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Different mechanisms have been proposed with respect to and hyperemia/congestion are some of the histopathological
the occurrence of arrhythmias. One of the important theories changes observed in the heart.24,25 During acute cardiac complica-
related to arrhythmias suggests that the number of impulse- tions or sudden death scenarios, tissue does not undergo necrosis
generating foci or a single rapid firing focus gives rise to fibril- or apoptosis because it usually takes time to reach that point of tis-
lation.12 This theory is known as ectopic foci theory, where sue damage. In these cases, preliminary changes such as hyper-
there are abnormal pacemaker sites within the heart apart from emia or congestion can occur. Both hyperemia and congestion
sino-atrial node that display automaticity.13 Thus, ectopic foci can are the result of excessive blood in a tissue. Hyperemia, if ob-
be a region other than sinus node where impulse formation can served under microscope, gives bright red and swollen appear-
lead to cardiac arrhythmias.14 Abnormal focal activity or ectopic ance. Tissue damage may not be evident, but capillaries are
foci can arise from afterdepolarization. Afterdepolarization refers engorged with blood. Physiologically, hyperemia/congestion can
to increase in intracellular calcium concentration and decrease in occur, because there is increase in demand of nutrients because
potassium and magnesium concentrations.15 Apart from electro- of cardiac work or stimulus associated with irritation of tissue.
lyte changes, cardiomyocyte damage can be other reason that Later, tissue may develop edema, thrombus, weakened ventricular
can affect changes in normal rhythm.14 muscle, and necrosis.
Another important mechanism for arrhythmias is the re-entry Based on recent increase in DFE abuse and related complica-
theory. Re-entry can take place within a small local region in the tions to it, we established a rat model to simulate the clinical con-
heart.13 Re-entry arrhythmias can be caused by electrophysiolog- ditions of inhalation abuse that resulted in sudden death. In this
ical abnormalities, but arrhythmias resulting from this mechanism study, the effects of multiple doses of DFE on the cardiovascular
do not need to depend on it.15 Many antiarrhythmic drugs alter the system were monitored.
effective refractory period or conduction velocity and thus reverse 1,1-Difluoroethane is abused by direct inhalation. Thus, we
the effects of re-entry mechanisms. mimicked the same route of administration in rats and evaluated
Electrolyte imbalance plays an important role in the genesis the ability of DFE to elicit arrhythmias. Furthermore, to elucidate
of arrhythmias. Electrolyte imbalance can alter cardiac ionic cur- mechanisms behind these arrhythmias, electrolyte levels, biomarker
rents, which can increase arrhythmogenic effects.16 Experiments levels, oxidative stress markers, and pathological changes were
have shown that combination of increased potassium levels in se- evaluated. It was hypothesized that these arrhythmogenic effects
rum and decreased calcium levels have cumulative effects on car- of 1,1-difluoroethane were due to electrolyte changes, enzyme
diac conduction and may result in ventricular tachycardia and leakage, stress parameters, and pathological changes in vivo.
ventricular fibrillation.17,18 Magnesium is the second most abun-
dant intracellular ion found in cardiomyocytes after potassium.16
Magnesium has opposing effects to calcium because it has been MATERIALS AND METHODS
shown that magnesium blocks the calcium ion channel. Thus, an
increase in serum magnesium levels is associated with decreased Chemicals and Instrumentation
calcium levels.
Cardiac biomarkers are biological parameters that can be 1,1-Difluoroethane of 98% (600 g) was obtained from Sigma-
measured and quantified as an indicator of changes occurring Aldrich chemical (St. Louis, Mo). Propranolol, epinephrine, lido-
in the heart. These substances are first released into blood when caine, amiodarone, verapamil, and metoprolol were purchased
there is cardiac damage or stress. The markers can be indicators from VWR chemicals. Isoflurane 5 % acted as control for all the
of myocardial infarction, acute coronary syndrome, cardiac ische- experiments. Grass polygraph, electrocardiogram (ECG) pream-
mia, and necrosis.19 Critical markers of early tissue necrosis or plifier, and subdermal needle electrodes were obtained from Grass
myocardial ischemia are cardiac troponin I (cTnI), creatine ki- Technology (West Warwick, RI). Microtome for pathology exper-
nase (CK), and lactate dehydrogenase (LDH). Troponin is con- iments was obtained from Reichert-Jung (Depew, NY).
