivo and in Vitro. Cancer Lett. 1984, 25, 49−54. (26) United States Pharmacopeia, United States Pharmacopeia 32/ National Formulary 27[M]. United States Pharmacopeia, USA: 2009. (27) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; et al. Gaussian 09, Revision A.01; Gaussian, Inc: Wallingford, CT, 2009. (28) Becke, A. D. Density-functional thermochemistry. III. The role of exact exchange. J. Chem. Phys. 1993, 98, 5648−5652. (29) Lee, C.; Yang, W.; Parr, R. G. Development of the Colle-Salvetti Correlation-energy Formula into a Functional of the Electron Density. Phys. Rev. B: Condens. Matter Mater. Phys. 1988, 37, 785−789. (30) Frisch, M. J.; Pople, J. A.; Binkley, J. S. Self-consistent Molecular-orbital Methods 25.supplementary Functions for Gaussianbasis Sets. J. Chem. Phys. 1984, 80, 3265−3269. (31) Curtiss, L. A.; Redfern, P. C.; Raghavachari, K. Gaussian-4 Theory. J. Chem. Phys. 2007, 126, 636−641. (32) Gonzalez, C.; Schlegel, H. B. An improved Algorithm for Reaction-path Following. J. Chem. Phys. 1989, 90, 2154−2161. (33) Trogolo, D.; Mishra, B. K.; Heeb, M. B.; Von Gunten, U.; Arey, J. S. Molecular Mechanism of NDMA Formation from N,Ndimethylsulfamide during Ozonation: Quantum Chemical Insights into a Bromide-catalyzed Pathway. Environ. Sci. Technol. 2015, 49, 4163−4175. (34) Karton, A.; O’Reilly, R. J.; Radom, L. Assessment of Theoretical Procedures for Calculating Barrier Heights for a Diverse Set of WaterCatalyzed Proton-Transfer Reactions. J. Phys. Chem. A 2012, 116, 4211−4221. (35) Smith, D. G. A.; Patkowski, K. Interactions between Methane and Polycyclic Aromatic Hydrocarbons: A High Accuracy Benchmark Study. J. Chem. Theory Comput. 2013, 9, 370−389. (36) Marenich, A. V.; Cramer, C. J.; Truhlar, D. G. Universal Solvation Model Based on Solute Electron Density and on a Continuum Model of the Solvent Defined by the Bulk Dielectric Constant and Atomic Surface Tensions. J. Phys. Chem. B 2009, 113, 6378−6396. (37) Zhao, Y.; Schultz, N. E.; Truhlar, D. G. Exchange-correlation Functional with Broad Accuracy for Metallic and Nonmetallic Compounds, Kinetics, and Noncovalent Interactions. J. Chem. Phys. 2005, 123, 161103. (38) Davidson, E. R. Comment on “Comment on Dunning’s Correlation-consistent Basis Sets. Chem. Phys. Lett. 1996, 260, 514− 518. (39) Dunning, T. H. Gaussian Basis Sets for use in Correlated Molecular Calculations. I. The Atoms Boron through Neon and Hydrogen. J. Chem. Phys. 1989, 90, 1007−1023. (40) Dunning, T. H.; Peterson, K. A.; Wilson, A. K. Gaussian basis sets for use in correlated molecular calculations. X. The Atoms Aluminum through Argon Revisited. J. Chem. Phys. 2001, 114, 9244− 9253. (41) Kendall, R. A.; Dunning, T. H.; Harrison, R. J. Electron Affinities of the First-row Atoms Revisited. Systematic Basis Sets and Wave Functions. J. Chem. Phys. 1992, 96, 6796−6806. 4513


Experimental and Theoretical Investigation of Effects of Ethanol and

Recommend Documents