A draining lymph node assay (DLNA) for assessing the sensitizing potential of proteins.

There is a need for a simple and predictive model to identify the respiratory sensitization potential of (novel) proteins. The present study examined the use of a mouse draining lymph node assay (DLNA) approach, employing several routes of exposure, as a possible starting point for assessing protein sensitization potential. Consistent with the experimental procedure for the standard local lymph node assay (LLNA), female BALB/c mice were dosed dermally (topical), intranasally (IN) or by oropharyngeal aspiration (OP) on days 1, 2 and 3, and proliferation in the relevant draining lymph nodes was measured on day 6. For each route, the auricular, superficial cervical and tracheobronchial lymph nodes (TBLN) were evaluated following treatment with Subtilisin Carlsberg (SUB; a potent sensitizer/allergen), ovalbumin (OVA; a potent food allergen), beta-lactoglobulin (BLG; a moderate food allergen), and keyhole limpet hemocyanin (KLH; a strong immunogen with no reports of respiratory sensitization). Initial studies with OVA indicated that dermal administration did not stimulate lymph node proliferation. Responses in the tracheobronchial lymph node were most dramatic (stimulation indices up to 100) and reproducible for both the IN and OP routes. In a comparative experiment, all proteins induced lymph node proliferation with a rank order potency of SUB>KLH>OVA>BLG. The influence of the endotoxin content on lymph node proliferation was determined to be minimal, and did not impact the rank order potency. Molecular characterization of the TBLN at an equipotent proliferative dose was conducted for select gene transcripts based on research examining chemical sensitizers. Expression profiles differed among the four proteins, but the relevance of these responses was not clear and they did not further discriminate their allergic potential. These data illustrate both the opportunities and challenges associated with the examination of the draining lymph node proliferative response to assess the allergenic potential of proteins.

Evaluation of a toxicogenomic approach to the local lymph node assay (LLNA).

Genomic technologies have the potential to enhance and complement existing toxicology endpoints; however, assessment of these approaches requires a systematic evaluation including a robust experimental design with genomic endpoints anchored to traditional toxicology endpoints. The present study was conducted to assess the sensitivity of genomic responses when compared with the traditional local lymph node assay (LLNA) endpoint of lymph node cell proliferation and to evaluate the responses for their ability to provide insights into mode of action. Female BALB/c mice were treated with the sensitizer trimellitic anhydride (TMA), following the standard LLNA dosing regimen, at doses of 0.1, 1, or 10% and traditional tritiated thymidine ((3)HTdR) incorporation and gene expression responses were monitored in the auricular lymph nodes. Additional mice dosed with either vehicle or 10% TMA and sacrificed on day 4 or 10, were also included to examine temporal effects on gene expression. Analysis of (3)HTdR incorporation revealed TMA-induced stimulation indices of 2.8, 22.9, and 61.0 relative to vehicle with an EC(3) of 0.11%. Examination of the dose-response gene expression responses identified 9, 833, and 2122 differentially expressed genes relative to vehicle for the 0.1, 1, and 10% TMA dose groups, respectively. Calculation of EC(3) values for differentially expressed genes did not identify a response that was more sensitive than the (3)HTdR value, although a number of genes displayed comparable sensitivity. Examination of temporal responses revealed 1760, 1870, and 953 differentially expressed genes at the 4-, 6-, and 10-day time points respectively. Functional analysis revealed many responses displayed dose- and time-specific induction patterns within the functional categories of cellular proliferation and immune response, including numerous immunoglobin genes which were highly induced at the day 10 time point. Overall, these experiments have systematically illustrated the potential utility of genomic endpoints to enhance the LLNA and support further exploration of this approach through examination of a more diverse array of chemicals.

Human sulphotransferases are involved in the activation of aristolochic acids and are expressed in renal target tissue.

Use of herbal preparations containing Aristolochia species has led to progressive nephropathy and urothelial cancer in humans. Analysis of DNA adducts formed in human target tissues and studies in animal models have pointed out a major role of the secondary plant metabolites, aristolochic acids, in these effects. Only a minority of the users of Aristolochia-containing products developed nephropathy and cancer, suggesting differences in individual susceptibility. Differences in metabolic activation and inactivation frequently affect the susceptibility towards chemicals. Others have shown that the activation of aristolochic acids to DNA-reactive and mutagenic metabolites requires reduction of their aryl nitro group. The biological activity of numerous nitro- and aminoarenes, after appropriate phase I metabolism, is strongly enhanced in the presence of acetyltransferases or sulphotransferases (SULTs). In the present study, we demonstrate that expression of human SULTs in bacterial and mammalian target cells reinforces the mutagenic activity of aristolochic acids. Using Salmonella typhimurium TA1538 as the recipient organism, we identified the expression of all 12 human SULT forms. SULT1A1 led to the strongest increase in the mutagenicity of aristolochic acids. Some activation was also observed with SULT1B1, but not with the remaining forms. The role of SULT1A1 in the activation of aristolochic acids was corroborated using S. typhimurium TA100- and Chinese hamster V79-derived target cells engineered for expression of human SULT1A1 when compared with control cells. Furthermore, pentachlorophenol, an inhibitor of SULT1A1, strongly reduced the mutagenic effect of aristolochic acids in V79-hCYP2E1-hSULT1A1 cells. Moreover, we demonstrate that SULT1A1 and SULT1B1 are expressed in human kidney using immunoblot analysis, but their levels are substantially lower than in liver. Finally, we discuss the possibility that reactive sulphuric acid conjugates produced in other tissues are transferred to kidney and ureter.