Applications of cytotoxicity assays and pre-lethal mechanistic assays for assessment of human hepatotoxicity potential

While drug toxicity (especially hepatotoxicity) is the most frequent reason cited for withdrawal of an approved drug, no simple solution exists to adequately predict such adverse events. Simple cytotoxicity assays in HepG2 cells are relatively insensitive to human hepatotoxic drugs in a retrospective analysis of marketed pharmaceuticals. In comparison, a panel of pre-lethal mechanistic cellular assays hold the promise to deliver a more sensitive approach to detect endpoint-specific drug toxicities. The panel of assays covered by this review includes steatosis, cholestasis, phospholipidosis, reactive intermediates, mitochondria membrane function, oxidative stress, and drug interactions. In addition, the use of metabolically competent cells or the introduction of major human hepatocytes in these in vitro studies allow a more complete picture of potential drug side effect. Since inter-individual therapeutic index (TI) may differ from patient to patient, the rational use of one or more of these cellular assay and targeted in vivo exposure data may allow pharmaceutical scientists to select drug candidates with a higher TI potential in the drug discovery phase.     

Popliteal lymph node (PLN) assay to study adjuvant effects on respiratory allergy

Different variants of the popliteal lymph node (PLN) assay have been published. Here we describe the adjuvant popliteal lymph node assay, an immune response assay to study the adjuvant activity of soluble substances as well as particulate matter. The substance to be studied for adjuvant activity is injected into the hind footpad of mice or rats together with an antigen. Adjuvant activity is determined as the increase in PLN weight and cell numbers in animals receiving antigen together with the substance under study, compared with PLN weight and cell numbers in animals given the antigen without the substance in question, and animals given the putative adjuvant alone. Because lymph node weight and cell numbers are immunologically non-specific parameters, specific immune response assays like serum antibody responses or antibody-forming cell numbers should additionally be performed. Different antigens and immune response assays may be used, depending on the research question asked. In relation to respiratory (or food) allergy, the assays should as a minimum include determination of specific IgE in serum, and preferably also IgG1 (mouse). Serum specific IgG2a antibody determination may be added to get an indication of the Th1-Th2-balance of the response. The adjuvant PLN assay, with cellular response assays performed in the draining popliteal lymph node and antibody determinations in serum, requires small amounts of test material. The assay offers a practical, sensitive and reproducible method to determine the adjuvant activity of soluble substances as well as particulate material, with the possibility to also perform mechanistic studies.     

Contact sensitization: A new approach to risk assessment

The murine local lymph node assay is a novel predictive test for the identification of skin sensitizing chemicals. The method measures sensitization potential of a chemical in mice as a function of proliferative activity induced in lymph nodes draining the site of topical exposure to that test chemical. Here we describe the use of the local lymph node assay for evaluation of the relative potency of skin sensitizing chemicals via derivation of the concentration required to produce a threshold positive reaction. Subsequently, the development of risk assessments based on comparisons with index contact allergens is outlined.     

Systematic analysis of genes involved in oral cancer metastasis to lymph nodes

Oral cancer remains a deadly disease worldwide. Lymph node metastasis and invasion is one of the causes of death from oral cancer. Elucidating the mechanism of oral cancer lymph node metastasis and identifying critical regulatory genes are important for the treatment of this disease. This study aimed to identify differentially expressed genes (gene signature) and pathways that contribute to oral cancer metastasis to lymph nodes. The GSE70604-associated study compared gene profiles in lymph nodes with metastasis of oral cancer to those of normal lymph nodes. The GSE2280-associated study compared gene profiles in primary tumor of oral cancer with lymph node metastasis to those in tumors without lymph node metastasis. There are 28 common differentially expressed genes (DEGs) showing consistent changes in both datasets in overlapping analysis. GO biological process and KEGG pathway analysis of these 28 DEGs identified the gene signature CCND1, JUN and SPP1, which are categorized as key regulatory genes involved in the focal adhesion pathway. Silencing expression of CCND1, JUN and SPP1 in the human oral cancer cell line OECM-1 confirmed that those genes play essential roles in oral cancer cell invasion. Analysis of clinical samples of oral cancer found a strong correlation of these genes with short survival, especially JUN expression associated with metastasis. Our study identified a unique gene signature – CCND1, JUN and SPP1 – which may be involved in oral cancer lymph node metastasis.     

