Evaluation of radioisotopic and non-radioisotopic versions of local lymph node assays for subcategorization of skin sensitizers compliant to UN GHS rev 4.

Recently UN GHS has introduced the sub-categorization of skin sensitizers for which ECt (concentration estimated to induce stimulation index above threshold) of the murine local lymph node assay (LLNA) is used as criteria. Non-radioisotopic variants of LLNA, LLNA: DA, LLNA: BrdU-ELISA, LNCC and LLNA: BrdU-FCM were developed yet their utilities for potency sub-categorization are not established. Here we assessed the agreement of LLNA variants with LLNA or human data in potency sub-categorization for 22 reference substances of OECD TG429. Concordance of sub-categorization with LLNA was highest for LLNA: BrdU-FCM(91%, κ = 0.833, weighted kappa) followed by LLNA: BrdU-ELISA (82%, κ = 0.744) and LLNA: DA (73%, κ = 0.656) whereas LNCC only showed a modest association (64%, κ = 0.441). With human data, LLNA agreed best (77%) followed by LLNA: DA and LLNA: BrdU-FCM(73%), LLNA: BrdU-ELISA (68%) and LNCC(55%). Bland-Altman plot revealed that ECt's of LLNA variants largely agreed with LLNA where most values fell within 95% limit of agreement. Correlation between ECt's of LLNA and LLNA variants were high except for LNCC(pair-wise with LLNA, LLNA: DA, r = 0.848, LLNA: BrdU-ELISA, r = 0.744, LLNA: BrdU-FCM, r=0.786, and LNCC, r = 0.561 by Pearson). Collectively, these results demonstrated that LLNA variants exhibit performance comparable to LLNA in the potency sub-categorization although additional substances shall be analyzed in the future.

Comparison of BALB/c and CBA/J mice for the local lymph node assay using bromodeoxyuridine with flow cytometry (LLNA: BrdU-FCM).

The local lymph node assay using 5-bromo-2-deoxyuridine (BrdU) with flow cytometry (LLNA: BrdU-FCM) is a modified LLNA that is used to identify skin sensitizers by counting BrdU-incorporated lymph node cells (LNCs) with flow cytometry. Unlike other LLNA methods (OECD TG 429, 442A and 442B) in which the CBA/J mouse strain is used, LLNA: BrdU-FCM was originally designed to be compatible with BALB/c, a mouse strain that is more widely used in many countries. To justify the substitution of CBA/J for BALB/c, the equivalence of the test results between two strains shall be established prior to the official implementation of LLNA: BrdU-FCM. This study aims to compare the test results of LLNA: BrdU-FCM produced in BALB/c mice with those in CBA/J mice for 18 reference substances, including 13 sensitizers and 5 non-sensitizers, listed in OECD Test Guideline 429. Based on the LLNA: BrdU-FCM test procedure, we selected an appropriate solvent and then performed preliminary tests to determine the non-irritating dose ranges for the main study, which revealed the difference in the irritation responses to 8 of the 18 chemicals between the two strains. In the main study, we measured the changes in the number of total LNCs, which indicated differences in the responses to test chemicals between the two strains. However, the stimulation index obtained with the counts of BrdU-incorporated LNCs with 7-AAD using flow cytometry yielded comparable results and 100% concordance between the BALB/c and CBA/J mouse strains was achieved, suggesting that the performance of LLNA: BrdU-FCM using BALB/c mice was equivalent to that with CBA/J mice.

Performance standard-based validation study for local lymph node assay: 5-bromo-2-deoxyuridine-flow cytometry method.

Local lymph node assay: 5-bromo-2-deoxyuridine-flow cytometry method (LLNA: BrdU-FCM) is a modified non-radioisotopic technique with the additional advantages of accommodating multiple endpoints with the introduction of FCM, and refinement and reduction of animal use by using a sophisticated prescreening scheme. Reliability and accuracy of the LLNA: BrdU-FCM was determined according to OECD Test Guideline (TG) No. 429 (Skin Sensitization: Local Lymph Node Assay) performance standards (PS), with the participation of four laboratories. Transferability was demonstrated through successfully producing stimulation index (SI) values for 25% hexyl cinnamic aldehyde (HCA) consistently greater than 3, a predetermined threshold, by all participating laboratories. Within- and between-laboratory reproducibility was shown using HCA and 2,4-dinitrochlorobenzene, in which EC2.7 values (the estimated concentrations eliciting an SI of 2.7, the threshold for LLNA: BrdU-FCM) fell consistently within the acceptance ranges, 0.025-0.1% and 5-20%, respectively. Predictive capacity was tested using the final protocol version 1.3 for the 18 reference chemicals listed in OECD TG 429, of which results showed 84.6% sensitivity, 100% specificity, and 88.9% accuracy compared with the original LLNA. The data presented are considered to meet the performance criteria for the PS, and its predictive capacity was also sufficiently validated.

Surface functionalization-specific binding of coagulation factors by zinc oxide nanoparticles delays coagulation time and reduces thrombin generation potential in vitro.

