Department of Pharmacology, Toxicology and Neuroscience, Louisiana State University Health Sciences Center, Shreveport, USA.
Cardiovasc Toxicol. 2011 Dec;11(4):316-24. doi: 10.1007/s12012-011-9123-1.
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.
4,4'-亚甲基二苯胺（DAPM）是一种芳香族二胺，直接用于生产聚氨酯泡沫和环氧树脂，或作为制造某些聚氨酯的 MDI 的前体。在我们之前的实验中，我们表明，雌性大鼠慢性间歇性 DAPM 处理会导致肺血管中层的动脉增生。此外，用 DAPM 处理培养的血管平滑肌细胞（VSMC）会以被 N-乙酰半胱氨酸共同处理所抑制的方式增加增殖率，但与氧化应激无关。因此，我们假设 NAC 治疗通过与 DAPM 代谢形成的反应性中间体竞争结合来抑制 DAPM 毒性。由于过氧化物酶酶环加氧酶在 VSMC 中持续表达，并且已知环加氧酶将类似的芳香胺代谢为亲电中间体，我们进一步假设 DAPM 诱导的 VSMC 增殖依赖于 COX-1/2 介导的生物激活。为了验证这一假设，我们用 DAPM 处理 VSMC，并测量细胞增殖、COX-2 表达、COX-1/2 活性和共价结合水平。DAPM 处理导致增殖呈剂量依赖性增加，而 COX-2 选择性抑制剂塞来昔布的共同处理则消除了这种增加。此外，DAPM 暴露增加了来自野生型而非 COX-2（-/-）小鼠的 VSMC 中的增殖率。矛盾的是，DAPM 处理降低了细胞产生的 PGE（2）和 PGF（2α），但剂量依赖性地增加了 COX-2 蛋白水平。与 COX-2（-/-）细胞相比，DAPM 与 VSMC 生物分子的共价结合更多。然而，细胞色素 P450 的非选择性抑制剂的共同处理并未改变 [(14)C]-DAPM 的共价结合。因此，这些研究表明，DAPM 诱导的 VSMC 增殖可能是由于 DAPM 的生物激活，可能通过环加氧酶的作用。此外，由于我们观察到与雄性大鼠相比，来自雌性大鼠的细胞中 DAPM 诱导的 VSMC 增殖增加，因此似乎有必要进一步研究 DAPM、雌激素受体和 COX-2 之间的潜在相互作用。
