The antimicrobial agents triclosan (TCS;2,4,4´-trichloro-2´-hydroxydiphenyl ether)and triclocarban (TCC; 3,4,4´-trichloro-carbanilide) are high-production-volumechemicals that are widely used as “valueadded” chemicals in personal care products. Ina limited retail survey, approximately 45% ofliquid and bar soaps on the market containedthese antimicrobials; TCS and TCC were thepredominant antimicrobials in liquid soapsand bar soaps, respectively (Perencevich et al.2001). According to the U.S. EnvironmentalProtection Agency (2007), U.S. consumersspend nearly $1 billion/year on these products.TCS is a broad-spectrum bacteriostatic germi-cide that is now used in consumer productssuch as liquid hand soap, toothpaste, mouthrinse, cosmetics, pharmaceutical products, fab-rics, plastics, textiles, and plastic kitchenwaresince its original introduction as an activeingredient in a surgical scrub for professionalhealth care in 1972 (Tierno 1999). It is a pow-erful antibacterial agent that inhibits the activ-ity of the enzyme enoyl-acyl carrier-proteinreductase, which catalyzes an essential step inmembranes of many bacteria and fungi (Heathet al. 1999; McMurry et al. 1998b). TCC,another antimicrobial agent, is more oftenadded to consumer bar soaps and deodorants,and it is active predominantly against gram-positive bacteria (McDonnell and Russell1999). The carbanilide analog 3-trifluo-romethyl-4,4´-dichlorocarbanilide (TFC) isalso used as an antibacterial agent (Jeffcoatet al. 1977).Although these compounds are broadlyclassified as halogenated aromatic hydro-carbons, TCS has functional moieties represen-tative of phenols, diphenyl ethers, andpolychlorinated biphenyls (PCBs), whereasTCC is structurally related to carbanilide com-pounds, including some drugs and pesticides,and sterically and electronically related to avariety of other chemicals (Figure 1). BothTCS and TCC have been detected at micro-gram per liter levels in waterways in the UnitedStates and Switzerland, indicating extensivecontamination of aquatic ecosystems (Haldenand Paull 2005; Kolpin et al. 2002; Lindstromet al. 2002). The potential of these compoundsfor bioaccumulation has raised public concernregarding their possible effects on humanhealth (Coogan et al. 2007; Darbre 2006;Daughton and Ternes 1999) and microbialresistance (McMurry et al. 1998a). Recentreports note that TCS levels as high as2,000 μg/kg lipid have been detected in humanbreast milk (Dayan 2007), and concentrationsin human fl uids such as plasma and milk arepositively correlated to levels of exposure(Adolfsson-Erici et al. 2002; Allmyr et al. 2006;Dayan 2007; Hovander et al. 2002).Because of these concerns, we screenedTCS, TCC, and a series of TCC analogs forbiological activity in several mechanisticallyderived cell-based assay systems. Mammalianligand-dependent nuclear receptors serve asbiomarkers that evaluate the potential of anenvironmental toxicant to affect endocrineand non–endocrine-signaling systems. Oneset of assays used in the present study is basedon the chemically activated luciferase geneexpression (CALUX) bioassays. The recombi-nant cells used in the CALUX bioassaysinclude a stably transfected aryl hydrocarbonreceptor (AhR)-, androgen receptor (AR)-, orestrogen receptor (ER)-responsive fireflyluciferase reporter gene that responds tochemicals that can bind to and/or activate therespective receptor, leading to the inductionof luciferase reporter gene expression.Environmental Health Perspectives • VOLUME 116 | NUMBER 9 | September 2008 1203ResearchAddress correspondence to B.D. Hammock,Department of Entomology, University ofCalifornia, Davis, Davis, CA 95616 USA.Telephone: (530) 752-7519. Fax: (530) 752-1537.E-mail: bdhammock@ucdavis.eduWe thank T.A. Ta for some of the [ 3 H]Ry bindinganalysis.The Research