Zinc oxide nanoparticles (ZnO NPs) have many biomedical applications such as chemotherapy agents, vaccine adjuvants, and biosensors but its hemocompatibility is still poorly understood, especially in the event of direct contact of NPs with blood components. Here, we investigated the impact of size and surface functional groups on the platelet homeostasis. ZnO NPs were synthesized in two different sizes (20 and 100 nm) and with three different functional surface groups (pristine, citrate, and L-serine). ZnO NPs were incubated with plasma collected from healthy rats to evaluate the coagulation time, kinetics of thrombin generation, and profile of levels of coagulation factors in the supernatant and coronated onto the ZnO NPs. Measurements of plasma coagulation time showed that all types of ZnO NPs prolonged both active partial thromboplastin time and prothrombin time in a dose-dependent manner but there was no size- or surface functionalization-specific pattern. The kinetics data of thrombin generation showed that ZnO NPs reduced the thrombin generation potential with functionalization-specificity in the order of pristine > citrate > L-serine but there was no size-specificity. The profile of levels of coagulation factors in the supernatant and coronated onto the ZnO NPs after incubation of platelet-poor plasma with ZnO NPs showed that ZnO NPs reduced the levels of coagulation factors in the supernatant with functionalization-specificity. Interestingly, the pattern of coagulation factors in the supernatant was consistent with the levels of coagulation factors adsorbed onto the NPs, which might imply that ZnO NPs simply adsorb coagulation factors rather than stimulating these factors. The reduced levels of coagulation factors in the supernatant were consistent with the delayed coagulation time and reduced potential for thrombin generation, which imply that the adsorbed coagulation factors are not functional.

Stouffer's test in a large scale simultaneous hypothesis testing.

In microarray data analysis, we are often required to combine several dependent partial test results. To overcome this, many suggestions have been made in previous literature; Tippett's test and Fisher's omnibus test are most popular. Both tests have known null distributions when the partial tests are independent. However, for dependent tests, their (even, asymptotic) null distributions are unknown and additional numerical procedures are required. In this paper, we revisited Stouffer's test based on z-scores and showed its advantage over the two aforementioned methods in the analysis of large-scale microarray data. The combined statistic in Stouffer's test has a normal distribution with mean 0 from the normality of the z-scores. Its variance can be estimated from the scores of genes in the experiment without an additional numerical procedure. We numerically compared the errors of Stouffer's test and the two p-value based methods, Tippett's test and Fisher's omnibus test. We also analyzed our microarray data to find differentially expressed genes by non-genotoxic and genotoxic carcinogen compounds. Both numerical study and the real application showed that Stouffer's test performed better than Tippett's method and Fisher's omnibus method with additional permutation steps.

Distinguishing between genotoxic and non-genotoxic hepatocarcinogens by gene expression profiling and bioinformatic pathway analysis.

A rapid and sensitive method to determine the characteristics of carcinogens is needed. In this study, we used a microarray-based genomics approach, with a short-term in vivo model, in combination with insights from statistical and mechanistic analyses to determine the characteristics of carcinogens. Carcinogens were evaluated based on the different mechanisms involved in the responses to genotoxic carcinogens and non-genotoxic carcinogens. Gene profiling was performed at two time points after treatment with six training and four test carcinogens. We mapped the DEG (differentially expressed gene)-related pathways to analyze cellular processes, and we discovered significant mechanisms that involve critical cellular components. Classification results were further supported by Comet and Micronucleus assays. Mechanistic studies based on gene expression profiling enhanced our understanding of the characteristics of different carcinogens. Moreover, the efficiency of this study was demonstrated by the short-term nature of the animal experiments that were conducted.

B cell increases and ex vivo IL-2 production as secondary endpoints for the detection of sensitizers in non-radioisotopic local lymph node assay using flow cytometry.

Non-radioisotopic local lymph node assay (LLNA) using 5-bromo-2'-deoxyuridine (BrdU) with flow cytometry (FCM) is gaining attention since it is free from the regulatory issues in traditional LLNA (tLLNA) accompanying in vivo uses of radioisotope, (3)H-thymidine. However, there is also concern over compromised performance of non-radioisotopic LLNA, raising needs for additional endpoints to improve the accuracy. With the full 22 reference substances enlisted in OECD Test Guideline No. 429, we evaluated the performance of LLNA:BrdU-FCM along with the concomitant measurements of B/T cell ratio and ex vivo cytokine production from isolated lymph node cells (LNCs) to examine the utility of these markers as secondary endpoints. Mice (Balb/c, female) were topically treated with substances on both ears for 3 days and then, BrdU was intraperitoneally injected on day 5. After a day, lymph nodes were isolated and undergone FCM to determine BrdU incorporation and B/T cell sub-typing with B220+ and CD3e+. Ex vivo cytokine production by LNCs was measured such as IL-2, IL-4, IL-6, IL-12, IFN-γ, MCP-1, GM-CSF and TNFα. Mice treated with sensitizers showed preferential increases in B cell population and the selective production of IL-2, which matched well with the increases in BrdU incorporation. When compared with guinea pig or human data, BrdU incorporation, B cell increase and IL-2 production ex vivo could successfully identify sensitizers with the accuracy comparable to tLLNA, suggesting that these markers may be useful for improving the accuracy of LLNA:BrdU-FCM or as stand-alone non-radioisotopic endpoints.