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Abstracts / Toxicology L

205-028valuation of cytokine production in auricular lymph nodes asn endpoint during murine contact allergy

. Karakaya, O.C. Ulker

Ankara University, Faculty of Pharmacy, Department of Toxicology,urkey

llergic contact dermatitis is a serious health problem. The murineocal lymph node assay (LLNA) has been developed to detecthemical allergens, and international validation studies have beenonducted. Although the validity of the LLNA, attention wasrawn to two disadvantages; radioactive in vivo measurementf lymph node cell proliferation (3H-thymidine labeling) andossibility of false positive results caused by non-specific cellctivation as a result of inflammatory processes in the skin (irri-ation). In the present study, we aimed to investigate the followingon-radioactive endpoints of auricular lymph node proliferativectivity; 5-bromo-2-deoxyuridine (BrdU) incorporation ex vivo andn vivo, IL-2, IFN-gamma, IL-4 and IL-5 production. 8–12 weeksld female BALB/c mice were treated by the topical application oftrong sensitizer 2,4-dinitrochlorobenzene (DNCB) in acetone:oliveil (4:1, v/v) (AOO) at the concentrations of %0.025, %0.05, %0.1,0.25. Ear thickness was also measured to determine the differenti-tion index (DI) which demonstrates the proportion of non-specificctivation due to irritating properties of test compound. At the con-entration of %0.05 stimulation index value was found 3 for DNCB.n vivo NR-LLNA and also ex vivo NR- LLNA results were good agree-

ent with previous radioactive LLNA data. We found significantncreases in Th1 cytokines (IL-2, IFN-gamma) at the concentrationf %0.05 DNCB and Th2 cytokines (IL-4, IL-5) at the concentration of0.1 DNCB. As the differentiation index was found bigger than one,

rritant effect was not detected for all applied concentrations. Theresent study reports that the use of these non-radioactive end-oints can be used to evaluate allergic contact dermatitis causedy chemicals in the laboratory where it can be difficult to handleadioisotopes.

oi:10.1016/j.toxlet.2010.03.679

205-029ffects of aflatoxin M1 and aflatoxin M2 on J774A.1 murineacrophage cell line

. Russo 1, G. Bianco 2, S. Marzocco 2, A. De Simone 1, G. Autore 2,. Severino 1

University of Naples Federico II, Italy, 2 University of Salerno, Italy

ntroduction: Aflatoxins (AF), contaminants of food and feed, areenotoxic, cancerogenic and immunotoxic; AFB1 represents theost potent hepatocancerogen for both humans and animals.FM1 and AFM2, hydroxylated metabolites of AFB1 and AFB2espectively, are excreted with milk. Despite several studies haveeen carried out about the immune effects of AFB1 (Meissonniert al., 2008; Hinton et al., 2003), only few studies have beenerformed about the effects of AFM1 on immune functions; noork is available regarding the immunotoxicity of AFM2. The aim

f the current study is to evaluate the immunotoxic effects ofoth AFM1 and AFM2, alone and in combination, on macrophagic

unctions.

Materials and methods: J774A.1 macrophages were culturednder standard conditions and exposed to serial dilutions of AFM1nd AFM2 (0.05–32 �M), alone and in combination, for 24, 48

196S (2010) S37–S351 S201

and 72 h. At first, we evaluated cell viability through MTT assay.Moreover, we also evaluated the activation of macrophagic func-tions measuring the amount of nitric oxide (NO) produced in cellsexposed to AFM1 and AFM2 (0.05–32 �M) and stimulated with LPS(1 �g/ml) for 24 h. Data were statistically examined.

Results and discussion: Both AFM1 and AFM2, alone or incombination, do not reduce significantly cell viability in J774A.1macrophages. Nevertheless, both AFM1 and AFM2 reduced(P < 0.01) the NO production in J774A.1 macrophages stimulatedwith LPS (1 �g/ml) at the highest concentration.

In conclusion, our results highlighted that AFM1 and AFM2 areimmunomodulating mycotoxins and their action exerts through areduction of NO production in macrophages stimulated with LPSwithout modify cellular viability.

Further investigations will be necessary in order to better clarifythe biological activity of AFM1 and AFM2 on macrophagic cells andother cellular models.

Reference

Meissonnier, et al., 2008. Toxicol. Appl. Pharm. 231, 142–149.Hinton, et al., 2003. Toxicol. Sci. 73 (2), 362–377.

doi:10.1016/j.toxlet.2010.03.680

P205-030CO-exposure to mercury accelerates autoimmunity induced bytrichloroethylene

K. Gilbert 1, B. Rowley 2, L. Hennings 3, S. Blossom 1

1 Arkansas Children’s Hospital Research Institute, United States,2 University of Central Arkansas, 3 University of Arkansas for MedicalSciences

Certain environmental toxicants are thought to help triggerautoimmunity in humans. Such toxicants include the industrialsolvent and persistent water pollutant trichloroethylene (TCE).We have shown that chronic (32-week) exposure to TCE inducedautoimmune hepatitis and CD4+ T cell activation in MRL+/+ mice.

In real-life individuals are rarely exposed to only one chemical.However, little is known about how chemical mixtures impact theimmune system. This study examined how co-exposure to anotherpersistent environmental pollutant, mercury, altered TCE-inducedautoimmunity. Female MRL+/+ mice were treated for only 8 weekswith TCE and/or mercuric chloride (HgCl2). TCE was administeredin the drinking water at levels of about 10 or 187 mg/kg/day (13%or 246% respectively of the current federal Permissible ExposureLimit). HgCl2 was administered twice weekly at 260 �g/kg sc.

Mice exposed to both toxicants for 8 weeks developed an earlystage of autoimmune hepatitis manifested by lymphocyte infil-tration into the liver. In contrast, mice exposed to TCE or HgCl2alone for 8 weeks showed no evidence of autoimmune disease. Themice were also examined for anti-liver antibodies. Similar to con-trol mice, mice exposed to HgCl2 alone generated little anti-liverantibodies. Exposure to TCE alone generated antibodies specificfor a 35 kDa liver protein. Interestingly, this antibody disappearedin mice exposed to both TCE and HgCl2. Instead the co-exposedmice generated antibodies specific for larger (between 75 and100 kDa) and smaller (30 kDa) liver proteins. In all cases the sera

reacted with liver tissue from either control or treated mice, sug-gesting that toxicant-induced reactivity was not hapten specific.Thus, the autoimmune-promoting capacity of TCE was enhanced oraccelerated by co-exposure to another immunotoxicant, mercury.In addition, the binary mixture generated a unique autoantibody