Testicular Toxicity in F344 Rats by Aminophenylnorharman, Formed from Norharman and Aniline Yukari Totsuka,* ,1 Toshihiko Kawamori,* Shigeru Hisada,² Kunitoshi Mitsumori,‡ Junko Ishihara,* Takashi Sugimura,* and Keiji Wakabayashi* *Cancer Prevention Division, National Cancer Center Research Institute, 1-1 Tsukiji 5-chome, Chuo-ku, Tokyo 104-0045, Japan; ² Safety Research Department, Teikoku Hormone Mfg. Co., Ltd., 1604 Shimo-sakunobe, Takatsu-ku, Kawasaki 213-0033, Japan; and ‡Laboratory of Veterinary Pathology, Faculty of Agriculture, Tokyo University of Aguriculture and Technology, 3-5-8 Saiwai-cho, Fuchu-shi, Tokyo 183-8509, Japan Received December 13, 2000; accepted June 20, 2001 Testicular Toxicity in F344 Rats by Aminophenylnorharman, Formed from Norharman and Aniline. Totsuka, Y., Kawamori, T., Hisada, S., Mitsumori, K., Ishihara, J., Sugimura, T., and Wakabayashi, K. (2001). Toxicol. Appl. Pharmacol. 175, 169 –175. 9-(4-Aminophenyl)-9H-pyrido[3,4-b]indole [aminophenylnor- harman (APNH)] is a novel mutagenic heterocyclic amine, pro- duced by the reaction of norharman with aniline in the presence of S9 mix. In the present study, the acute toxicity of this compound was investigated in male F344 rats. Ten-week-old animals were treated with a single intragastric injection of APNH at doses of 45 or 90 mg/kg body wt and euthanized 1, 3, or 6 days afterward. When APNH was administered at a dose of 90 mg/kg, vacuolation of Sertoli cells in the testis was seen at 1 day after treatment. The testicular damage had markedly progressed by day 6, with multinucleated giant cells and loss of round spermatids in the seminiferous tubules observed in groups 1 and 2 of the four histological categories of spermatogenesis. Numbers of spermato- gonia were also decreased by APNH treatment. No toxic changes were observed in Leydig cells under these conditions and serum follicle-stimulating hormone and luteinizing hormone concentra- tions were also unchanged from control values. Such severe tes- ticular damage was not observed at any time point at the 45 mg/kg dose level of APNH. Moreover, neither norharman nor aniline alone exerted acute testicular toxicity at doses equivalent to 90 mg/kg of APNH. In addition to the testicular lesions, erosive changes of urinary bladder, thymic atrophy, and panmyelophthi- sis were evident in rats given APNH at 90 mg/kg. © 2001 Academic Press Key Words: testicular toxicity; aminophenylnorharman; F344 rat; Sertoli cell; norharman; aniline; heterocyclic amine. Norharman (9H-pyrido[3,4-b]indole), produced when L-tryptophan is heated (Sugimura et al., 1977), is not itself a mutagen to Salmonella typhimurium TA98 and TA100 with or without S9 mix, which is a metabolic activation system derived from rat liver. However, norharman becomes mutagenic to S. typhimurium TA98 and YG1024 in the presence of S9 mix, when mixed with nonmutagenic aromatic amines including aniline (Nagao et al., 1977, 1978; Sugimura et al., 1982; Mori et al., 1996). Therefore, we called norharman a “comutagen”. Norharman is widely distributed in our environment, because of its existence in cigarette smoke and cooked foodstuffs (Gross et al., 1993; Poindexter and Carpenter, 1962; Totsuka et al., 1999). Its levels in cigarette smoke condensate samples are markedly high, being 900 – 4240 ng per cigarette, more than 100-fold higher than those of known mutagenic and carcino- genic heterocyclic amines (HCAs) (Totsuka et al., 1999). Nor- harman has also been detected in human urine samples, at levels much greater than those of HCAs (Ushiyama et al., 1995). Furthermore, aniline is present in cigarette smoke, along with norharman, at levels of 0.1–18.1 g per cigarette (Luceri et al., 1993), and is also detectable in some vegetables (IARC, 1982). Moreover, aniline is found in human urine and milk samples (Riffelmann et al., 1995; Bayoumy et al., 1986; De- Bruin et al., 1999). Thus, it is likely that humans are continu- ously exposed to combinations of norharman and aniline in daily life. Recently, the mechanism of comutagenic action of norhar- man with aniline in the presence of S9 mix was demonstrated to be due to the production of a coupled compound, 9-(4'- aminophenyl)-9H-pyrido[3,4-b]indole [aminophenylnorharman (APNH)], as shown in Fig. 1 (Totsuka et al., 1998). APNH can be further metabolically activated to the hydroxyamino derivative and then converted to ultimate reactive forms, such as N-acetoxy- APNH by esterification enzymes, that yield DNA adducts. APNH was found to induce 187,000 and 1,783,000 revertants per micro- gram with S9 mix, respectively, in TA98 and YG1024 (Totsuka et al., 1998), numbers comparable to those for HCAs such as 2-ami- no-3,8-dimethylimidazo[4,5-f ]quinoxaline (Wakabayashi et al., 1992). Since humans are simultaneously exposed to norharman and aniline, possible toxic and carcinogenic effects of APNH in vivo may be a problem. To preliminarily assess this question, in the present study, the compound was chemically synthesized from norharman and p-bromonitrobenzene as starting materials 1 To whom correspondence should be addressed. Fax: (81) 3-3543-9305; E-mail: ytotsuka@gan2.ncc.go.jp. Toxicology and Applied Pharmacology 175, 169 –175 (2001) doi:10.1006/taap.2001.9236, available online at http://www.idealibrary.com on 169 0041-008X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.