Vascular Medial Hyperplasia Following Chronic, Intermittent Exposure to 4,4'-Methylenedianiline

Abstract

4,4'-Methylenedianiline (DAPM) is an aromatic amine used in the synthesis of polyurethanes and epoxy resins. Acute exposure to DAPM produces hepatobiliary toxicity in humans as well as animal models. However, the toxic effects of intermittent DAPM exposure have not been explored. We treated male and female rats with 25 mg DAPM/kg or vehicle once per week for 17-22 wk. Though concentric fibrosis around bile ducts of the liver was noted, vascular medial hyperplasia was also prominent. Morphometric analysis of histologic sections revealed that in male rats, vessel wall area increased relative to lumen area in hepatic arteries by 22 wk. However, in female rats, wall areas of both hepatic and pulmonary arteries increased relative to lumen area by 17 wk. In both male and female rats, increased wall thickness was localized to the medial layer; no intimal changes were noted. In vitro treatment of vascular smooth muscle cells (VSMC) with 25-100 microM DAPM resulted in increased DNA synthesis and VSMC proliferation. To test whether the observed alterations in cell cycle control involved VSMC-mediated metabolism of DAPM to electrophilic intermediates, cells were treated with DAPM or DAPM plus 50 microM N-acetylcysteine (NAC). Coincubation with NAC afforded dramatic protection against DAPM-induced VSMC proliferation. Though DAPM had no appreciable effect on levels of reduced glutathione, oxidized glutathione, or oxidant production, DAPM increased glutathione-S-transferase activity in VSMC. These data indicate that DAPM can initiate VSMC proliferation, possibly via VSMC-mediated metabolism of DAPM to reactive intermediates.

Role of COX-2 in the Bioactivation of Methylenedianiline and in Its Proliferative Effects in Vascular Smooth Muscle Cells

“…In our prior experiments, similar increases in BrDU incorporation in DAPM-treated cells resulted in ~20% increases in cell number (6). In experiments aimed at testing whether COX-2 had a causal role in DAPM-mediated VSMC proliferation, we treated cells with 25 or 50 μM DAPM ± the COX-2 selective inhibitor celecoxib.…”

“…While the heptobiliary toxicity of methylenedianiline is well-known (3–5), in our prior studies, we demonstrated that once weekly treatment of female rats for 16–20 weeks produced medial hyperplasia of pulmonary arteries (6), suggesting a DAPM-induced proliferation of vascular smooth muscle cells (VSMC). The observed pathology was reminiscent of the human pathological condition pulmonary hypertension.…”

“…Primary cultures of VSMC were isolated as described previously (6) from four male Sprague-Dawley rats (~6 wks old). Cells from passages 2–7 were used for experiments.…”

“…For these studies, we utilized the same isolation protocol we reported earlier for male rats (6), except that in this case, the duration of the elastase/collagenase digestion procedure was not longer than 30 min. All other cell culture and DAPM treatment paradigms were precisely as described for VSMC isolated from male rats.…”

“…VSMC proliferation could also be induced by in vitro treatment with DAPM (6), suggesting that hepatic metabolism may not be required for DAPM-induced VSMC proliferation. Treatment with N-acetylcysteine (NAC), an antioxidant thiol, attenuated VSMC proliferation, but its protective effects did not involve a reduction in cellular oxidative stress, since DAPM did not itself produce an oxidant injury (6). Thus, we hypothesized that the protective effects of the NAC may be due to detoxication of reactive DAPM metabolites, e.g.…”