Affiliations of authors: Laboratories of Respiratory Biology (AKB, LMD, H-YC, CRW, SRK) and Experimental Pathology (DD), National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC; Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Health Sciences Center, Denver, CO (AMM)
Correspondence to: Alison K. Bauer, PhD, Laboratory of Respiratory Biology, National Institute of Environmental Health Sciences, National Institutes of Health, 111 T.W. Alexander Drive, Building 101, Rm. E214, Research Triangle Park, NC 27709 (e-mail: bauer1{at}niehs.nih.gov).
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ABSTRACT |
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Butylated hydroxytoluene (BHT) is not carcinogenic (10) and is metabolized in the lungs of mice to oxidative species that cause reversible lung injury and inflammation and promote lung tumorigenesis following protooncogene activation (1113). Promotion is dependent on inflammation elicited by BHT metabolites (14). C3H/HeJ (HeJ) mice have a dominant negative proline-to-histidine substitution (15) in Tlr4, whereas the coisogenic C3H/HeOuJ (OuJ) strain has functional Tlr4 (15). C.C3H-TlrLpsd (BALBLpsd ) mice are congenic for a 10 centimorgan region of HeJ chromosome 4 that contains Tlr4 and thus lack Tlr4 function compared with wild-type BALB/cJ (BALB) mice (16).
Two protocols were used in this study based on a previous study that demonstrated a correlation, but not a causal relationship, between BHT-induced inflammation and promotion (14). To study the effects of Tlr4 on inflammation (protocol 1), HeJ, OuJ, BALBLpsd, and BALB mice (68 weeks of age; Jackson Laboratories, Bar Harbor, ME) were injected intraperitoneally with BHT (Sigma, St. Louis, MO) once a week for 4 weeks (125150 mg/kg for the first dose, followed by 200 mg/kg for the next three doses). Mice were killed 1 day after the last injection to assess vascular leakage of proteins into bronchoalveolar lavage fluid and 3 days after BHT injection to assess leukocyte infiltration. The right lung was lavaged, total protein concentration was determined as an indicator of hyperpermeability, and leukocyte infiltration was estimated as described previously (17). To assess the role of Tlr4 in BHT-induced lung tumor promotion (protocol 2), mice were injected intraperitoneally with 10 µg 3-methylcholanthrene (MCA; Sigma)/gm of body weight or with corn oil vehicle. At this dose, MCA did not cause lung inflammation (data not shown). One week later, 150 mg of BHT/kg was administered intraperitoneally, followed by five weekly injections of 200 mg/kg each. The two additional BHT injections were given (compared with four in protocol 1) to maximize promotional efficacy; the number of tumors that arise is a linear function of the number of BHT injections (18). Mice were killed 20 weeks after the MCA injection, lungs were removed en bloc and fixed in Tellyesniczky's fixative (19) for 48 hours, and tumors were counted using a dissecting microscope. The National Institute of Environmental Health Sciences Animal Care and Use Committee approved all protocols. Differences between groups were tested by two-way analysis of variance and Tukey a posteriori comparisons of means (SigmaStat, Jandel Scientific Software, San Rafael, CA). All statistical tests were two-sided; P<.05 was considered statistically significant.
To identify the potential mechanisms by which functional Tlr4 inibits BHT-induced injury, we examined signaling elements myeloid differentiation factor 88 (MyD88), nuclear factor kappa B (NFB), and activator protein 1 (AP-1), all of which act downstream of TLR4 in HeJ and OuJ mice. Immunoblotting was performed using 100 µg of protein obtained from total lung homogenates, and blots were quantified (BioRad Quantity Image One Software; BioRad, Hercules, CA) as previously described (12). TLR4 (goat polyclonal; L14) and MyD88 (goat polyclonal; N-19) antibodies were obtained (Santa Cruz Biotechnology, Santa Cruz, CA), and nuclear protein was prepared according to the manufacturer's instructions (Nuclear Extraction Kit; Active Motif, Carlsbad, CA). To determine transcription factor DNA binding activity, 7 µg of nuclear protein was incubated with consensus sequences for either NF
B or AP-1, and specific activity was determined with p65 NF
B (rabbit polyclonal), phosphorylated (p)-c-jun, or c-fos (both rabbit polyclonal) antibodies by colorimetric analysis (TransAm NF
B and AP-1 kits; Active Motif).
Functional Tlr4 inhibited BHT-induced weight loss, inflammation, and MCA/BHT-induced tumor formation. Statistically significantly greater weight loss was observed in Tlr4-mutated HeJ and BALBLpsd mice compared with their respective Tlr4 normal controls (HeJ versus OuJ, difference = 6.7%, 95% confidence interval [CI] = 10.2% to 3.1%, P = .006; BALBLpsd versus BALB, difference = 7.9%, 95% CI = 11.2% to 4.6%, P<.002) (Fig. 1, A.) Survival rates were 100% for OuJ, BALB, and BALBLpsd mice and 62% for HeJ mice (95% CI = 39.0% to 85.0%; P = .032). In addition, bronchoalveolar lavage fluid from HeJ and BALBLpsd mice contained more protein, alveolar macrophages, and lymphocytes than that from OuJ and BALB mice, respectively (Fig. 1, B), suggesting a protective role for TLR4 in lung inflammation and permeability. Lung tumors did not develop in MCA/BHT-treated OuJ or HeJ mice because these strains contain a tumor resistance allele at the Kras locus (20). Tumor-sensitive BALB mice contain one copy of a 37-bp intronic sequence in Kras (21), whereas two copies are found in resistant strains, such as OuJ and HeJ (22). The results described herein confirm earlier findings in C3H mice (23). Tumor multiplicity increased 60% in BALBLpsd mice compared with that in BALB mice (means = 22.3 and 13.9 tumors/mouse, respectively; difference = 8.4 tumors/mouse, 95% CI = 4.6 to 12.1 tumors/mouse; P = .025) (Fig. 1, C), but no differences in overall tumor size or morphology were noted. Interestingly, increases in numbers of pulmonary lymphocytic aggregates were found in Tlr4-mutated mice compared with wild-type mice following MCA or MCA/BHT treatment, although the importance of these cellular aggregates remains unclear.
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NOTES |
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Manuscript received April 5, 2005; revised September 14, 2005; accepted September 29, 2005.
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