Blood cell oxidative stress precedes hemolysis in whole blood-liver slice co-cultures of rat, dog, and human tissues.
A novel in vitro model to investigate time-dependent and concentration-dependent responses in blood cells and hemolytic events is studied for rat, dog, and human tissues. Whole blood is co-cultured with a precision-cut liver slice. Methimazole (MMI) was selected as a reference compound, since metabolism of its imidazole thione moiety is linked with hematologic disorders and hepatotoxicity. An oxidative stress response occurred in all three species, marked by a decline in blood GSH levels by 24 h that progressed, and preceded hemolysis, which occurred at high MMI concentrations in the presence of a liver slice with rat (>or=1000 microM at 48 h) and human tissues (>or=1000 microM at 48 h, >or=750 microM at 72 h) but not dog. Human blood-only cultures exhibited a decline of GSH levels but minimal to no hemolysis. The up-regulation of liver genes for heme degradation (Hmox1 and Prdx1), iron cellular transport (Slc40a1), and GSH synthesis and utilization (mGST1 and Gclc) were early markers of the oxidative stress response. The up-regulation of the Kupffer cell lectin Lgals3 gene expression indicated a response to damaged red blood cells, and Hp (haptoglobin) up-regulation is indicative of increased hemoglobin uptake. Up-regulation of liver IL-6 and IL-8 gene expression suggested an activation of an inflammatory response by liver endothelial cells. In summary, MMI exposure led to an oxidative stress response in blood cells, and an up-regulation of liver genes involved with oxidative stress and heme homeostasis, which was clearly separate and preceded frank hemolysis.
Drug Safety Evaluation, Allergan, 2525 Dupont Dr, Irvine, CA 92612, USA. vickers_alison@allergan.com
Alison E M
AE
Vickers
John R
JR
Sinclair
Robyn L
RL
Fisher
Stephen R
SR
Morris
William
W
Way
D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D027682:Cation Transport Proteins / Q000378:metabolism; D018920:Coculture Techniques; D004285:Dogs; D004912:Erythrocytes / Q000187:drug effects / Q000378:metabolism*; D005260:Female; D015870:Gene Expression / Q000187:drug effects; D051547:Heme Oxygenase-1 / Q000378:metabolism; D006461:Hemolysis / Q000187:drug effects*; D006801:Humans; D008099:Liver / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D008713:Methimazole / Q000737:chemistry / Q000633:toxicity; D008954:Models, Biological; D018384:Oxidative Stress / Q000187:drug effects*; D054464:Peroxiredoxins / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D014019:Tissue Donors; D015854:Up-Regulation / Q000187:drug effects; D000098742:Ferroportin
D015415:Biomarkers; D027682:Cation Transport Proteins; D000098742:Ferroportin; D008713:Methimazole; C515940:PRDX1 protein, human; D054464:Peroxiredoxins; C496541:HMOX1 protein, human; D051547:Heme Oxygenase-1