The nasal toxicity of the herbicide 2,6-dichlorobenzonitrile: Mechanisms and biomarkers.

摘要

The overall goal of this study is to investigate mechanisms of the potent nasal specific toxicity of the herbicide 2,6-dichlorobenzonitrile (DCBN) in rodents, and to determine whether DCBN could induce similar nasal toxicity in humans. The central hypotheses are (1) that the nasal specific toxicity of DCBN in rodents is mediated by its electrophilic intermediates that are formed through metabolic activation catalyzed by target tissue cytochrome P450 enzymes (P450) and can interfere with stem cell regeneration and differentiation in the olfactory epithelium (OE); and (2) that human nasal tissues are also capable of catalyzing bioactivation of DCBN. The specific aims are (1) to identify the mouse P450 enzyme(s) responsible for DCBN metabolic activation in vivo, and to determine whether hepatic P450-generated DCBN metabolites play a significant role in DCBN toxicity in the olfactory mucosa (OM); (2) to examine the capability of human nasal tissues to activate DCBN, and to identify potential biomarkers for monitoring DCBN exposure and nasal toxicity; and (3) to determine the role of inflammatory cytokines in the permanent loss of olfactory receptor neurons (ORNs) induced by DCBN in mice.;In the first aim, definitive evidence has been provided to conclude that CYP2A5, the most abundant P450 in mouse OM, plays an essential role in mediating DCBN toxicity in the OM, and that hepatic P450 enzymes, although essential for DCBN clearance, are not necessary for DCBN-induced OM toxicity. In the second aim, a sensitive LC-MS/MS method has been established for detection and quantification of the glutathione conjugate of DCBN (GS-DCBN) in biological matrices. Then it has been demonstrated that human fetal nasal mucosa microsomes catalyzed the formation of GS-DCBN, with a Km value comparable to that for GS-DCBN formation in adult mouse nasal microsomes. The involvement of CYP2A enzymes in this bioactivation in human nasal microsomes was suggested by the finding of an inhibition of the activity by 8-methoxyporsalen, a known CYP2A-selective inhibitor. Furthermore, GS-DCBN and its metabolites were detected in the nasal mucosa and nasal-wash fluid obtained from DCBN-exposed mice; the detected amounts of GS-DCBN and its metabolites increased, with increases in DCBN dose, and they were detectable at as early as 30 min after DCBN exposure. Two metabolites, the cysteine conjugate and the mercapturic acid, were still detectable at 24 hours after DCBN exposure. Thus, this study presented the first evidence that human nasal tissue is capable of in situ metabolic activation of DCBN, and that GS-DCBN (or its derivatives) in nasal-wash fluid may serve as biomarkers of DCBN exposure and potential nasal toxicity in humans.;In the third aim, we tested the hypothesis that the inflammatory cytokine interleukin-6 (IL-6) induced by DCBN treatment plays a critical role in preventing olfactory neural stem cell differentiation and OE regeneration. A significant increase of IL-6 levels in mouse nasal-wash fluid has been detected after DCBN injection. The extent of this increase was significantly higher than the increase induced by another olfactory toxicant, methimazole (MMZ), which does not cause permanent loss of ORNs. It is then demonstrated that subcutaneous administration of an anti-inflammatory agent, dexamethasone, can inhibit the acute increase of IL-6 induced by DCBN in mouse nasalwash fluid, without inhibiting DCBN-induced acute OE damages. However, the inhibition of the acute IL-6 increase did not prevent DCBN-induced eventual permanent loss of ORNs. Based on these findings, it is concluded that acute increase of IL-6 in the nasal tissue induced by DCBN is not critical in causing permanent loss of ORNs. Thus, the mechanisms underlying DCBN's ability to cause permanent loss of ORNs remain to be identified. In that regard, it is discovered that OE stem cells are detached and lost in the dorsal medial region of DCBN-treated mice. The regenerated basal cells, which appeared to differ from the original ones in morphology, failed to differentiate into mature ORNs.