748 Chem. Res. Toxicol., Vol. 11, No. 7, 1998
Skordos et al.
C-2 through an addition-rearrangement pathway (13)
to form the enol which also tautomerizes to the keto form
(3-methyloxindole). In both cases, the oxygen incorpo-
rated into the oxindole does, in fact, arise from molecular
nine plays a role in the toxicity of 3-methylindole, then
aldehyde oxidase is probably involved in detoxification
processes and may be protective of hepatic cells. How-
ever, the relative order of species sensitivities to the
pneumotoxic effects of 3-methylindole does not correlate
to a relative lack of aldehyde oxidase activity in pulmo-
nary tissues of sensitive species.
Presented here is an indirect confirmation that a
cytochrome P450-dependent 2,3-epoxide of 3-methylin-
dole exists and can be characterized by monitoring its
decomposition products. Additional work is required to
elucidate the epoxide’s role and the role of 3-hydroxy-3-
methylindolenine in 3-methylindole-mediated pneumo-
toxicity. An analysis of the protein and/or nucleic acid
alkylation sites for each of the proposed reactive inter-
mediates is essential to an understanding of the cascade
of events that likely lead to the observed toxicity. Work
describing the nature of thiol conjugates of these reactive
intermediates is underway.
2
oxygen, but H at position 2 would be lost.
3-Hydroxy-3-methylindolenine is another reactive in-
termediate of 3-methylindole that likely results from
epoxide ring opening leaving the oxygen at position 3.
This transient electrophilic intermediate appears to be
oxidized to the stable metabolite, 3-hydroxy-3-methylox-
indole, by the cytosolic enzyme aldehyde oxidase. Both
the 18O incorporation characteristics and the putative
detoxification pathway were investigated in this work.
The incorporation of molecular oxygen into 3-hydroxy-
3-methyloxindole was higher than that of 3-methyloxin-
dole, 87% versus 74%. The ratios of the 18O0, 18O1, and
18O2 isotopomers indicate that, except for a minor amount
of incorporation of two oxygen atoms that originated from
molecular oxygen, the vast majority of the compound was
formed by incorporation of only one atom from molecular
oxygen. There are several possible scenarios for oxygen
incorporation into the metabolite, but the one that best
fits the data is one in which one site incorporates oxygen
from molecular oxygen and the other incorporates oxygen
from water. In this case the expected ratios of the
isotopomers 18O0, 18O1, and 18O2 in 3-hydroxy-3-methyl-
oxindole, from either molecular oxygen or water, would
be 1:1:0. This scenario best fits the data as presented in
Tables 1 and 2. The very small amount of the 18O2
isotopomer was probably produced by an uncharacterized
mechanism that involves incorporation of two oxygen
atoms from molecular oxygen.
Ack n ow led gm en t. This work was supported by
United States Public Health Service Grant HL13645 from
the National Heart, Lung and Blood Institute and by a
United States Public Health Service Research Career
Development Award (Grant HL02119) to G.S.Y. The
authors gratefully acknowledge the technical assistance
of Sandra J . Lehman and Dr. Pradipta J eshti. We are
grateful to Drs. Douglas E. Rollins and Roger L. Foltz of
the Center for Human Toxicology for providing mass
spectrometry instrumentation.
Refer en ces
The mass spectral data (Figure 1) also support a
mechanism for 3-hydroxy-3-methyloxindole formation
that involves incorporation of only one oxygen from
molecular oxygen and that the site of incorporation is the
alcohol oxygen. This also supports a mechanism involv-
ing the epoxide that ring-opens to position 3. The only
fragmentation mechanism resulting in the M - 28 ions
at m/z 135/137 is loss of CO; thus, the retention of
approximately an equal isotopomeric ratio (18O atom %
of H2O was 50%) in the m/z 135/137 fragment cluster
indicates that the site of incorporation must be the
alcohol at C 3 (Figure 1).
Aldehyde oxidase appears to catalyze the oxidation of
3-hydroxy-3-methylindolenine to 3-hydroxy-3-methylox-
indole; therefore, it is reasonable to hypothesize that the
enzyme plays an important role in the detoxification of
this 3-methylindole reactive intermediate. Presumably,
animal species with higher pulmonary aldehyde oxidase
activities for this substrate, e.g., rabbits (14), would be
less susceptible to 3-methylindole-induced pneumotox-
icity. When the model substrate (N-methylnicotinamide)
for this enzyme was utilized, the pattern of enzyme
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the oxidation of 3-hydroxy-3-methylindolenine may be
different from the isozyme that catalyzes the oxidation
of N-methylnicotinamide. If 3-hydroxy-3-methylindole-
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