sidered to be the standard for diagnosis of any acute injury with
additional information from patient history and electrocardiogram Experimental Animals
(EKG) recordings.20 Male Sprague Dawley rats (275300 g) were purchased from
Cardiac arrhythmias can cause sudden cardiac death, and Taconic farms, Germantown, New York. The animals were main-
some mechanistic studies and pathological processes link the tained at the St. John's University Animal Facility. All procedures
cause to oxidative stress caused by reactive oxygen species (ROSs). mentioned were approved by the Institutional Animal Care and
In excitable cardiomyocytes, ROSs regulate both cellular metabo- Use Committee of St. John's University. The research was con-
lism and ion homeostasis.21 Reactive oxygen species such as sin- ducted in compliance with the Animal Welfare Act and other
glet oxygen, superoxide radical, and hydroxyl radical are highly federal statutes.
unstable and highly reactive. A study on isolated heart showed that
exposure to oxygen radicals for brief time intervals caused a de-
crease in high-energy phosphates, loss of contractile function of Plasma Sample and Heart Collection
heart, and some structural abnormalities.22 To counter the effects Blood samples from rats were collected by cardiac puncture
of free radicals, enzymatic and nonenzymatic antioxidants are either under isoflurane anesthesia for control group or DFE for
usually present within the cell. Three important cellular enzymes treatment group and were immediately stored in lithium heparin
are superoxide dismutase (SOD), glutathione peroxidase (Gpx), tubes, followed by centrifugation at 1500g and 4C for 10 minutes
and catalase (CAT). Some ex vivo studies showed that there was to separate plasma fraction from blood. Plasma was transferred to a
impaired energy production, depressed contractile function, and capped polypropylene 2-mL tubes and stored at 80C until needed.
increased lipid peroxidation when isolated heart was exposed to
free radicals.23
Enzyme leakage (LDH, CK, and troponin) and oxidative 1,1-Difluoroethane Exposure
stress markers can be due to cardiomyocyte damage or histopath- Rats were exposed to a fixed amount of DFE using a flow
ological changes in the heart. Apoptosis, necrosis, disorientation meter designed to deliver gas to a plastic chamber with an area
of cells, uneven pigmentation of cells, appearance of granules, of 2300 cm3.2 The chamber had an inlet through which DFE
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was administered and an outlet connected to charcoal canister Assay for Potassium
for safety. A total of 0.05 mL of specimen is added to trichloroacetic
The DFE flow was regulated by a flow meter, which was set acid solution, followed by centrifugation at high speed for 5 to
at a rate of 20 L/min, and this flow rate was kept constant for all 10 minutes. The supernatant added to mixture of potassium boron
the experiments. All the animals were given a constant multiple and sodium hydroxide. Sample is transferred to cuvette and incu-
DFE doses for 30 seconds at 20-L/min flow rate with 1-minute bated at room temperature for 5 minutes. The cuvette is read
break between each dose.2 After multiple exposures, the animals at 580 nm.
were immediately removed and blood was collected by cardiac
puncture. For pathology experiments, the hearts were placed in Assay for Magnesium
10% formalin buffer and fixed using paraffin after 3 to 4 days.
A total of 0.01 mL of sample or standard is mixed with 1-mL
Isoflurane acted as control for all the experiments, because we
Xylidyl Blue-1, and the mixture is incubated at room temperature
wanted to use a halogenated compound, which has no cardiac ef-
of 10 minutes and read at 520 nm.
fects and can also be used as anesthesia.
Cardiac Biomarkers
Electrocardiogram Monitoring and Onset of Death
Cardiac biomarkers, LDH and CK, were measured using
In an EKG test, the electrical impulses made while the heart Stanbio assay kits, whereas troponin I was measured by perform-
is beating are recorded and usually shown on a piece of paper ing enzyme-linked immunosorbent assay (ELISA), using Life
known as electrocardiograph. Rats were divided into 7 groups. Diagnostic ELISA kit.
