4,4'-Methylenedianiline (
DAPM) is an aromatic
diamine used directly in the production of
polyurethane foams and
epoxy resins, or as a precursor to MDI in the manufacture of some
polyurethanes. In our prior experiments, we showed that chronic, intermittent treatment of female rats with
DAPM resulted in vascular medial
hyperplasia of pulmonary arteries. In addition, treatment of vascular smooth muscle cells (VSMC) in culture with
DAPM increased the rates of proliferation in a manner that was inhibited by co-treatment with
N-acetylcysteine but was not associated with oxidative stress. We thus hypothesized that NAC treatment inhibited
DAPM toxicity by competing for binding reactive intermediates formed through
DAPM metabolism. Because the
peroxidase enzyme cyclooxygenase is constitutively expressed in VSMC, and because
cyclooxygenase is known to metabolize similar aromatic
amines to electrophilic intermediates, we further hypothesized that
DAPM-induced VSMC proliferation was dependent upon COX-1/2-mediated bioactivation. To test this hypothesis, we treated VSMC with
DAPM and measured cell proliferation, COX-2 expression, COX-1/2 activity, and levels of covalent binding.
DAPM treatment resulted in a dose-dependent increase in proliferation that was abolished by co-treatment with the COX-2-selective inhibitor
celecoxib. In addition,
DAPM exposure increased the rates of proliferation in VSMC isolated from wild-type but not COX-2 (-/-) mice. Paradoxically, treatment with
DAPM reduced the cellular production of
PGE(2) and
PGF(2α), but dose-dependently increased the COX-2
protein levels. Covalent binding of [(14)C]-
DAPM to VSMC biomolecules was greater in wild-type than in COX-2 (-/-) cells. However, covalent binding of [(14)C]-
DAPM was not altered by co-treatment with a nonselective inhibitor of
cytochromes P450. These studies thus suggest that
DAPM-induced VSMC proliferation may be due to bioactivation of
DAPM, perhaps through the action of
cyclooxygenase. The data furthermore suggest that
DAPM's mechanism of action may possibly involve inhibition or suicide inactivation of COX-2. In addition, because we observed an increase in
DAPM-induced VSMC proliferation in cells isolated from female compared to male rats, further studies into the potential interplay between
DAPM, the
estrogen receptor, and COX-2 seem warranted.