Applied In Vitro Toxicology

This study aimed at evaluating the anticancer activity of selenium and zinc in human colorectal adenocarcinoma (HT-29) cell line exposed to sulfasalazine (SSZ).

Lipid peroxidation through the thiobarbituric acid reagent, oxygen-free radicals with the dichloro-dihydro-fluorescein diacetate reagent, glutathione (GSH) reserves through the 5,5’-dithiobis-(2-nitrobenzoic acid) reagent, and mitochondrial activity with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide reagent were evaluated in the HT-29 cell line. The activity of superoxide dismutase (SOD) and glutathione peroxidase (GPx) enzymes and the activity of caspase-3 were determined using an ELISA kit. Genetic toxicity was also assessed by Comet assay.

Combination therapy of SSZ with zinc or selenium in HT-29 reduced their growth, GSH, and the activity of SOD and GPx enzyme and increased reactive oxygen species and lipid peroxidation and the activity of caspase-3. The amount of tail moment in the comet test in the presence of these substances has also increased, indicating damage to cancer cells' DNA. In general, the best effect of selenium and zinc at concentrations of 100 and 200 μM (p < 0.05) (p < 0.01) was observed.

Due to zinc and selenium's mechanism of action in inhibiting cancer cells' growth, these two substances can be used as a supplement to improve the effect of SSZ in treating diseases (e.g., colorectal cancer) and inhibiting drug toxicity.

Perfluoroalkylated substances (PFAS) such as carboxylic acids, and sulfonic acids were manufactured in high quantities and are ubiquitous environmental contaminants. These chemicals persist in the environment and tend to bioaccumulate.

In the current study, the estrogenic potential of a series of perfluoro carboxylic acids and select perfluoro sulfonic acids were assessed in an in vitro rainbow trout estrogen receptor (rtER) binding assay and an ex vivo rtER dependent vitellogenin (Vtg) expression rainbow trout liver slice assay.

Perfluoro carboxylic acids with perfluoroalkyl chain lengths of four to six did not significantly bind to the rtER or induce Vtg expression in liver slices. Perfluoro carboxylic acids with chain lengths of seven to ten, and sulfonic acids with seven and eight carbon chains bound to the rtER, but with low relative binding affinities. While affinity for the rtER increased with increasing chain length the highest affinity measured was only 0.0025% relative to the endogenous hormone 17β-estradiol at 100%. Both the eight-carbon carboxylic acid and eight-carbon sulfonic acid induced Vtg expression in ex vivo liver slices. However, toxicity did not allow expression to achieve maximum efficacy relative to estradiol.

Seven carbon and longer perfluoro carboxylic acids and sulfonic acids are capable of binding to the rtER and inducing ER dependent gene expression in rainbow trout liver slices, but toxicity seems have a greater effect than any adverse endocrine effect.

Teratogenic events such as fetal heart malformations are serious and tragic drug-induced toxicities. Preclinical studies conducted during drug safety evaluation are intended to identify teratogenic risks using rodent and nonrodent models, largely based on the experience with thalidomide, which is teratogenic in humans and rabbits but refractory in rodents.

To study the teratogenic properties of thalidomide in rabbit, a novel method was designed to develop cardiomyocytes (CMs) from rabbit induced pluripotent stem cells (rbiPSCs). Throughout the course of differentiation, rbiPSC-CMs were examined by real time polymerase chain reaction, high-content analysis, calcium flux measurements, and electrophysiology to confirm the cell's mature CM phenotype. The effects of thalidomide were then measured in each of the endpoints when present throughout differentiation.

As early as differentiation day (DD) 3 of the differentiation, rbiPSCs begin to express early CM markers (e.g., NXK2.5) and mesodermal markers (e.g., FOXF1). By DD 21, rbiPSC-CMs expressed messenger RNA (mRNA) and protein of mature CM genes, including TNNT2, HCN4, and KCNJ2. Spontaneous beating was observed by microscopy and electrophysiology as early as DD 8, and live cell calcium probes demonstrated pulsatile calcium flux. Thalidomide treatment to rbiPSC-CMs downregulated mRNA and protein of early and mature cardiac markers, suggesting suppressed differentiation. In addition, thalidomide treatment abrogated the onset of spontaneously beating cells.

Taken together, these data indicate that rbiPSC-CMs can recapitulate thalidomide-induced cardiac teratogenicity observed in rabbits. Moving forward, this model can be deployed to investigate the underlying mechanisms of rabbit teratogenicity and better understand species-specific differences in drug-induced fetal heart malformations.