The first group was treated with multiple DFE doses as discussed
earlier. The remaining groups were treated with propranolol + DFE, Assay for LDH
verapamil + DFE, amiodarone + DFE, lidocaine + DFE, and met-
oprolol + DFE, the last group was given a dose of isoflurane, Specimens mixed with working reagent prepared from LDH
which acted as control for the experiment. buffer and nicotinamide adenine dinucleotide (NAD). The cuvette
For DFE administration, 1 rat was placed at a time in the is incubated at 37C for a minute and absorbance is recorded at
chamber and was given a dose of 20-L/min DFE for 30 seconds. 1 minute. Furthermore, continuing the incubation absorbance is
Immediately, pin electrodes were inserted subcutaneously in 4 again recorded at 2- and 3-minute mark. Average absorbance is
limbs of the rat, and ECG machine was started, which was determined per minute (A/min) and multiplied by the factor
followed by ECG recording for 30 seconds. After 30-second re- 3376 to get amount of LDH in unit per liter.
cording, the rats were given a second dose of DFE. Similarly, this
procedure was repeated 4 to 5 times or until death occurred. Thus, Assay for CK
30-second EKG recording was obtained after every DFE. Thus, Specimens mixed with working reagent are prepared from
every animal had a total of 2- to 3-minute overall recording on imidazole buffer and CK enzyme reagent containing nicotinamide
the electrocardiograph. In addition, time of death for all the animal adenine dinucleotide phosphate (NADP) and adenosine diphos-
groups was recorded. phate. The cuvette is incubated at 37C for a minute, and absor-
For the remaining groups, the same procedure for DFE ad- bance is recorded at 1 minute. Furthermore, continuing the
ministration was followed except that antiarrhythmic agents were incubation absorbance is again recorded at 2- and 3-minute mark.
administered via intraperitoneal route before DFE administration. Average absorbance is determined per minute (A/min) and mul-
Antiarrhythmic agents such as propranolol, metoprolol, verapamil, tiplied by the factor 3376 to get amount of LDH in unit per liter.
lidocaine, and amiodarone were administered at 5-, 25-, 10-, 1-, and
50-mg/kg doses, respectively. Last group acted as a control, and Assay for cTnI
isoflurane 5 % was used for all the animals. The ELISA uses 2 different affinity purified antibodies. First
is used for immobilization on well plate, and the second is conju-
gated to horseradish peroxidase (HRP). Plasma sample is diluted
Electrolyte Panel with plasma diluent and allowed to react simultaneously with the
Sodium, calcium, potassium, and magnesium levels were mea- 2 antibodies, resulting in cTnI being sandwiched between solid
sured using kits purchased from Stanbio Laboratory (Boerne, Tex). phase and HRP-conjugated antibodies. After 1 hour of incubation
on the shaker, the plates are washed to remove any unbound HRP-
conjugated antibodies. A solution of HRP substrate, tetramethyl-
Assay for Sodium benzidine, is added, and plates are incubated for 20 minutes
For measuring the sodium levels, 0.5-mL plasma or standard resulting in development of blue color. The color development is
was added to test tube and mixed with 0.5-mL precipitating re- stopped by addition of 1 N HCl, and the concentration on cTnI
agent (aqueous trichloraacetic acid). The mixture is centrifuged is proportional to the absorbance at 450 nm. The amount of cTnI
at high speed for 5 to 10 minutes. A total of 0.5 mL of supernatant is calculated by using a standard curve and graph.
is then added to 2.5-mL coloring reagent (uranyl acetate). The
mixture is incubated at room temperature for 10 minutes and cen-
trifuged at high speed for 5 minutes. Supernatant is transferred to Oxidative Stress Assay
cuvette and read at 420 nm against blank.
Assay for CAT
The CAT activity was measured according to a well-known
Assay for Calcium method, which is based on the rate of disappearance of exogenous
For calcium levels, 0.5 mL of specimen is added to coloring H2O2 added to the sample.26
reagent containing orthocresolphthalein complexone. The mixture One-milliliter sample was mixed with 1 mL of 30 mM H2O2
is incubated at room temperature for 5 minutes. Absorbance is and 1 mL of 0.01 M PBS pH 7.4, in a spectrophotometric quartz
read at 550 nm. cuvette. The changes in absorbance of reaction mixture was
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measured at 240 nm twice, once immediately after mixing and Adenosine Triphosphate Measurement
again 1 minute later.