Changes in homing receptor expression on murine lymphokine-activated killer cells during IL-2 exposure

The effects of IL-2 on the expression of homing receptors by lymphocytes of NK or lymphokine activated killer (LAK) cell derivation has not yet been evaluated. We developed a murine model to evaluate the potential of LAK cells to localize into peripheral lymph nodes since LAK cells are used to treat human cancers which have metastasized to these tissues. Using a frozen section binding assay, LAK cell adhesion to the lymph node microvasculature was easily demonstrable. Inhibition studies demonstrated that LAK cell binding to lymph nodes was mediated by mechanisms previously described in T cells. LAK cell surface expression of the 85- to 95-kDa homing receptor recognized by the antibody MEL-14 on LAK cells was assessed by indirect immunofluorescence. The percentage of cells which bound MEL-14 decreased slightly over 3 days of IL-2 exposure (from 73 to 60%), particularly in the large granular lymphocyte (cytotoxic effector) subpopulation (45% MEL-14+). The expression of another homing-related molecule, leukocyte function-associated Ag-1, markedly increased during activation of LAK cells. Despite the expression of these homing receptors, we observed almost no LAK cell localization into lymph nodes in vivo. Furthermore, IL-2 pretreatment of recipient animals did not increase the adhesion of LAK cells to lymph node microvasculature or enhance their extravasation. IL-2 activation of non-T, non-B lymphocytes results in significant changes in both the expression and function of cell surface homing receptors. Our results indicate that in vitro analysis does not always predict in vivo localization potential.     

Reproductive and developmental toxicity of formaldehyde: a systematic review

Formaldehyde, the recently classified carcinogen and ubiquitous environmental contaminant, has long been suspected of causing adverse reproductive and developmental effects, but previous reviews were inconclusive, due in part, to limitations in the design of many of the human population studies. In the current review, we systematically evaluated evidence of an association between formaldehyde exposure and adverse reproductive and developmental effects, in human populations and in vivo animal studies, in the peer-reviewed literature. The mostly retrospective human studies provided evidence of an association of maternal exposure with adverse reproductive and developmental effects. Further assessment of this association by meta-analysis revealed an increased risk of spontaneous abortion (1.76, 95% CI 1.20-2.59, p=0.002) and of all adverse pregnancy outcomes combined (1.54, 95% CI 1.27-1.88, p<0.001), in formaldehyde-exposed women, although differential recall, selection bias, or confounding cannot be ruled out. Evaluation of the animal studies including all routes of exposure, doses and dosing regimens studied, suggested positive associations between formaldehyde exposure and reproductive toxicity, mostly in males. Potential mechanisms underlying formaldehyde-induced reproductive and developmental toxicities, including chromosome and DNA damage (genotoxicity), oxidative stress, altered level and/or function of enzymes, hormones and proteins, apoptosis, toxicogenomic and epigenomic effects (such as DNA methylation), were identified. To clarify these associations, well-designed molecular epidemiologic studies, that include quantitative exposure assessment and diminish confounding factors, should examine both reproductive and developmental outcomes associated with exposure in males and females. Together with mechanistic and animal studies, this will allow us to better understand the systemic effect of formaldehyde exposure.     

DNA intercalative potential of marketed drugs testing positive in in vitro cytogenetics assays

