DNA Damages and White Blood Cell Death Processes in Victims with Severe Injury

Objective. To study the mechanisms of posttraumatic changes in the blood cells, by investigating DNA damages associat ed with hypoxia caused by massive blood loss (BL) in severe injury.

Subjects and methods. Ninetyfive patients aged 40.6±16.5 years (from 20 to 79 years) who had sustained severe mechanical injury with different BL volumes (BLV) (from 100 to 4000 ml) and hemodynamic disorders were examined to study DNA damages and white blood cell necrotic and apop totic processes. In terms of the victims' weight, the mean BL was 21.5±16.5 ml/kg (from 1.4 to 61.5 ml/kg). The victims
were divided into 4 groups according to BLV: 1) 26 victims whose BLV was less than 750 ml (5.93±2.41 ml/kg) (grade I BL); 2) 23 victims whose BLV was 750—1500 ml (11.5±1.5 ml/kg) (grade 2 BL); 3) 23 victims whose BLV was 1500—2000 ml (23.8±4.0 ml/kg) (grade 3 BL); 4) 23 victims whose BLV was over 2000 ml (45.6±10.1 ml/kg) (grade 4 BL), according to the type of injury: 1) severe skeletal injury (SSI) (n=17); 2) brain injury (BI) (n=43); 3) a concurrence of SSI and BI (SSI+BI) (n=35); according to the development of infectious complications: 1) 69 victims who developed infectious com plications on days 5—7 postinjury; 2) 26 victims who did not. To evaluate the impact of hypoxia on DNA damages, white blood cell apoptotic and necrotic processes, the victims were divided into 2 groups: 1) hypoxia (18 of the 95 victims who had 4 altered indicators, such as capillary blood pO2, plasma lactate levels, pH, and BE); 2) no hypoxia (10 of the 95 victims whose indicators were within the normal range). DNA damages and necrotic and apoptotic changes in the white blood cells were assessed by the DNA comet assay. The plasma concentration of extracellular DNA was fluorometrically deter
mined using a QuantiTTM HS DNA Assay Kit (Invitrogen, USA). That of 8hydroxy2deoxyguanosine was estimated by enzyme immunoassay employing an 8hydroxy2deoxyGuanosine EIA Kit (Cayman Chemical, USA). The levels of cas pase3, caspase9, superoxide dismutase, and sAPO1/FAS were measured by enzyme immunoassay using test systems
(Bender MedSystems, Austria).

Results. There were significantly higher plasma levels of free DNA in the victims than in the controls throughout the followup, which is due to its exit from the cells damaged from tissue injury. In the first two weeks after injury, there were increases in DNA damages and white blood cell alteration processes by a necrotic and apoptotic mechanism in the victims with different types of injury, which may be associated with the active participation of leuko cytes in the processes of cellular breakdown product removal in the tissues damaged during injury and the in those of prevention of infectious complications. In the victims, white blood cell alteration in the necrotic pathway does not depend on BLV and hypoxia degree while that in the apoptotic pathway showed a relationship of leukocyte alteration to hypoxia in these patients. The sum of the values of necrotic DNA comets, apoptotic DNA comets, and single and doublestrand breaks on day 3 postinjury may serve as a predictor of the likely development of infectious complications in victims with injury, blood loss, and marked hypoxia. There were differences in the levels of DNA damages and white blood cell apoptosis and necrosis in the victims with BI and SSI. The injury victims showed a threefold decrease in plasma 8hydroxy2 deoxyguanosine concentrations, which was accompanied and, possibly, caused by an increase in the amount of superoxide dismutase.

Conclusion. There was a relationship between the degree of DNA damages, apoptosis, and necrosis in the white blood cells of victims with injury and hypoxiainduced blood loss.

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