Toxicology Letters 180 (2008) 38–45 Contents lists available at ScienceDirect Toxicology Letters journal homepage: www.elsevier.com/locate/toxlet Mechanism of trifluralin-induced thyroid tumors in rats Shakil A. Saghir ∗ , Grantley D. Charles, Michael J. Bartels, Lynn H.L. Kan, Mark D. Dryzga, Kathy A. Brzak, Amy J. Clark Toxicology & Environmental Research & Consulting, The Dow Chemical Company, Midland, MI 48674, USA article info Article history: Received 15 April 2008 Received in revised form 27 May 2008 Accepted 27 May 2008 Available online 6 June 2008 Keywords: Trifluralin Dinitroanilines Thyroxine (T4) Triiodothyronine (T3) Thyroid stimulating hormone (TSH) Thyroid tumor in rats abstract Trifluralin, an herbicide, has been reported to cause a significant increase in thyroid follicular cell tumors in male Fischer 344 rats. This study was designed to determine the mechanism of thyroid hyperactivity after trifluralin exposure. A group of 15 male Fischer 344 rats were exposed to trifluralin-fortified (6500ppm) diet for 2 weeks. The time weighted average daily intake of trifluralin was 441 ± 77 mg/kg/day. Ten rats of the group were sacrificed and the sera analyzed for T3, T4, and TSH levels. The livers were also analyzed for selected T4-specific UGT gene expression and total UGT enzyme activity. In the trifluralin treated rats, the serum T3 and T4 levels decreased by 17% and 90%, respectively and TSH increased by 37% more than the control rats. Trifluralin-induced total hepatic UGT enzymes (2.4-fold) and mRNA expression of selected hepatic UGT isozymes (UGT1A1, 1.4-fold; UGT1A6, 6.4-fold; UGT2B1, 3.7-fold). For the remaining 5 rats in the group, bile was collected for 2h and analyzed for free and conjugated T3 and T4. The total amount of T4 in bile more than doubled in trifluralin treated rats. Trifluralin treatment increased bile flow, caused a 3.2-fold increase in biliary elimination of conjugated T4 and 63% increase in conjugated T3. Based on these data, the decrease in total serum T3 and T4 levels in the trifluralin treated rats was due to enhanced peripheral metabolism and an increase in bile flow that results in a compensatory increase in TSH synthesis and secretion. The increased levels of TSH with chronic exposure to trifluralin would exert a continuous stimulation of the thyroid gland leading to cellular hypertrophy and proliferation predisposing to the development of follicular cell tumors in rats. © 2008 Published by Elsevier Ireland Ltd. 1. Introduction Trifluralin is one of the 2,6-dinitroaniline herbicides used to control many germinating annual grass and broadleaf weeds. The dinitroaniline herbicides prevent cells in roots and shoots from dividing and developing (Grover et al., 1997). Some of the other members of the 2,6-dinitroaniline herbicide family include ben- fluralin, ethalfluralin, oryzalin and pendimethalin. Trifluralin has been reported to cause a significant increase in thyroid follicular cell tumors in male Fischer 344 rats only at the highest dietary dose of 6500 ppm in a 2-year chronic study (Emmerson et al., 1980). Other dinitroaniline herbicides have also been reported to cause thyroid tumors in rats (USEPA, 1997, 2003, 2004). There are no specific stud- ies investigating the mechanism by which trifluralin causes these thyroid cell tumors. ∗ Corresponding author at: Toxicology & Environmental Research & Consulting, The Dow Chemical Company, 1803 Building, Midland, MI 48674, USA Tel.: +1 989 636 8708; fax: +1 989 638 9863. E-mail address: ssaghir@dow.com (S.A. Saghir). Thyroid gland secretes thyroid hormones (TH) thyroxine (T4) and triiodothyronine (T3) in response to thyroid stimulating hor- mone (TSH) from the anterior pituitary gland, as illustrated in Fig. 1. Thyroid hormones bind strongly, but not covalently, to serum pro- tein and are critical in regulating normal growth, development and metabolism. Humans, primates and dogs possess a high-affinity TH-binding protein, thyroxine-binding globulin (TBG). Rats lack TBG; however, both humans and rats possess low affinity carrier proteins for TH, thyroxine-binding prealbumin (transthyretin) and albumin (Hill et al., 1989, 1998). Binding affinity of TH to albu- min and transthyretin is 3 and 5 orders of magnitude lower than TBG, respectively (McClain, 1992). The lack of TBG makes rats more susceptible to TH removal from blood through metabolism and excretion, which is evident from the shorter serum half-lives of T4 and T3 in rats. The serum half-lives of T4 and T3 in rats are 0.5–1 day and 0.25 day, respectively; whereas in humans, the half-lives are 5–9 days and 1 day, respectively (Choksi et al., 2003; Hill et al., 1998; McClain, 1992). This results in higher stimulation of the thyroid gland by TSH in rats, which is evident from the relatively small folli- cles in rat thyroid glands, often surrounded by cuboidal epithelium (McClain, 1992; Hill et al., 1998). Whereas, thyroid follicular cell in humans are large and less active with abundant colloid surrounded 0378-4274/$ – see front matter © 2008 Published by Elsevier Ireland Ltd. doi:10.1016/j.toxlet.2008.05.019