Chemical Research in Toxicology

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Article

Figure 14. (a) Probable pathways P450 activation mechanism in N-nitroso piperazine (b) Probable pathways P450 activation mechanism in Nnitroso thiomorpholine.

that the electron-withdrawing group at the para position
influences the steps in the metabolic pathway. Interestingly, the
activation energy for the hydrolysis reaction is quite high for all
the aromatic reactions except for compound 35 indicating that
2-substituted nitroso pyridine easily undergoes reaction with
water. Furthermore, the activation energies for interaction with
DNA bases are also observed to be higher for the aromatic
molecules but less when compared with the hydrolysis reaction
which indicates that aromatic diazonium is more reactive
toward DNA bases as compared to water which supports
higher potency for these molecular categories when compared
with the cyclic saturated nitrosamines.
Within the aryl nitrosamine subclass, N-nitroso-N-methyl
pyridines (NMPYs) (33−35) present another interesting case
study. Even with a subtle difference in structure (i.e., isomers
of pyridines), the carcinogenicity changes dramatically from 2substituted compound 35 being carcinogenic (TD50 of 0.214
mg/kg/day) to 3- and 4-pyridine (34, 33 respectively), both
being noncarcinogenic. It is well-known that these molecules
can undergo a variety of metabolic reactions, one of them
being the N-oxidation reaction.28 A study from Eisenbrand40,41
showed that in the case of substituted N-nitroso-Nmethylpyridines, multiple metabolites are observed. From in
vitro studies, they showed that 2-nitroso methylpyridine (2NMPY) predominantly undergoes α-hydroxylation and 2hydroxy pyridine is also observed. Our results also indicate that
when compared to 3-NMPY (34) and 4-NMPY (33), the
activation energies for the hydrolysis reaction (14.4 kcal/mol
for 2-NMPY compared to 28.4 and 31.7 kcal/mol for 34 and
33, respectively) and DNA base substantially low for 2-NMPY
(35) as well as for the reaction with DNA base. Breton42 also
observed that 2-pyridyldiazoium cation is more stable than 3pyridyldiazonium and 4-pyridyl diazonium ion These results
clearly differentiate 33-35 in terms of the reactivity toward
hydrolysis and DNA base. In short, the presence of competing
and more accessible alternative CYP450 oxidative pathway
results in few of the aromatic nitrosamines being noncarcinogenic, whereas molecules that follow the typical
nitrosamine metabolic pathway are carcinogenic, consistent
with the mechanism of action and the adverse outcome
pathway for nitrosamines.

Figure 15. Aromatic nitrosamines considered in the present study.

0.142−1.300 mg/kg/day and two noncarcinogenic molecules
32 and 33. Thus, the CPCA potency category is significantly
conservative for this class of molecules. Nonetheless, we have
attempted to understand the reactivity of these aromatics
through a carcinogenic metabolic pathway mechanism.
The activation energies (ΔG‡AB) for all of the stepwise
reactions involved in the carcinogen metabolic pathway are
shown in Table 5. The calculated ΔG‡AB is in the range of 19.4
(29)−21.2 (30) kcal/mol for 29−34 and considerably high for
compound 35 (22.2 kcal/mol), probably because of the steric
effect from the N-electron pair. This result clearly indicates
Table 5. Gibbs Activation Free Energies (in kcal/mol) for
All of the Mechanistic Steps Involved in the Aromatic
Nitrosamines
nitrosamine

ΔG‡AB

ΔG‡BC

ΔG‡DE

ΔG‡DG

29
30
31
31
33
34
35

19.4
21.2
20.6
20.1
20.5
20.3
22.2

22.4
22.3
23.1
23.8
23.3
22.9
24.1

34.0
39.0
36.3
35.6
28.4
31.7
14.4

28.9
35.1
31.8
30.7
22.8
27.8
11.8
1020

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https://doi.org/10.1021/acs.chemrestox.4c00087
Chem. Res. Toxicol. 2024, 37, 1011−1022