We have previously noted that the Physicians' Desk Reference (PDR) contains over 80 instances in which a drug elicited a positive genotoxic response in one or more in vitro assays, despite having no obvious structural features predictive of covalent drug/DNA interactive potential or known mechanistic basis. Furthermore, in most cases, these drugs were "missed" by computational genotoxicity-predicting models such as DEREK, MCASE and TOPKAT. We have previously reported the application of a V79 cell-based model and a 3D DNA docking model for predicting non-covalent chemical/DNA interactions. Those studies suggested that molecules that are very widely structurally diverse may be capable of intercalating into DNA. To determine whether such non-covalent drug/DNA interactions might be involved in unexpected drug genotoxicity, we evaluated, using both models where possible, 56 marketed pharmaceuticals, 40 of which were reported as being clastogenic in in vitro cytogenetics assays (chromosome aberrations/mouse lymphoma assay). As seen before, the two approaches showed good concordance (62%) and 26 of the 40 (65%) drugs exhibiting in vitro clastogenicity were predicted as intercalators by one or both methods. This finding provides support for the hypothesis that non-covalent DNA interaction may be a common mechanism of clastogenicity for many drugs having no obvious structural alerts for covalent DNA interaction.     

Applicability of Preoperative Nuclear Morphometry to Evaluating Risk for Cervical Lymph Node Metastasis in Oral Squamous Cell Carcinoma

Background

We previously reported the utility of preoperative nuclear morphometry for evaluating risk for cervical lymph node metastases in tongue squamous cell carcinoma. The risk for lymph node metastasis in oral squamous cell carcinoma, however, is known to differ depending on the anatomical site of the primary tumor, such as the tongue, gingiva, mouth floor, and buccal mucosa. In this study, we evaluated the applicability of this morphometric technique to evaluating the risk for cervical lymph node metastasis in oral squamous cell carcinoma.

Methods

A digital image system was used to measure the mean nuclear area, mean nuclear perimeter, nuclear circular rate, ratio of nuclear length to width (aspect ratio), and nuclear area coefficient of variation (NACV). Relationships between these parameters and nodal status were evaluated by t-test and logistic regression analysis.

Results

Eighty-eight cases of squamous cell carcinoma (52 of the tongue, 25 of the gingiva, 4 of the buccal mucosa, and 7 of the mouth floor) were included: 46 with positive node classification and 42 with negative node classification. Nuclear area and perimeter were significantly larger in node-positive cases than in node-negative cases; however, there were no significant differences in circular rate, aspect ratio, or NACV. We derived two risk models based on the results of multivariate analysis: Model 1, which identified age and mean nuclear area and Model 2, which identified age and mean nuclear perimeter. It should be noted that primary tumor site was not associated the pN-positive status. There were no significant differences in pathological nodal status by aspect ratio, NACV, or primary tumor site.

Conclusion

Our method of preoperative nuclear morphometry may contribute valuable information to evaluations of the risk for lymph node metastasis in oral squamous cell carcinoma.     

A neuronal cell-based botulinum neurotoxin assay for highly sensitive and specific detection of neutralizing serum antibodies

Clostridium botulinum neurotoxin (BoNT) serotypes A and B are widely used as pharmaceuticals to treat various neurological disorders and in cosmetic applications. The major adverse effect of these treatments has been resistance to treatment after multiple injections. Currently, patients receiving BoNT therapies and patients enrolled in clinical trials for new applications and/or new formulations of BoNTs are not routinely monitored for the formation of neutralizing antibodies, since no assay other than the mouse protection procedure is commercially available that reliably tests for the presence of such antibodies. This report presents a highly sensitive and specific neuronal cell-based assay that provides sensitive and specific detection of neutralizing antibodies to BoNT/A.     

口腔癌中肿瘤出芽与VEGF—C表达及淋巴结转移的相关关系

目的：通过检测肿瘤出芽、淋巴结转移以及血管内皮生长因子-C（VEGF—C）表达水平,分析口腔癌中肿瘤出芽与VEGF—C表达及淋巴结转移的相关关系,为临床治疗提供理论参考。方法：选取2009年1月-2013年1月4年间在我院接受诊治且资料完整63例口腔癌患者作为研究对象,观察肿瘤出芽、VEGF-C表达和淋巴结转移情况,分析相互之间的相关关系。结果：本次纳入研究的患者中,检出肿瘤出芽患者40例,所占比例为63．5％,VEGF—C表达阳性患者39例,阳性率率为61．9％,淋巴结转移患者40例,转移率为63．5％;肿瘤出芽与淋巴结转移的符合率为84．1％,肿瘤出芽与VEGF—C的表达符合率为79．4％,VEGF-C的表达与淋巴结转移发生的符合率为76．2％。肿瘤出芽与淋巴结转移呈正相关,经Spear相关分析,r=0．932,P〈0．05,与VEGF-C的表达也呈正相关,经Spear相关分析,r=0．897,P〈0．05。结论：肿瘤出芽与VEGF—C的表达水平和淋巴结转移均呈正相关关系,可用于预测判断口腔癌淋巴结转移情况。     