The enzyme activity was calculated as nM of H2O2 reduced/ Adenosine triphosphate (ATP) is primary energy currency of
mg protein/min. the living system. Virtually, all energy-dependent processes use
the chemical energy stored in phosphate bond of ATP. Principle
in ATP ELISA involves a simple method, which uses the phos-
Assay of GPx phorylation of glycerol to product that is easily quantified by
Glutathione peroxidase catalyzes the conversion of glutathi- colorimetric method.
one to glutathione disulfide. In the presence of an excess of The plasma samples are first deproteinized. Ten microliter of
-NADPH and glutathione reductase, glutathione disulfide is re- deproteinized sample is added to 96 well plate, and volume is ad-
duced to GSH and NADPH is reoxidized to NADP+. The rate of justed to 50 L by ATP assay buffer. A reaction mixture consisting
decrease in absorbance at 340 nm as a result of the conversion of ATP probe, ATP converter, and developer is made, and 50 L of
of NADPH to NADP+ reflects the activity of GPx. The GPx the reaction mixture is added to all the plates. Simultaneously,
activity was measured according to the method of Paglia and Val- ATP standard curve is prepared by following the same procedure.
entine27 as modified by Greenwald.28 To a 1-mL spectrophoto- Samples and standards are incubated for 30 minutes and protected
metric quartz cuvette, 500 L of 0.01 M PBS pH 7.4, 100 L of from light. Absorbance is measured at 570 nm in a micro-plate
10 U/mL glutathione reductase solution, and 100 L of 10 mM reader. Amount in ATP was calculated by the following equation:
GSH were added in succession. After mixing and standing at ATP concentration (C) = B/V D (nmol/L), where B is ATP
room temperature for 10 minutes, the reaction mixture was mixed amount from standard curve, V is sample volume added into sam-
with 100 L of 15 mM -NADPH and incubated at 37C for ple wells, and D is dilution factor.
3 minutes. After the addition of 100 L of 5 M H2O2, the decrease
in absorbance of the reaction mixture was monitored at 340 nm
for 1 minute. The enzyme activity was calculated as nM product Epinephrine Measurement
oxidized/mg of protein/min.
Epinephrine was assayed using competitive ELISA kit by
Abnova. Upon acylation and extraction as per the assay protocol,
Assay of SOD the samples and standards were added to 25-L enzyme solution
The activity SOD was measured by the spectrophotometric in 96 well plate. Furthermore, 50-L adrenaline antiserum was
method.28 This method measures the ability of SOD to prevent added, and the samples and standards were incubated on a shaker
the autoxidation of epinephrine by O2 to a pink adrenochrome at room temperature for 2 hours. The contents of the well were
showing a strong absorbance at 480 nm. Hence, the extent of discarded, and plate was washed thrice using wash solution. One
inhibition of autoxidation, as reflected by the dismutation of hundred microliter of enzyme conjugate was added, and plate
O2 by SOD, is taken as measure of catalytic activity. A 100-L al- was incubated for 30 minutes on a shaker at room temperature.
iquot of sample was diluted with 1.8 mL of NaHCO3 Na2CO3 After another wash, 100 L of substrate was added to all the
buffer pH 10.2, mixed with 100 L of 0.01 M ()-epinephrine, wells and incubated for 30 minutes at room temperature. Finally,
and allowed to stand at room temperature for 1 minute. The 100-L stop solution was added and absorbance was read be-
change in absorbance of the reaction mixture was read at 480 nm tween 620 and 650 nm within 10 minutes. Values for unknown
for 1 minute. The enzyme activity was calculated as nM product samples were obtained after using nonlinear regression curve
oxidized/mg of protein/min. fitting for standards.
FIGURE 1. Effect of DFE on EKG monitoring. A, Regular sinus rhythm after isoflurane treatment. B, Premature ventricular contractions
after DFE treatment. Ventricular tachycardia (C) and ventricular fibrillation (D) after DFE treatment.
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FIGURE 3. Effect of DFE on electrolyte levels plasma sodium (A), plasma calcium (B), plasma potassium (C), and plasma magnesium (D)
for treatment and control groups. Data are expressed as mean SEM, N = 6 rats per group, P 0.05 was considered statistically significant
and was denoted as * for EPI + isoflurane and + for isoflurane group.
