Reproductive Toxicology 16 (2002) 795–800 Effects of nicotine and cotinine on bovine theca interna and granulosa cells Sara R. Sanders a , S. Peder Cuneo b , Adele M. Turzillo a,∗ a Department of Physiology, University of Arizona, PO Box 245051, Tucson, AZ 85724-4347, USA b Department of Veterinary Science and Microbiology, University of Arizona, Tucson, AZ 85724-4347, USA Received 10 April 2002; received in revised form 6 May 2002; accepted 11 June 2002 Abstract The purpose of this study was to determine if nicotine or cotinine inhibits steroidogenesis in the ovarian follicle. Theca interna and granu- losa cells were isolated from bovine follicles, cultured with nicotine or cotinine for 24 h, and culture media were assayed for androstenedione or estradiol. Treatment of theca interna with 6, 60, and 600 M nicotine decreased (P ≤ 0.002) production of androstenedione to 55, 53, and 24% of control levels, respectively. Levels of androstenedione in theca interna treated with cotinine were not different from control values. In granulosa cells, nicotine inhibited production of estradiol at the highest dose tested. Treatment with 600 M nicotine decreased (P ≤ 0.001) estradiol concentration to 12% of control values, attributable to a general cytotoxic effect. Cotinine had no effect on estradiol production by granulosa cells. These results provide novel evidence for inhibitory effects of nicotine on androgen production by theca interna. © 2002 Elsevier Science Inc. All rights reserved. Keywords: Nicotine; Cotinine; Granulosa cells; Theca cells; Ovary; Steroidogenesis; Estradiol; Androstenedione 1. Introduction Cigarette smoke is a complex mixture of toxic chemicals including nicotine, carbon monoxide, and several recognized carcinogens and mutagens [1]. These toxicants are absorbed through the pulmonary vasculature and transported via the bloodstream [2], causing cytotoxicity, genotoxicity, and tu- morigenicity throughout the body [3]. Nicotine is metabo- lized primarily by the liver, and to a lesser extent, the lung and kidney [4], with the primary metabolite being cotinine [5–7]. In addition to the deleterious effects on cardiovascular and pulmonary physiology, cigarette smoking affects the re- productive system and imposes a number of unique risks specific to women, both pre- and postmenopause. In pre- menopausal women, smoking is associated with infertility [2,8,9], ectopic pregnancy [10–12], spontaneous abortion [13], menstrual abnormalities [8,2,14], and early onset of menopause [8,15,16]. Interestingly, women who smoke have a decreased risk of breast cancer [17] and endometrial can- cer [18] but an increased risk of osteoporosis [19]. Because these phenomena are all estrogen dependent, it has been sug- gested that smoking has antiestrogenic effects [20,21]. In ∗ Corresponding author. Tel.: +1-520-626-4347; fax: +1-520-626-2382. E-mail address: turzillo@u.arizona.edu (A.M. Turzillo). fact, female smokers had significantly lower levels of estriol, estradiol, and estrone during the luteal phase of menstrual cycles and tended to have lower levels of these estrogens during the follicular phase compared to nonsmokers [20]. One possible mechanism underlying the antiestrogenic effects of smoking is inhibition of ovarian estrogen pro- duction by one or more chemical constituents of cigarette smoke. Treatment of rats with nicotine was associated with a decrease in estrogen-dependent parameters including uter- ine weight and diameter and thickness of the myometrium and endometrium [22]. In cultured human granulosa cells, aqueous extracts of cigarette smoke or nicotine inhibited aromatase activity by 50% [23]. In contrast, other studies have shown either a stimulation [24,25] or no effect [26] of nicotine on estradiol production by cultured human granu- losa cells. Clearly, additional investigations are needed to clarify nicotine’s effects on follicular steroidogenesis. In women and most other mammals, ovarian production of estradiol occurs in a cooperative fashion between granu- losa and theca interna cells. Within the theca interna, choles- terol is enzymatically converted to progesterone, which in turn is converted to androstenedione. Androstenedione is transported across the follicular basement membrane to the granulosa cells where it is enzymatically converted to testosterone. Testosterone is then aromatized to form estradiol-17 (estradiol). Granulosa cells do not express the 0890-6238/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII:S0890-6238(02)00049-7