In vitro cytotoxicity assay to screen compounds for apoptosis-inducing potential on lymphocytes and neutrophils.

Abstract

-In vitro cytotoxicity assay to screen compounds for apoptosis-inducing potential on lymphocytes and neutrophils was investigated. Mouse, rat, dog, and human whole blood were incubated for 4 and 6 hr with actinomycin D, camptothecin, cortisone acetate, cycloheximide, doxorubicin, etoposide, 5-FU, mitomycin C and puromycin. Apoptotic lymphocytes and neutrophils were counted. All test compounds induced in vitro apoptosis of lymphocytes and/or neutrophils, but there were different potencies among the test compounds and there were also species differences in susceptibility. To investigate the in vivo effects of etoposide and cycloheximide which induced apoptosis of rat lymphocytes and that of rat lymphocytes and neutrophils, respectively, in in vitro assay, rats were intravenously administered either etoposide at 12.5, 25 or 50 mg/kg or cycloheximide at 1.25, 2.5 or 5 mg/kg. Etoposide caused decreases of circulating lymphocytes at 3 hr after administration in a dose-dependent manner, -16, -25 and -51%. Although cycloheximide caused neither decreased lymphocyte nor neutrophil counts, apoptosis in 30% of neutrophils was observed in rats receiving 5 mg/kg at 3 hr after administration. Etoposide at 50 mg/kg and cycloheximide at 5 mg/kg caused lymphocyte apoptosis in the spleen, thymus, mesenteric lymph nodes, bone marrow, and Peyer's patch from 1 to 6 hr after administration, with the maximum changes at 3 hr. In addition to apoptosis of these organs, cycloheximide at 5 mg/kg caused apoptosis of polymorphonuclear cells in the lamina propria of the small intestine. Therefore, it was found that the changes seen in the in vivo experiments considerably reflected the changes seen in the in vitro experiments.From these results, apoptosis is probably one of the major mechanisms for leukocyte toxicity induced by cytotoxic compounds, and the in vitro assay to screen compounds for acute apoptosis-inducing potential on lymphocytes and neutrophils would be useful as a primary screening method for animal toxicity studies. It may also be useful for risk assessments in advance of clinical trials.

Estresse oxidativo e aumento da apoptose em neutrófilos de cães com azotemia pré-renal

“…2). Este efeito indutor de apoptose da CAM já havia sido descrito em neutrófilos de humanos (Nagami et al, 2002), porém não na espécie canina.…”

Apoptosis is a natural stimulus of IL6R shedding and contributes to the proinflammatory trans-signaling function of neutrophils

“…30,37 Interestingly, the amount of apoptotic neutrophils after Fas stimulation was not altered in the presence of IL6 or IL6/sIL6R ( Figure S2A). Our finding indicates that during apoptosis sIL6R-shedding does not necessarily render the cells unresponsive to IL6 but it rather seems to induce IL6 trans-signaling in neighboring nonapoptotic cells.…”

“…30,39 Whereas macrophages are important for the resolution of inflammation, neutrophils are essential for initiation and execution of the acute inflammatory response and are the predominant immune cells in the early stage of infection. Although the inflammatory response is highly beneficial to the host, coordinated termination is critical to limit tissue damage.…”

“…sIL6R is released from apoptotic primary human neutrophils and has the capacity to direct transition from neutrophil to mononuclear cell infiltration during acute inflammation Neutrophils undergo apoptosis during resolution of inflammation, which is regulated by proapoptotic mediators such as TNF␣ and Fas ligand. 30 Human neutrophils were isolated from blood of healthy donors and apoptosis was subsequently induced by Fas stimulation, UV irradiation, or doxorubicin treatment. Fas stimulation and UV treatment induced apoptosis of neutrophils with 45% and 90% of the cells being annexin V positive after 6 hours ( Figure S2A).…”

Benzene and 2-ethyl-phthalate induce proliferation in normal rat pituitary cells

“…Incubation with 250 µg/ml cycloheximide (CHX) (Sigma, Saint Louis MO, USA) served as representative control for cytotoxicity given that high doses of the protein synthesis inhibitor have been shown to be cytotoxic [28, 29]; wells incubated with DMEM + 0.1% BSA represented untreated control. Treatments were repeated in four separate experiments on quadruplicate wells.…”