Translation Core of the University ofCalifornia, Davis Superfund Basic Research Programwas instrumental in coordinating the studies. This research was supported by the NationalInstitute of Environmental Health Sciences (NIEHS)Superfund Basic Research Program (5P42 ES04699);NIEHS Center for Environmental Health Sciences(P30 ES05707); NIEHS grant R37 ES02710; theUniversity of California, Davis Center for Children’sEnvironmental Health and Disease Prevention (1PO1ES11269); the U.S. Environmental ProtectionAgency through the Science to Achieve Results(STAR) program (R829388); and National Instituteof Occupational Safety and Health Center forAgricultural Disease and Research, Education andPrevention grant 1 U50 OH07550. The authors declare they have no competingf i nancial interests.Received 20 December 2007; accepted 15 May 2008.In Vitro Biologic Activities of the Antimicrobials Triclocarban, Its Analogs,and Triclosan in Bioassay Screens: Receptor-Based Bioassay ScreensKi Chang Ahn, 1 Bin Zhao, 2 Jiangang Chen, 3 Gennady Cherednichenko, 4 Enio Sanmarti, 5 Michael S. Denison, 2Bill Lasley, 3 Isaac N. Pessah, 4 Dietmar Kültz, 5 Daniel P.Y. Chang, 6 Shirley J. Gee, 1 and Bruce D. Hammock 11 Department of Entomology and Cancer Research Center, 2 Department of Environmental Toxicology, 3 Center for Health andEnvironment, 4 Department of Molecular Biosciences and Center for Children’s Environmental Health and Disease Prevention,5 Department of Animal Science, and 6 Department of Civil and Environmental Engineering, University of California, Davis, Davis,California, USAB ACKGROUND : Concerns have been raised about the biological and toxicologic effects of the anti-microbials triclocarban (TCC) and triclosan (TCS) in personal care products. Few studies haveevaluated their biological activities in mammalian cells to assess their potential for adverse effects.O BJECTIVES : In this study, we assessed the activity of TCC, its analogs, and TCS in in vitronuclear-receptor–responsive and calcium signaling bioassays.M ATERIALS AND METHODS : We determined the biological activities of the compounds in in vitro,cell-based, and nuclear-receptor–responsive bioassays for receptors for aryl hydrocarbon (AhR),estrogen (ER), androgen (AR), and ryanodine (RyR1).R ESULTS : Some carbanilide compounds, including TCC (1–10 µM), enhanced estradiol (E 2 )-dependent or testosterone-dependent activation of ER- and AR-responsive gene expression up to2.5-fold but exhibited little or no agonistic activity alone. Some carbanilides and TCS exhibitedweak agonistic and/or antagonistic activity in the AhR-responsive bioassay. TCS exhibited antago-nistic activity in both ER- and AR-responsive bioassays. TCS (0.1–10 µM) signif i cantly enhancedthe binding of [ 3 H]ryanodine to RyR1 and caused elevation of resting cytosolic [Ca 2+ ] in primaryskeletal myotubes, but carbanilides had no effect.C ONCLUSIONS : Carbanilides, including TCC, enhanced hormone-dependent induction of ER- andAR-dependent gene expression but had little agonist activity, suggesting a new mechanism of action ofendocrine-disrupting compounds. TCS, structurally similar to noncoplanar ortho-substituted poly-chlorinated biphenyls, exhibited weak AhR activity but interacted with RyR1 and stimulated Ca 2+mobilization. These observations have potential implications for human and animal health. Furtherinvestigations are needed into the biological and toxicologic effects of TCC, its analogs, and TCS. K EY WORDS : androgen receptor, antimicrobial, aryl hydrocarbon receptor, bioactivity, carbanilideanalog, estrogen receptor, ryanodine receptor, sensitization, signal amplif i cation, triclocarban, tri-closan. Environ Health Perspect 116:1203–1210 (2008). doi:10.1289/ehp.11200 available viahttp://dx.doi.org/ [Online 16 May 2008]