Xenotransplantation Models to Study the Effects of Toxicants on Human Fetal Tissues

Many diseases that manifest throughout the lifetime are influenced by factors affecting fetal development. Fetal exposure to xenobiotics, in particular, may influence the development of adult diseases. Established animal models provide systems for characterizing both developmental biology and developmental toxicology. However, animal model systems do not allow researchers to assess the mechanistic effects of toxicants on developing human tissue. Human fetal tissue xenotransplantation models have recently been implemented to provide human‐relevant mechanistic data on the many tissue‐level functions that may be affected by fetal exposure to toxicants. This review describes the development of human fetal tissue xenotransplant models for testis, prostate, lung, liver, and adipose tissue, aimed at studying the effects of xenobiotics on tissue development, including implications for testicular dysgenesis, prostate disease, lung disease, and metabolic syndrome. The mechanistic data obtained from these models can complement data from epidemiology, traditional animal models, and in vitro studies to quantify the risks of toxicant exposures during human development     

Image processing algorithms to facilitate and enhance sentinel node detection using a hand-held gamma ray camera in surgical breast cancer staging

We have developed a miniature scintillation camera to be used in surgical cancer staging. The availability of such a compact hand-held gamma camera may in certain cases improve localization of the sentinel lymph node and reduce the duration of a surgical breast cancer staging procedure. We have investigated image processing algorithms applied to planar images that may improve node detection capabilities for breast cancer staging. We have also studied contrast enhancement methods that may be able to identify nodes that would otherwise be missed. Exposure duration for a given camera position can be adaptively shortened or increased by using an optical flow algorithm to estimate camera motion with respect to the current frame. By determining if the camera is in motion or not, the exposure time may be increased to allow more image counts to accumulate at a given camera position. Adaptive exposure time may improve the ease of use of the hand-held camera, and allow regions of interest to be imaged more effectively. We feel that these image processing techniques can improve the utility of a hand-held gamma ray imager for sentinel lymph node detection during breast cancer staging.     

ANO1对口腔鳞癌SCC-25细胞株的影响

采用免疫组化检测160例口腔鳞癌组织及相应正常组织的ANO1表达,并进行多项体外买验,以明确ANO1对SCC-25细胞迁移的影响。结果显示,正常组织中的ANO1阳性表达明显低于口腔鳞癌组织;有淋巴结转移的口腔鳞癌组织的ANO1阳性表达显著高于无转移的口腔鳞癌组织;多项体外实验表明,ANO1过表达有利于SCC-25细胞的迁移;尼氟灭酸能减缓细胞的迁移速度。综上所述,ANO1促进了口腔鳞癌患者体内的肿瘤转移,并增加了SCC-25细胞的体外移动、侵袭、伸展、剥脱能力,有望成为治疗口腔鳞癌的新的靶点。     

Effective delivery of a lipophilic 6-chloro-2',3'-dideoxyguanosine (6-Cl-ddG) into rat lymphoid tissues.

Lipophilic 6-chloro-2',3'-dideoxyguanosine (6-Cl-ddG) was evaluated for its improved lymph node delivery by comparison with the parental nucleoside (ddG) in vitro and in vivo. The in vitro studies with rat plasma, lymph node homogenate and stomach content indicated that 6-Cl-ddG converted to ddG more effectively in the lymph node homogenate and that 6-Cl-ddG was more stable than ddG in the stomach content. In an in vivo study, plasma and lymph nodes were collected from rats after a subcutaneous or oral administration of 6-Cl-ddG or ddG. With the subcutaneous administrations of the drugs, the area under the concentration time-curve (AUC) value in the plasma for converted ddG following a 6-Cl-ddG administration was less than half the value for ddG following a ddG administration but the converted ddG AUC values in the lymph nodes due to 6-Cl-ddG administration were 1.4- to 2.0-fold higher than the ddG AUC values due to ddG administration. Moreover, with the oral administrations, the converted ddG AUC value in plasma after a 6-Cl-ddG administration was 3-fold higher than ddG after a ddG administration, and high levels of converted ddG were detected in the lymph nodes, but no ddG was detected in the lymph node following ddG administration. These results suggest that lipophilic 6-Cl-ddG is a useful prodrug for delivering ddG into the lymph nodes by oral administration.     

