Application of High‐Content Analysis in Clinical Cytology for Translational Safety Biomarkers of Drug‐Induced Toxicity for Lymphoma Chemotherapy

Abstract

Drug cytotoxicity detected by high-content analysis (HCA) of live, cultured, human hepatocytes concurs with human toxic potential. We assessed HCA effectiveness in vivo using human, lymphoma line (HuT-78) and blood cells from healthy dogs and lymphomatous dogs treated with anticancer drugs: cytarabine, arsenic trioxide (AT), doxorubicin and mitoxantrone. DNA was stained with Hoechst-33342, calcium with fluo-4, mitochondria with TMRM and cell permeability with toto-3. HuT-78 and canine blood lymphocytes were treated 24 hr with 10 times increasing drug concentrations to determine concentrationresponse relationships to test agents. These showed hormesis (biphasic response) for TMRM and nuclear area. Fluorescence intensity was decreased with AT and mitoantrone but increased with cytarabine for TMRM, decreased from cell injury or anthracycline interaction for Hoechst-33342 and decreased with AT and interfered with by doxorubicin for fluo-4. Nuclear area increased with anthracyclines and AT. Mitochondria, calcium and nuclear area were affected in peripheral blood lymphocytes of lymphomatous dogs receiving chemotherapy: mitochondria were inhibited by acute treatment, but increased 2 weeks after treatment, as were ionized calcium, nuclear area and DNA. We conclude (i) concentration-response relationships are determinable in vitro for human and canine lymphocytes by HCA, (ii) cytotoxicity is detectable in vivo during and after anticancer drug treatment using canine blood lymphocytes: mitochondria are most affected, with smaller changes in calcium and nuclei and (iii) blood cell viability may be a biomarker for anticancer drug toxicity.

A new approach for the assessment of the toxicity of polyphenol-rich compounds with the use of high content screening analysis

“…Also, a role is played by the hormesis phenomenon (biphasic response), which is commonly manifested in both biological and toxicological settings with a frequency of about 40% [ 33 – 36 ]. Being a protective mechanism derived from metabolic adaptation to environmental stresses, hormesis has been shown to influence cell number, mitochondrial activity, nuclear area, intracellular calcium level, membrane permeability and other activities [ 7 , 37 , 38 ]. For example, hormetic effects have been observed in response to endogenous agonists, drugs and natural compounds, like daidzein, genistein, curcumin, naringin or resveratrol [ 36 ].…”

“…Similarly, Lieggi et al reported an apparent equivalence between human HepG2 and human T-lymphocytes for the identification of cytotoxic agents, when comparing those cell lines for the assessment of anti-cancer drug cytotoxicity [ 37 ]. In addition, quite recently canine peripheral blood lymphocytes have also been demonstrated to be suitable in the detection and monitoring of in vivo drug toxicity by high-content analysis [ 7 ].…”

“…Studies using human hepatocytes have shown that HCS is more effective than past methods for identifying cytotoxic substances in humans [4]. The ability of HCS to quickly analyze the toxicological effects of a large number of compounds has also been confirmed in other studies, using various cell types, such as macrophages [9,10], human bronchial epithelial cells [10], human monocytes [11], intestinal epithelial cells [11], neuronal cell lines [12], lymphocytes [7], human osteosarcoma cells [13], and bovine kidney cells [14]. As a result of global acceptance of HCS technology in drug discovery and basic biomedical research [6], recent years have seen a growing number of reports dealing with the evaluation of the toxicity of various compounds, including drugs with known clinical hepatotoxicity profiles [15], fungal toxins occurring in foods [14], nanoparticles [16] and plant extracts [17,18].…”

“…High content screening is defined as a combination of modern cell biology, automated high-resolution microscopy and flow cytometry integrated to simultaneously detect multiple parameters, such as nuclear area/intensity, intracellular calcium level, mitochondrial membrane potential, plasma membrane permeability and cell number. The process allows the characterization of new therapeutic lead compounds and the identification of the mechanisms of drug toxicity, including mitochondrial dysfunction, oxidative stress, calcium dyshomeostasis, apoptosis, phospholipidosis and steatosis [ 6 , 7 ]. In addition, HCS may serve as an important predictive tool for the optimization and prioritization of the safety of a compound [ 4 , 8 ].…”

Workshop of Scandinavian Society for Cell Toxicology 25‐27 September 2013 in Denmark

“…Per Artursson also presented weaknesses of the current in vitro methodologies. To overcome these challenges, he presented recent approaches towards more quantitative predictions of drug exposure: for example, models for estimation of free drug concentration, proteomics-informed quantification of drug transport and metabolism, and in vitro predictions of drug-induced liver injury.The scientific programme included presentations of new technologies in cell toxicity testing including efficient and sensitive techniques of high-content screening (HCS) based on in vitro monitoring of biochemical and morphological responses of individual live cells to chemical stress in microtitre plates [2][3][4]. This technique can be further refined and developed for use in preclinical testing, diagnostic and prognostic testing as well as monitoring.…”