FLOW CYTOMETRY IN PRECLINICAL TOXICOLOGY/SAFETY ASSESSMENT - Flow Cytometry in Drug Discovery and Development - page 123

plots is very cumbersome and requires the person performing the data analysis to
identify all populations of interest. Although there have been some interesting
attempts at automating portions of data analysis [2-5], there is no approach available
that is an accepted replacement for a trained cytometrist.
There are also a few lesser challenges that are worth mention. One such challenge
is simply getting colleagues in preclinical toxicology to accept flow cytometry-based
approaches to analyses that were done using different techniques in the past.
Returning to the bone marrow analysis mentioned earlier, where manual evaluation
of smears is the commonly accepted practice, it may be difficult to get clinical
pathologists and hematologists to endorse a flow cytometric approach. Another
challenge could simply be a lack of technical expertise in flow cytometry within
the department. An experienced flow cytometrist, especially one with cell-sorter
operation experience, is an invaluable asset if flow cytometry is to be a core technique
within your organization.
The remainder of this chapter will be devoted to three examples of research
performed within the department of safety assessment at a major pharmaceutical
company. The first details a multiparameter flow cytometric analysis of oxidative
stress for use as an in vitro screening tool. The second involves the search for
a biomarker for drug-induced vascular injury and will describe how flow cytometry
and cell sorting contributed to this effort. The last example describes the development
of a flow cytometric method for bone marrow assessment in mice.

6.5 IN VITRO OXIDATIVE STRESS ASSAY

Oxidative stress, characterized by disturbances in cellular redox status, is a frequent
mechanism by which drugs and chemicals induce toxicity [6]. Therefore, measure-
ment of drug-induced oxidative stress in vitro can be useful in assessing toxicity of
potential drug candidates. Flow cytometry is the perfect choice for this application
since it provides simultaneous data acquisition from multiple parameters allowing
analysis of diverse functional changes on an individual cell basis [7]. In addition,
a wide variety of fluorescent probes are available for studying pathophysiological
changes in cells associated with drug toxicity. The objective of this study was to
develop a multiparameter flow cytometric method to assess oxidative stress induction
in cultured cells by simultaneously measuring relative intracellular glutathione
level, mitochondrial membrane potential, lipid peroxidation, apoptosis, and plasma
membrane integrity using fluorescent reporters for these end points.

6.5.1 Methods
6.5.1.1 Cell Lines and Maintenance The promyelocytic leukemic cell line
HL-60 was employed as it is commonly used for in vitro toxicity assays within
the department. HL-60 cells obtained from American Type Culture Collection
(ATCC, Gaithersburg, MD), were maintained in RPMI-1640 medium
supplemented with 10% heat-inactivated fetal bovine serum, 100 I.U. penicillin,