Applied In Vitro Toxicology

Three-Dimensional (3D) liver microtissues, specifically prepared from primary human hepatocytes (PHH) in coculture with nonparenchymal cells (NPCs), have been shown to be a valuable tool for in vitro toxicology. However, a lack of thorough characterization on a functional, transcriptomic, and proteomic level of such models during long-term cultivation is evident. By integrating multiple omics technologies, we provide in this study an in-depth long-term characterization of 3D microtissues composed of PHH from three different donors cocultured with primary NPCs. The 3D human liver microtissues (hLiMTs) exhibited stable adenosine triphosphate (ATP) content and albumin secretion over 5 weeks. Histological analysis indicated a healthy liver tissue with polarized expression of bile salt export pump (BSEP) and multidrug resistance protein 2 (MRP2) in a structure reminiscent of bile canaliculi. The 3D microtissues exhibited stable basal and inducible cytochrome P450 activities up to 5 weeks in culture. Analysis of 40,716 transcripts using RNA arrays revealed distinct similarities to native human liver gene expression. Long-term culture showed a stable phenotype up to 5 weeks, with differences in liver gene expression primarily attributed to individual donors. Proteomic profiling of 2200 unique proteins by label-free LC-MS/MS revealed a relatively stable protein expression where only 7.3% were up- or downregulated more than twofold from day 7 to 35 in culture. Taken together, these results suggest that hLiMTs represent a responsive and physiologically relevant in vitro liver model that maintains stable function over 5 weeks and is therefore well suited for repeated-dose toxicity testing.

Understanding biotransformation pathways in aquatic species is an integral part of ecological risk assessment with respect to the potential bioactivation of chemicals to more toxic metabolites. The long-range goal is to gain sufficient understanding of fish metabolic transformation reactions to be able to accurately predict fish xenobiotic metabolism. While some metabolism data exist, there are few fish in vivo exposure studies where metabolites have been identified and the metabolic pathways proposed. Previous biotransformation work has focused on in vitro studies, which have the advantage of high throughput but may have limited metabolic capabilities, and in vivo studies, which have full metabolic capacity but are low throughput. An aquatic model system with full metabolic capacity in which a large number of chemicals could be tested would be a valuable tool. The current study evaluated the ex vivo rainbow trout liver slice model, which has the advantages of high throughput as found in in vitro models and non-dedifferentiated cells and cell to cell communication found in in vivo systems. The pesticide diazinon, which has been previously tested both in vitro and in vivo in a number of mammalian and aquatic species, including rainbow trout, was used to evaluate the ex vivo slice model as a tool to study biotransformation pathways. While somewhat limited by the analytical chemistry method used, results of the liver slice model, mainly that hydroxypyrimidine was the major diazinon metabolite, are in line with the results of previous rainbow trout in vivo studies. Therefore, the rainbow trout liver slice model is a useful tool for the study of metabolism in aquatic species.

Arsenic, a naturally occurring environmental contaminant, is harmful to humans at elevated concentrations. Increased levels of arsenic in the environment occur as a result of human activities and from natural geologically sourced leaching into ground and surface water. These sources pose an exposure risk above the USEPA standard to individuals whose food and water sources become contaminated. Arsenic exposure negatively impacts organ function and increases the risk for developing pathologies, including cancer. Some of the effects of arsenic on cancer translate to normal cell function in wound healing. To evaluate whether arsenic influences wound healing, an in vitro scratch assay was employed to study the effects of arsenic on cellular migration, which is a key component in the normal wound-healing process. In this study, skin cells were exposed to environmentally relevant concentrations of arsenic, and wound closure was evaluated. Results indicated that arsenic significantly decreased the rate of cellular migration in the scratch assay when compared with controls. In addition, estradiol, which has been shown to positively influence cellular and tissue processes involved in wound healing, reversed the slowing effects of arsenic on wound closure. These results suggest that arsenic contamination may inhibit, and estrogen may provide a therapeutic benefit for individuals with arsenic-contaminated wounds.

Cosmetics legislation in Europe has driven the validation and acceptance of non-animal alternatives, most recently in the area of skin sensitization. Despite use of these methods to meet regulatory needs, it is also essential that they allow evaluation regarding human safety. For cosmetic product safety, it is necessary to understand how they can be used and with what limitations, and thereby reveal what remains to be addressed. A dataset of 165 ingredients (137 cosmetic ingredients +28 reference substances) has been identified, curated, and subjected to testing using accepted in vitro methods, with additional information, including physicochemical data and in silico results. The inputs from multiple determinants of skin sensitizing activity have been used in five individual supervised classification models (or machine learning approaches), which were then collated in a robust statistical manner, a stacking meta-model, to deliver a prediction with an optimized level of confidence. For the training set, with the probability cutoffs at 70% and 30%, predictive sensitivity was 97%, specificity was 88%, the overall accuracy was 93%, and kappa was 85%. A further 52 substances were used to test the effectiveness of the model: the predictive sensitivity was 89%, specificity was 95%, overall accuracy was 91%, and kappa was 82%. In conclusion, this stacking meta-model delivers improved performance and therefore enhanced confidence in the discrimination of skin sensitizers from nonsensitizers. The key remaining gap, prediction of skin sensitization potency, may benefit from a similar approach, maximizing use of evidence from individual strands of prediction, while minimizing the impact of the limitations from any particular one.

Teratogenic potential of human drugs is assessed in embryo-fetal development (EFD) studies in two species as per regulatory guidelines. In vitro developmental toxicity assays can be vital in early drug development efforts to distinguish teratogenic potential of drugs, while reducing animal use. Results from two developmental toxicity in vitro assays (rat whole embryo culture assay and mouse embryonic stem cell test), were evaluated for their ability to predict the teratogenic potential of 83 compounds with known EFD outcome in rats. With an integrated model, the sensitivity and specificity for teratogens and/or embryo-lethal drugs in the presence (89% and 54%, respectively) or absence (96% and 52%, respectively) of maternal toxicity were calculated. Based on these results, we propose that a battery of assays be used to screen for potential EFD toxicity and, in combination with reduced in vivo rat EFD studies, be a part of an integrated regulatory risk assessment.

We propose silkworm as an animal model for screening the drug molecules, especially antibiotics, because they exhibit a conserved immune response and similar pharmacokinetics (PK) as mammals and importantly no ethical issues are associated. To evaluate the PK parameters and effectiveness of ofloxacin in silkworm. Silkworms with their ideal body weight of 3.8 g were injected with 40 μL of 2 mg/mL ofloxacin. The hemolymph was collected at different time intervals 0 to 300 minutes. The samples were used for estimating the retention of ofloxacin in silkworm hemolymph by high performance liquid chromatography (HPLC), ultra violet visible (UV-VIS) spectrophotometry, liquid chromatography mass spectroscopy (LC-MS), and Fourier transform infrared spectroscopy (FTIR). PK analysis was performed by using the PK solver MS-Excel add-on software. The HPLC method was standardized for the quantification of ofloxacin in silkworm. Further with reference, the structure of ofloxacin was elucidated on the basis of LC-MS spectral data. We reconfirmed the presence and activity of the ofloxacin in silkworm by UV-VIS spectrophotometry and FTIR spectroscopy. The noncompartmental analysis technique was used to quantitatively evaluate and predict the in vivo fate of drug by modeling the concentration time–data with suitable model. The features of the silkworm described suggest that they are a suitable alternative evaluation model for determining the therapeutic effectiveness of candidate drugs. The availability of a large number of silkworms at low cost and no associated ethical issues makes it a better animal model suitable to screen novel antibiotics for their therapeutic effectiveness.