Combined cytotoxicity of aflatoxin B1 and deoxynivalenol to hepatoma HepG2/C3A cells

Co-occurrence of aflatoxin B1 (AFB1) and deoxynivalenol (DON) is one of the most common mycotoxin contaminations in cereal crops and food ingredients. However, the mechanism of their combined toxicity is poorly understood. In the current investigation, the hepatoma HepG2/C3A cell line was used to explore the combined cytotoxicity of AFB1 and DON. The values of IC50, based on a sulforhodamine B (SRB) assay, were 4.5 mM and 18.7 mM for DON and AFB1, respectively. The analysis of cytotoxicity endpoints using the combination index (CI) theory revealed that the changes of mitochondrial membrane permeability and ATP resulted from an additive cytotoxic effect (CI≈1) of AFB1 and DON. However, the endpoints of double strand DNA (ds-DNA), reactive oxygen species (ROS) and cell viability (SRB) were synergistically (CI<1) affected in a dose-dependent manner. RNA-seq analysis demonstrated a number of uniquely expressed genes in their combination (AFB1: 2.5 mM, DON: 0.56 mM), indicating a synergistic interaction between AFB1 and DON at a molecular level. Additional transcriptomics analysis showed that the endoplasmic reticulum stress-associated JNK/p38/MAPK pathway was induced by DON, whereas the p53 signalling pathway was activated by AFB1. The expression profiles of apoptosis-related genes caspase-3, Bax and Bcl-2 suggested a mitochondria-mediated apoptosis pathway that was shared between AFB1 and DON. Thus, different cytotoxicity pathways and their converging at the apoptotic process might be the mechanism of the additive/synergistic cytotoxicity of AFB1 and DON to HepG2/C3A cells.

Differential Cytotoxicity Responses by Dog and Rat Hepatocytes to Phospholipogenic Treatments

Dog and rat hepatocytes were treated with phospholipogenics to identify the more sensitive species and to determine whether lysosomal or mitochondrial changes were the primary cause of cytotoxicity. Endpoints included cell death, lysosome membrane integrity, mitochondrial membrane polarization, and fluorescent phospholipid (NBD-PE). Dog cells exhibited lower survival IC50values than did rat cells with all phospholipogenic treatments and exhibited a lower capacity to accumulate NBD-PE in 4 of 5 phospholipogenic test conditions. The lysosomal modulator Bafilomycin A1 (Baf) rescued dog cells from cytotoxicity caused by 3 phospholipogenic 5HT1bantagonists and hydroxychloroquine, but not fluoxetine, and rescued rat cells from hydroxychloroquine and NMTMB, a 5HT1bantagonist. Following NMTMB treatment, rat mitochondrial membrane hyperpolarization was observed at modestly cytotoxic concentrations and depolarization at the highest concentration. At the highest test concentration, lysosomal loss of acridine orange occurred by 30 min, mitochondrial polarity changes by 1 hr, and NBD-PE accumulation by 2 hr, respectively. Baf shifted mitochondrial polarity from a depolarized state to a hyperpolarized state. These data demonstrate that (a) dog hepatocytes were generally less capable of mounting an adaptive, protective phospholipidotic response than rat hepatocytes, (b) effects on mitochondria and survival were preventable by lysosomal protection, and (c) destabilizing changes in both organelles are involved causally in cytotoxicity.

A Comparison of Three Cytotoxicity Endpoints in the Corneal HCE-T Model

The prediction of ocular irritation potential from in vitro assays still presents a problem, despite a number of validation trials. A study with coded cosmetic formulations, for which historic in vivo data were available, has been conducted with a human corneal multilayered model system. This corneal model, the HCE-T model, was developed by using HCE-T cells, a transfected human corneal epithelial cell line. The relative effectiveness of three endpoints that provide a measure of cytotoxicity in the HCE-T model was evaluated. Cell viability immediately after exposure to the test materials was determined by using the MTT and Alamar Blue™ (AB) assays, and, 24 hours later, by using the MTT, AB and lactate assays. Viability measurements with the MTT, AB and lactate assays gave similar dose–response curves at the 24-hour endpoint. One formulation (an anti-dandruff shampoo) caused a less severe drop in viability in assays conducted immediately after the exposure than at the 24-hour time-point. There was little deterioration in viability with the other test materials. The ranking of the test formulations on the basis of relative loss of viability and release of lactate resulted in the same order as for the Modified Maximum Average Draize Test Score. Comparison of the HCE-T model cytotoxicity assay results with historic in vitro data from two different cytotoxicity assays, conducted by using fibroblast monolayer cultures and the same materials, indicated that the multilayered corneal model had a greater predictive ability. The results of a blind trial with the lactate assay in two laboratories indicated that the techniques required were transferable between laboratories. The lactate results were reproducible between laboratories, even when cultures derived from different passage human corneal cells were tested, provided that the passage number was below 20.

Alterations of ultrastructure and elemental composition in cultured human epidermal keratinocytes after treatment with nitrofurazone and silver sulfadiazine

Cell culture systems have become increasingly popular as a means of screening toxic agents and studying toxic mechanisms of drugs and other chemicals at the cellular and subcellular levels. These in vitro tests can be conducted rapidly in a broad range of relevant mammalian culture systems; a variety of biological and biochemical cytotoxicity endpoints can be examined. The following study utilized human keratinocytes to evaluate the relative cytotoxicities of nitrofurazone (NF) and silver sulfadiazine (SS), the active ingredients of FURACIN(R) Topical Cream and SILVADENE(R) Cream, respectively. These compounds are anti-infectives used in the treatment of burn patients. Cell ultrastructure and elemental composition were utilized as cytotoxicity endpoints.Normal Human Epidermal Keratinocytes (HK) were prepared from the EpiPackTM culture system (Clonetics Corporation, Boulder, CO). For scanning electron microscopy (SEM) and transmission electron microscopy (TEM), cells were seeded on sterile 35 mm Falcon plastic dishes; for elemental microanalysis, cells were plated on polished pyrolytic carbon discs (E. Fullam, Latham, NY) placed in the culture dishes.