Inhibition of erythroid cell growth by allogeneic murine lymphocytes. Evidence for a synergism between lymph node cells and thymocytes

Murine lymphoid cells from thymus and lymph nodes were tested for synergistic response in a graft-vs-host test. The test is based on the principle that allogeneic lymphocytes inhibit erythroid cell proliferation in the spleens of irradiated mice infused with syngeneic bone marrow cells.I was observed that mixtures of thymocytes and lymph node cells from the same parental strain yielded graft-vs-host responses in irradiated F₁-hybrids higher than expected by summing the responses of the two cell populations tested separately. A similar synergistic response was obtained using mixtures of thymocytes and lymph node cells obtained from the two parental strains of the hybrid, whereas such an effect was not detected using mixtures of lymph node cells or mixtures of thymocytes from the two parental strains. Nor could synergy be demonstrated between parental strain lymph node cells and thymocytes syngeneic with the bone marrow target cells. Thymocytes obtained from one parental strain which were injected into its irradiated F₁-hybrid transformed into a population of sensitized cells in the spleens of the recipients. This transformation was suppressed by the simultaneous injection of lymph node cells from the second parental strain. Since there is a synergistic immune response by such cell mixtures it is concluded that thymocytes may enhance the graft-vs-host response of lymph node cells. Parental strain thymocytes and lymph node cells, the latter being specifically immunologically tolerant to the bone marrow target cells, failed to give a synergistic response indicating that thymocytes do not transform unresponsive lymphocytes into responsive, but rather enhance the reactivity of existing, specifically responsive cells.The results thus show that thymocytes may enhance the response of lymph node cells in this specific graft-vs-host assay.     

Schistosoma mansoni: the effect of regional lymphadenectomy on the level of protection induced in mice by radiation-attenuated cercariae

The lymph nodes which drain the sites of percutaneous vaccination with optimally irradiated cercariae of Schistosoma mansoni were surgically excised in studies to determine their role in the induction of protective immunity. Lymphadenectomy of the axillary and inguinal nodes which drain the abdominal exposure site, or of the cervical node which drains the aural site of exposure, five days prior to vaccination reduced the levels of resistance by two-thirds. Excision of these nodes on Days 5, 10, 15, or 20 postvaccination also significantly reduced the levels of immunity induced, though ablation was less effective at later times. Removal of lymph nodes not draining the site of vaccination had no effect on the induction of resistance. We interpret the results as indicating that successful vaccination of mice against S. mansoni requires the presentation of antigen to lymphocytes in local lymph nodes draining the vaccination site, rather than distant lymphoid organs such as the spleen.     

Relationship between estrogen receptor-binding and estrogenic activities of environmental estrogens and suppression by flavonoids

In this study, we investigated the estrogenic activity of environmental estrogens by a competition binding assay using a human recombinant estrogens receptor (hERbeta) and by a proliferation assay using MCF-7 cells and a sulforhodamine-B assay. In the binding assay, pharmaceuticals had a stronger binding activity to hERbeta than that of some phytoestrogens (coumestrol, daidzein, genistein, luteolin, chrysin, flavone, and naringenin) or industrial chemicals, but phytoestrogens such as coumestrol had a binding activity as strong as pharmaceuticals such as 17alpha-ethynylestradiol (EE), tamoxifen (Tam), and mestranol. In the proliferation assay, pharmaceuticals such as diethylstilbestrol, EE, Tam, and clomiphene, and industrial chemicals such as 4-nonylphenol, bisphenol A, and 4-dihydroxybiphenyl had a proliferation-stimulating activity as strong as 17beta-estradiol (ES). In addition, we found that phytoestrogens such as coumestrol, daidzein, luteolin, and quercetin exerted a proliferation stimulating activity as strong as ES. Furthermore, we examined the suppression of proliferation-stimulating activity, induced by environmental estrogen, by flavonoids, such as daidzein, genistein, quercetin, and luteolin, and found that these flavonoids suppressed the induction of the proliferation-stimulating activity of environmental estrogens. The suppressive effect of flavonoids suggests that these compounds have anti-estrogenic and anti-cancer activities.     

