Assessment of cellular and molecular metrics for dose selection in an in vivo comet assay: A case study with MDI

The in vivo comet assay can evaluate the genotoxic potential of a chemical in theoretically any tissue that can be processed to a single cell suspension. This flexibility enables evaluation of point‐of‐contact tissues using a relevant route of test material administration; however, assessing cytotoxicity is essential for the interpretation of comet results. Histopathological evaluation is routinely utilized to assess cytotoxicity, but temporal‐ and cell‐specific considerations may compromise applicability to the comet assay. In the present study, 1,1′‐methylenebis(4‐isocyanatobenzene) (4,4'‐MDI) was administered to rats for 6 h by nose‐only inhalation, and the comet assay was conducted to evaluate genotoxicity in the site‐of‐contact tissue (bronchoalveolar lavage cells) and distal tissues (liver and glandular stomach). Given the reactive nature of MDI, cellular and molecular metrics at the site‐of‐contact‐ including inflammation, macrophage activation, apoptosis/necrosis, and oxidative stress‐ were used to set appropriate exposure concentrations, in addition to the standard systemic measures of toxicity. In the range‐finding study, a concentration of 4 mg/m(3) was considered the maximum noninflammatory concentration; hence target concentrations of 2, 5, and 11 mg/m(3) were selected for the comet study. In the lung lavage, MDI exposure substantially increased total protein and β‐glucuronidase, along with cellular apoptosis. Although MDI did not increase the comet assay response (% tail DNA) in any of the tissues examined, the positive control (ethyl methanesulfonate, EMS) significantly increased % tail DNA in all tissues. In total, these data indicate that appropriate cellular and molecular measurements may facilitate dose selection to discern cellular status in the comet assay.