Toxic effects of xylazine on endothelial cells in combination with cocaine and 6-monoacetylmorphine

Abstract

The use of xylazine as a drug of abuse has emerged worldwide in the last 7 years, including Puerto Rico. Clinical findings reported that xylazine users present greater physiological deterioration, than heroin users. The aim of this study was to assess the xylazine toxicity on endothelial cells, as this is one of the first tissues impact upon administration. Human umbilical vein endothelial cells in culture were treated with xylazine, cocaine, 6-monoacetylmorphine (heroin metabolite) and its combinations, at concentrations of 0.10-400 μM, for periods of 24, 48 and 72 h. IC50 were calculated and the Annexin V assay implemented to determine the cell death mechanism. Results indicated IC50 values at 24h as follow: xylazine 62 μM, cocaine 210 μM, 6-monoacetylmorphine 300 μM. When these drugs were combined the IC50 value was 57 μM. Annexin V results indicated cell death by an apoptosis mechanism in cells treated with xylazine or in combination. Results demonstrated that xylazine use inhibits the endothelial cell proliferation, at lower concentrations than cocaine and 6-monoacetylmorphine. These findings contribute to the understanding of the toxicity mechanisms induced by xylazine on endothelial cells.

Keywords: 6-Monoacetylmorphine; Apoptosis; Cocaine; Drug abuse; Xylazine.

Figure 1

Dose response comparisons of EA.hy926 cells; 1A: treated with camptothecin (positive control) and yohimbine, 1B: treated with xylazine and its combinations with yohimbine, 1C: treated with cocaine, 6-monoacetylmorphine (6-MAM) and its combinations with xylazine. Dose range from 0.10 µM to 400 µM, at treatment periods of 24, 48 and 72 hours.

Figure 1

Dose response comparisons of EA.hy926 cells; 1A: treated with camptothecin (positive control) and yohimbine, 1B: treated with xylazine and its combinations with yohimbine, 1C: treated with cocaine, 6-monoacetylmorphine (6-MAM) and its combinations with xylazine. Dose range from 0.10 µM to 400 µM, at treatment periods of 24, 48 and 72 hours.

Figure 1

Dose response comparisons of EA.hy926 cells; 1A: treated with camptothecin (positive control) and yohimbine, 1B: treated with xylazine and its combinations with yohimbine, 1C: treated with cocaine, 6-monoacetylmorphine (6-MAM) and its combinations with xylazine. Dose range from 0.10 µM to 400 µM, at treatment periods of 24, 48 and 72 hours.

Figure 2. Annexin V assay representation

The characteristic migration of PS to the exterior (extracellular) side of the cell membrane is typical of apoptosis. The translocation of PS from the cytoplasmic face to the external face of the plasma membrane can be detected using Annexin V during early apoptosis. Once on the cell surface, PS can be easily detected by staining with the fluorescent conjugate of Annexin V, a protein that has a high affinity for PS, detection could be analyzed by fluorescence microscopy. As apoptosis progresses the plasma membrane becomes compromised and loses membrane integrity later. Necrotic cells expose PS and lose membrana integrity concurently after process initiation. Propidium Iodine (PI), a DNA binding dye, can be used to discriminate necrotic cells from apoptotic. Necrotic cells that have lost of membrane integrity will show red staining (PI) throughout the nucleus and green (annexin v) in the membrane. While apoptotic cells will show only green staining (annexin v) in the membrane.

Figure 3. Annexin V staining assay results

Data is shown in percentage of apoptotic cells, treated with all drugs, their combinations, vehicle as negative control and camptothecin as positive control, exhibiting significant apoptotic activity. A; The percentages of apoptotic cells treated with the drugs were camptothecin (Positive Control) 64%, xylazine 63%, cocaine 56%, and 6-MAM 54%. B; Drugs combinations treatments were xylazine/cocaine 50%, xylazine/6-MAM 30% and 47% for xylazine/cocaine/6-MAM. Approximately 5×10⁵ cells were treated for 24 hours with xylazine (60 µM), Cocaine (160 µM), 6-MAM (160 µM), camptothecin (50 µM), xylazine/cocaine (50 µM), xylazine/6-MAM (50 µM) and xylazine/cocaine/6-MAM (40 µM); and vehicle. One-way ANOVA statistical analysis with Tukey post hoc test was performed. All values were compared with positive control to determine the significant difference among drugs, if P< 0.05. P value Summary: P<0.001= ***, P<0.01= **. NC = Negative Control, PC = Positive Control, XYL = Xylazine, COC = Cocaine, 6-MAM = 6-Monoacetylmorphine (Heroin metabolite).

Figure 3. Annexin V staining assay results

Data is shown in percentage of apoptotic cells, treated with all drugs, their combinations, vehicle as negative control and camptothecin as positive control, exhibiting significant apoptotic activity. A; The percentages of apoptotic cells treated with the drugs were camptothecin (Positive Control) 64%, xylazine 63%, cocaine 56%, and 6-MAM 54%. B; Drugs combinations treatments were xylazine/cocaine 50%, xylazine/6-MAM 30% and 47% for xylazine/cocaine/6-MAM. Approximately 5×10⁵ cells were treated for 24 hours with xylazine (60 µM), Cocaine (160 µM), 6-MAM (160 µM), camptothecin (50 µM), xylazine/cocaine (50 µM), xylazine/6-MAM (50 µM) and xylazine/cocaine/6-MAM (40 µM); and vehicle. One-way ANOVA statistical analysis with Tukey post hoc test was performed. All values were compared with positive control to determine the significant difference among drugs, if P< 0.05. P value Summary: P<0.001= ***, P<0.01= **. NC = Negative Control, PC = Positive Control, XYL = Xylazine, COC = Cocaine, 6-MAM = 6-Monoacetylmorphine (Heroin metabolite).

Figure 4

Illustration of structures envolved in the metabolization of heroin molecule (A-Diacetylmorphine) to 6-Monoacetylmorphine (B) and finally to Morphine (C), once it enters the human body.

Figure 5

Chemical structure illustration of phenothiazine drugs group (A) and xylazine (B).