Name: LUIZ RICARDO RODRIGUES SILVA
Publication date: 08/02/2024
Examining board:
Name |
Role |
|---|---|
| LUCIENE CRISTINA GASTALHO CAMPOS LUIZ | Examinador Externo |
| SILVANA DOS SANTOS MEYRELLES | Examinador Interno |
Summary: DNA, fundamental in genetic coding and metabolic regulation in living organisms,
traditionally confined within the nucleus of eukaryotic cells and mitochondria, is also
recognized in the extracellular environment as cell-free DNA (cf-DNA). Cf-DNA was first
identified in the plasma of healthy individuals in 1948. However, this highly fragmented
double-stranded DNA can be detected in various body fluids (plasma/serum, urine,
cerebrospinal fluid, etc.) under physiological conditions such as exercise and aging, as well
as pathological conditions due to cell death or active release of cf-DNA by living cells.
This study aimed to evaluate possible elevations in cf-DNA levels in experimental models
of cardiac damage. Male adult Wistar rats were divided into 8 groups: Ct (control, received
10 mL/kg saline i.p.), Fe250 (250 mg/kg iron-dextran i.p. in the same final volume), Fe500
(500mg/kg), Fe1000 (1000mg/kg), Dox20 (doxorubicin 20mg/kg i.p.), Dox30 (30mg/kg),
and Iso150 (isoproterenol 150mg/kg s.c. divided into two doses), and Iso300 (300mg/kg
s.c. divided into two doses). Forty-eight hours later, blood and tissue samples were
collected, and serum was obtained by blood centrifugation, in which iron status parameters,
creatinine, creatine-phosphokinase MB, lactate dehydrogenase, alkaline phosphatase,
aspartate transaminase, alanine transaminase, and troponin I were measured. Additionally,
tissue iron levels were assessed by inductively coupled plasma optical emission
spectrometry (ICP-OES) in the heart, liver, and kidneys. Cf-DNA levels were estimated by
fluorimetry using SYBR® nucleic acid stain, or by quantitative PCR (qPCR) using specific
primers for mitochondrial gene fragments NADH-ubiquinone oxidoreductase chain 6
(ND6) and cytochrome B (Cyt-B). Finally, oxidative stress were evaluated by quantifying
malondialdehyde (MDA) as an estimate of lipid peroxidation, and advanced oxidation
protein products (AOPP) as an estimate of oxidative damage to proteins. All Fe groups
showed an increase in serum iron levels, ferritin, and transferrin saturation, and elevation
of cardiac damage markers troponin I and LDH especially in the Fe500 and Fe1000, and
LDH positively correlated with serum and cardiac iron levels. Serum creatinine was
elevated in the Fe250 and Fe1000 groups, and its level correlated with serum iron and
kidney iron content. Moreover, only AST was significantly increased by iron intoxication
at higher doses, which positively correlated with serum iron and hepatic iron content.
Finally, there was an increase in TBARS and AOPP in all Fe groups, this increase being
proportional to the level of intoxication and showing positive correlations with serum iron
levels, LDH, creatinine, and AST. Despite these results, it was not possible to assess cf-
DNA in iron-intoxicated groups using fluorimetry and qPCR methods. However, cf-DNA
quantification by qPCR was successful in the Ct group and the other models used. In the
isoproterenol model, there was no change in serum iron profile or tissue iron content, but
there was an elevation of LDH and troponin I. In the doxorubicin toxicity model, despite
no change on serum and tissue iron profiles, there was an elevation of LDH, creatinine, and
ALT at the higher dose, and AST at both studied doses. In the cf-DNA analysis, these two
models showed greater amplification (1/CT) of Cyt-B and ND6 fragments in a dose-effect
relationship. This result was reinforced by the analysis of cf-DNA Cyt-B and ND6
concentration, which increased in both the Iso 150 group (45.91 ng ± 0.94 ng/ml and 47.30
± 0.58 ng/ml, respectively) and the Iso 300 group (46.97 ± 0.54 ng/ml and 48.47 ± 0.87
ng/ml, respectively) compared to the Ct group (41.21 ± 0.42 ng/ml and 43.91 ± 0.62 ng/ml).
Similarly, in the doxorubicin injection model, both the Dox20 and the higher dose Dox30
groups increased cf-DNA levels of Cyt-B and ND6 in the serum (47.86 ± 0.84 ng/ml and
50.89 ± 1.03 ng/ml, respectively). Furthermore, positive correlations were identified
between the levels of these fragments in circulation and some of the serum markers used:
Cyt-B positively correlated with LDH and troponin I in the isoproterenol model, while Cyt-
B and ND-6 levels positively correlated with LDH, ALT, AST, and creatinine in the
doxorubicin model. In conclusion, acute parenteral iron intoxication resulted in increased
serum and tissue iron levels, and modified serum damage markers that correlate with the
level of intoxication and oxidative stress. Additionally, while the other two cardiopathy
models showed distinct effects on different organs, both resulted in elevated mitochondrial
cf-DNA levels for the two genes studied (Cyt-B and ND-6), suggesting that the analysis of
these circulating DNA fragments may be a useful biomarker, as they positively correlate
with different tissue damage biomarkers in these conditions.