Pregnancy outcomes,embryonic and fetal development in maternal exposure to chinese medicines

Chinese medicine is a common name for a collection of Chinese Materia Medica with therapeutic properties for medical treatment and healing. Similar to Western pharmaceuticals, Chinese medicines are not free of risk, and have the potential to cause adverse pregnancy outcomes and affect embryonic and fetal development. However, most clinical data concerning safety of maternal exposure to Chinese medicines during pregnancy are not available and the conclusion remains elusive. Some individual clinical trials of Chinese medicines reported some minor adverse effects during pregnancy, whereas few animal studies identified some adverse maternal and perinatal effects, as well as embryotoxic potentials. Basic research and mechanistic studies of the teratogenicity of Chinese medicines are still lacking. There is an urgent need for testing the safety of Chinese medicines before recommendation and commercialization. Until more reliable and scientific research data become available, clinicians should consider both the risks and benefits before recommending Chinese medicines to pregnant women. More systematic investigations of the safety implications of the use of Chinese medicines are highly recommended, in addition to more clinical trials with a larger sample size to confirm its safety during pregnancy. This review includes a critical overview of available clinical and experimental data and provides directions to study the safety issue of Chinese medicines for pregnancy. Birth Defects Research (Part C) 99:275–291, 2013. © 2013 Wiley Periodicals, Inc.     

High-frequency Ultrasound Imaging of Mouse Cervical Lymph Nodes

High-frequency ultrasound (HFUS) is widely employed as a non-invasive method for imaging internal anatomic structures in experimental small animal systems. HFUS has the ability to detect structures as small as 30 µm, a property that has been utilized for visualizing superficial lymph nodes in rodents in brightness (B)-mode. Combining power Doppler with B-mode imaging allows for measuring circulatory blood flow within lymph nodes and other organs. While HFUS has been utilized for lymph node imaging in a number of mouse  model systems, a detailed protocol describing HFUS imaging and characterization of the cervical lymph nodes in mice has not been reported. Here, we show that HFUS can be adapted to detect and characterize cervical lymph nodes in mice. Combined B-mode and power Doppler imaging can be used to detect increases in blood flow in immunologically-enlarged cervical nodes. We also describe the use of B-mode imaging to conduct fine needle biopsies of cervical lymph nodes to retrieve lymph tissue for histological  analysis. Finally, software-aided steps are described to calculate changes in lymph node volume and to visualize changes in lymph node morphology following image reconstruction. The ability to visually monitor changes in cervical lymph node biology over time provides a simple and powerful technique for the non-invasive monitoring of cervical lymph node alterations in preclinical mouse models of oral cavity disease.     

Orally administered lead chloride induces bias of mucosal immunity

The hypothesis that lead disturbs gut immune functions upon oral ingestion was tested. Long-term exposure to oral PbCl(2)for 10 days caused persistent downregulation of TGF-beta mRNA levels in intestinal tissue. PbCl(2) also disturbed oral tolerance induction to the dietary antigen ovalbumin. Upon challenge with an immunizing dose of ovalbumin and rechallenge of draining lymph node cells in vitro, tolerance induction was partially suppressed in animals exposed to oral PbCl(2). This was shown by increased proliferation to antigenic stimulus, increased production of IFN-gamma and decreased secretion of TGF-beta. In conclusion, we show for the first time that oral exposure to PbCl(2)has a significant effect on the gut immune system, demonstrated by a bias of the cytokine pattern towards Th(1)and by disturbed oral tolerance mechanisms.