Decrease in 4-Aminobiphenyl-Induced Methemoglobinemia in Cyp1a2( -/ -) Knockout Mice 1 Howard G. Shertzer, 2 Timothy P. Dalton, Glenn Talaska, and Daniel W. Nebert Department of Environmental Health and Center for Environmental Genetics, University of Cincinnati Medical Center, Cincinnati, Ohio 45267-0056 Received December 4, 2001; accepted February 28, 2002 Decrease in 4-Aminobiphenyl-Induced Methemoglobinemia in Cyp1a2(/) Knockout Mice. Shertzer, H. G., Dalton, T. P., Talaska, G., and Nebert, D. W. (2002). Toxicol. Appl. Pharmacol. 181, 32–37. Methemoglobin formation, as well as hemoglobin or DNA adducts, are useful biomarkers of occupational exposure to certain arylamines. It has been suggested that, in liver from animals not treated with a cytochrome P450 (CYP) inducer, hepatic CYP1A2 is the major P450 involved in N-hydroxylation. This is the first step in the metabolic activation of many arylamines, such as the human urinary bladder carcinogen 4-aminobiphenyl (ABP). The product of this catalytic step, N-hydroxy-4-ABP, reacts in the blood with oxyhemoglobin to form methemoglobin and nitrosobiphenyl. We therefore examined the role of CYP1A2 in causing methemoglobinemia in ABP-treated Cyp1a2(/) knockout mice. Application of ABP (100 mol/kg body wt) to the skin resulted in a marked depletion in the levels of the hepatic thiols (reduced glutathione and cysteine) after 2 h, which rebounded to basal levels 24 h later, and we found no differences between the Cyp1a2(/) and wild-type Cyp1a2(/) animals. Un- expectedly, the methemoglobin levels were significantly (p < 0.05) higher in Cyp1a2(/) than Cyp1a2(/) mice at 2, 7, and 24 h following topical ABP. Treatment with dioxin, 24 h prior to ABP, decreased methemoglobin levels by about half at each of the time points in both the Cyp1a2(/) and Cyp1a2(/) mice. These data suggest that CYP1A2 does not play a positive role in methemoglobin formation via the activation of ABP;rather, the absence of CYP1A2 enhances ABP-induced methemoglobinemia. Because liver CYP1A2 levels are known to vary more than 60-fold between humans, our findings may be relevant to patients who are exposed to arylamines in the workplace. © 2002 Elsevier Science (USA) Key Words: methemoglobinemia; 4-aminobiphenyl; biomarkers of exposure; CYP1A2; dioxin; hepatic cysteine levels; reduced glutathione. Exposures to certain environmental N-heterocyclic and aro- matic amine compounds pose a threat to human health. The most common route for occupational human exposure to aro- matic amines such as 4-aminobiphenyl (ABP 3 ) is transdermal (Scott, 1962; NIOSH, 1990). ABP and other aromatic amines are efficiently absorbed through the skin and have been given a “skin notation” by the American Conference of Governmen- tal Industrial Hygienists. Workers exposed to these materials have been documented to have a tremendously excessive risk of urinary bladder cancer; observed relative risks range from 10-fold to approximately 90-fold, depending on the study (Vineis et al., 1994). In addition, aromatic amines such as ABP, 2-naphthylamine, and o-toluidine are known to be com- ponents of tobacco smoke. It has been documented (Talaska et al., 1991, 1993) that N-(deoxyguanosin-8-yl)-4-aminobiphenyl is the major DNA adduct in the urothelium of tobacco smokers. In mouse studies, topical ABP has been shown to produce ABP–DNA adducts in lung, liver, skin, and urinary bladder (Underwood et al., 1997). The major biotransformation pathways for ABP are illus- trated in Fig. 1. The metabolic balance between the detoxifi- cation and activation pathways for ABP are believed to mod- ulate its toxicity. ABP detoxification occurs primarily in the liver, with major pathways thought to involve N-acetyltrans- ferase-2 and cytochrome P450 (CYP)-mediated ring hydroxy- lation (Turesky et al., 1998; Probst-Hensch et al., 2000). N- hydroxylation is the primary initial step in the activation pathway, catalyzed by one or more forms of CYP. The N- hydroxy metabolite may be detoxified by N-acetylation or undergo further activation by O-acetylation, O-sulfation, or O-glucuronidation. The former two are considered to be highly unstable species that can react with protein and DNA under physiological conditions. All three of the O-conjugates are unstable under acidic conditions, such as may occur in the urinary bladder. These compounds may undergo acid-mediated decomposition to form the electrophilic nitrenium cation, which is able to form adducts with DNA, hemoglobin, and protein or nonprotein sulfhydryls. (Kadlubar and Badawi, 1995; Guengerich et al., 1995; Landi et al., 1996). Alterna- tively, the aryl hydroxylamine may enter the blood and un- 1 The views expressed in this article are those of the authors and do not reflect the official policy or position of the National Institutes of Health. 2 To whom correspondence should be addressed at Department of Envi- ronmental Health, P.O. Box 670056, University of Cincinnati Medical Center, Cincinnati, OH 45267-0056. Fax: (513) 558-0925; E-mail: shertzhg@ ucmail.uc.edu. 3 Abbreviations used: ABP, 4-aminobiphenyl; FMO, flavin-containing monooxygenase; OTC, ornithine carbamoyltransferase; TCDD, 2,3,7,8-tetra- chlorodibenzo-p-dioxin. Toxicology and Applied Pharmacology 181, 32–37 (2002) doi:10.1006/taap.2002.9398, available online at http://www.idealibrary.com on 32 0041-008X/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved.