Discrimination of haptens from prohaptens using the metabolically deficient Cpr low/low mouse

Abstract

The murine local lymph node assay (LLNA) is a validated, well accepted method for identification of chemical contact allergens. Both direct acting haptens and prohaptens (requiring metabolic activation) can be identified, but not differentiated by this assay. This study was used to assess the utility of a pan microsomal metabolic deficient mouse to distinguish between direct acting haptens and prohaptens in the LLNA. Hapten and prohapten induced cell proliferation was compared in C57BL/6J (B6) wild type (WT) versus homozygous (HO) knockout mice with a hypomorphic NADPH-Cytochrome P450 Reductase (CPR) gene ( termed Cpr low/low ) resulting in low CPR enzyme activity. Mice were dosed with known prohaptens; benzo(a)pyrene (BaP), carvone oxime (COx) and paracetamol (PCM) and haptens; oxazolone (OX), 4-ethoxymethylene-2-phenyl-2-oxazolin-5-one (EtOX), and N-acetylbenzoquinoneimine (NABQI) in this study. Skin microsomes from the WT, HO and heterozygous (HT) Cpr low/low mice were compared and evaluated for CPR activity. Lymphocyte proliferative responses to BaP, COx and PCM were significantly abrogated by 36.4%, 45.2% and 50.8%, respectively; in Cpr low/low knock out (KO) mice versus WT mice; while the lymphocyte proliferative responses to the direct acting haptens OX, EtOX and NABQI were comparable. CPR activity, determined as Units/mg protein, was determined to be significantly lower in the Cpr low/low mice compared to the WT. Results of the present study suggest potential utility of the Cpr low/low mice in the LLNA to differentiate prohaptens from direct acting haptens.