Protein Adducts from Perchloroethene Metabolism
Chem. Res. Toxicol., Vol. 11, No. 9, 1998 1003
however, the formed products are not stable and may not
be detected by the antibody or by mass spectrometry.
Intermediate formation of protein adducts from fluoro-
alkyl S-conjugates at the cysteine sulfur atom has been
observed (26).
The most intensive response of anti-TCA antibody was
observed in liver microsomes and cytosol of PER treated
rats. This may be due to the oxidative bioactivation of
PER by microsomal enzymes to trichloroacetyl chloride
which is capable of acylating cellular proteins. Like
dichlorothioketene, the acyl halide formed by oxidative
metabolism of PER is highly reactive and is expected to
react with amino acids close to the site of formation. In
addition to the reaction of the acyl halide with proteins,
reaction with lipids may also occur (3).
In liver microsomes, immunoreactive proteins with
molecular weights of approximately 40-45 kDa were
detected. Since cytochromes P450 are involved in the
oxidation of PER to trichloroacetyl chloride (27), a
reaction of the acyl halide with this protein is likely.
Several cytochrome P450 enzymes have molecular weights
of approximately 50 kDa; moreover, acylation of a specific
cytochrome P450, cytochrome P450 2E1, has been dem-
onstrated by acylating agents formed metabolically from
trichloroethene (28) and halothane. In liver cytosol, one
of the major immunoreactive proteins likely represents
albumin (66 kDa) detected as well by the anti-DCA
antibody in kidney cytosol. The identity of the lower
molecular weight proteins in liver cytosol may represent
glutathione S-transferases. Cytosolic glutathione S-
transferases have been identified as proteins modified
during the oxidative metabolism of halothane (29). The
reaction likely involves glutathione conjugate formation
of trichloroacetyl chloride. The formed thioester is also
an acylating agent and may acylate lysine in the vicinity
of the glutathione binding site of the glutathione S-
transferases. In kidney subcellular fractions, the anti-
DCA antibody recognized major immunoreactive proteins
in the molecular mass range of 28-47 kDa. Some of
these proteins may represent cysteine conjugate â-lyases,
which were reported to have molecular mass ranges of
(2) National Cancer Institute, NCI (1976) Bioassay of trichloroeth-
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(
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(
(
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4
3-55 kDa (21). In addition, enzyme subunits of dehy-
drogenase complexes such as pyruvate and 2-oxoglut-
arate dehydrogenase have the appropriate molecular
masses to be considered as possible candidates for adduct
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In summary, antibodies were developed with high
specificity for protein modifications induced by PER
metabolites. These antibodies may have broad utility to
study toxic effects of PER, but also for any other haloalk-
enes metabolized to identical reactive intermediates.
Moreover, the high sensitivity of this method for the
detection of modified proteins may be used to detect and
quantify modified proteins as a biochemical effect marker
in comparative studies on PER metabolism and toxicity.
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(
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Ack n ow led gm en t. Research described in this article
was funded by the United States Environmental Protec-
tion Agency (EPA) (CR824456-01-0) and the Biomed
Program of the European Union (Contract No. BMH4-
CT96-0184).
(
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