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

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 concentration–response 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.

[1]  Jorrit J Hornberg,et al.  A high content screening assay to predict human drug-induced liver injury during drug discovery. , 2013, Journal of pharmacological and toxicological methods.

[2]  I. Berzina,et al.  Rapid, effective and user-friendly immunophenotyping of canine lymphoma using a personal flow cytometer , 2013, Irish Veterinary Journal.

[3]  P. O'Brien,et al.  Translation of novel anti-cancer cytotoxicity biomarkers detected with high content analysis from an in vitro predictive model to an in vivo cell model. , 2010, Toxicology in vitro : an international journal published in association with BIBRA.

[4]  P. O'Brien.  Discovery Toxicology Screening: Predictive, In Vitro Cytotoxicity , 2009 .

[5]  Laszlo Urban,et al.  Hit and lead profiling : identification and optimization of drug-like molecules , 2009 .

[6]  P. O'Brien.  Cardiac troponin is the most effective translational safety biomarker for myocardial injury in cardiotoxicity. , 2008, Toxicology.

[7]  Harvey J Clewell,et al.  Hormesis and Adaptive Cellular Control Systems , 2008, Dose-Response.

[8]  P. O'Brien.  High Content Analysis of Sublethal Cytotoxicity in Human HepG2 Hepatocytes for Assessing Potential and Mechanism for Chemical‐ and Drug‐Induced Human Toxicity , 2007 .

[9]  P. Bernardi,et al.  High concordance of drug-induced human hepatotoxicity with in vitro cytotoxicity measured in a novel cell-based model using high content screening , 2006, Archives of Toxicology.

[10]  Heinrich Leonhardt,et al.  DNA labeling in living cells , 2005, Cytometry. Part A : the journal of the International Society for Analytical Cytology.

[11]  M. van den Berg,et al.  Cytochrome P450 1A1 and 1B1 in human blood lymphocytes are not suitable as biomarkers of exposure to dioxin-like compounds: polymorphisms and interindividual variation in expression and inducibility. , 2005, Toxicological sciences : an official journal of the Society of Toxicology.

[12]  J. Nickerson,et al.  Quantifying DNA concentrations using fluorometry: a comparison of fluorophores. , 2002, Molecular vision.

[13]  E. Calabrese,et al.  Applications of hormesis in toxicology, risk assessment and chemotherapeutics. , 2002, Trends in pharmacological sciences.

[14]  S. Polley,et al.  Advantages of glutamate dehydrogenase as a blood biomarker of acute hepatic injury in rats , 2002, Laboratory animals.

[15]  P. O'Brien,et al.  Effect of diquat on the antioxidant system and cell growth in human neuroblastoma cells. , 2002, Toxicology and applied pharmacology.

[16]  K. Fung,et al.  Inhibition of P-glycoprotein expression and reversal of drug resistance of human hepatoma HepG2 cells by multidrug resistance gene (mdr1) antisense RNA. , 2000, Life sciences.

[17]  J. Revillard,et al.  Anthracyclines trigger apoptosis of both G0 G1 and cycling peripheral blood lymphocytes and induce massive deletion of mature T and B cells , 1999, Cancer research.

[18]  R. Rosenfeld,et al.  Deciphering the fluorescence signature of daunomycin and doxorubicin. , 1998, Biophysical chemistry.

[19]  N. Shams,et al.  Study on the interaction between doxorubicin and Deoxyribonucleic acid with the use of methylene blue as a probe , 2009 .

[20]  S. Carpenter,et al.  Drug metabolizing enzymes in lymphocytes , 1999, Journal of biochemical and molecular toxicology.