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FIGURE 4. Effects on cardiac biological markers and ATP levels after DFE treatment LDH (A), CK (B), troponin I (C), and ATP levels (D)
for treatment and control groups. Data are expressed as mean SEM, N = 6 rats per group, P 0.05 was considered statistically significant
and was denoted as * for EPI + isoflurane and + for isoflurane group.
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FIGURE 5. Effect of DFE on oxidative stress markers SOD (A), CAT (B), and Gpx levels (C) for treatment and control groups. Data are expressed
as mean SEM, N = 6 rats per group, P 0.05 was considered statistically significant and was denoted as * for EPI + isoflurane and + for
isoflurane group.
shown in Figure 5, A and C whereas plasma CAT levels were sig- amiodarone showed that time for onset of death was increased
nificantly elevated in DFE- and EPI + DFEtreated groups as seen as compared with DFE-treated animals.
in Figure 5B.
DISCUSSION
Histopathological Analysis After DFE Treatment
In the last few years, clinical cases and reports related to DFE
The heart sagittal sections for treatment and control groups
abuse have gone up markedly. With its intended use, very little
were evaluated for histopathological changes. All the sections
risk is involved; on the contrary, DFE-containing products have
were analyzed after treating with hematoxylin and eosin stain.
become abuse substrates and have been diverted from their origi-
Changes with respect to congestion or hyperemia were checked
nal intended use. Toxicokinetic studies have been recently per-
for all the sections. Isoflurane and EPI + isoflurane sections did
formed,2 but the mechanism of DFE action still remains unknown.
not see any changes as described in Figure 6, A and C respectively,
The current study determines the arrhythmogenic effects of DFE
whereas DFE and EPI + DFE treatment showed congestion/
and reasons behind it, which might answer the cause of death
hyperemia as shown with black arrows in Figure 6, B and D re-
due to DFE and determine the mechanism of action occurring be-
spectively. Furthermore, the sections were graded on the basis of
cause of DFE intoxication. 1,1-Difluoroethane has also been seen
a scale from 0 to 3, with 0 being least severe damage and 3 being
to cause lethargy in accidents involving its abuse; hence, giving a
highest damage. As shown in Table 1, there was significant difference
clue to its central nervous system effects. In a pharmacokinetic
in the grading for treated and control groups. 1,1-Difluoroethane
study in vivo, tissue levels of DFE were determined at different
and EPI + DFE got grading of 2.8 and 2.7, respectively, whereas
time points after its exposure. The brain and heart showed DFE
control groups got a grading of 0.8.
in tissues at different levels, but DFE level in the heart remained
higher than the brain tissue after approximately 60-second post-
Protection Experiment DFE withdrawal.2 A previous report also had speculated that
To check if antiarrhythmic agents have an effect on onset of DFE elimination is rapid in the brain as compared with the heart.29
death, protection experiment was performed in vivo. As shown in This may further result in abuser inhaling more DFE to sustain eu-
Figure 7, antiarrhythmic agents, propranolol, metoprolol, and phoric effect because central nervous system effects are reduced
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FIGURE 6. Histopathological analysis after DFE treatment. Histopathology of heart. Isoflurane (A), DFE (B), isoflurane + EPI (C), and
EPI + DFE (D). Black arrows showing hyperemia/congestion.
and thus lead to accumulation in the heart. In another case report, a DFE-treated animals. 1,1-Difluoroethanetreated animals had sig-
24-year-old woman was pronounced dead after a vehicle accident nificantly higher amount of epinephrine release than the control
involving inhalation of DFE.9 Aortic blood showed highest DFE group, which confirms the process happening in the clinical cases
concentration, which was around 85.6 mg/L. Lower blood levels as shown in some studies. While performing in vivo studies, it has
were calculated in sites, which were further from the lung and been observed that insufficient epinephrine is usually released,
heart. This demonstrated the difference in absorption of DFE and hence, a common procedure is to administer epinephrine ex-
through the body tissues. As DFE or similar volatile compounds ternally to mimic conditions similar to clinical cases or accidents
have low solubility in water, it is highly unlikely to be absorbed during inhalation or volatile substance abuse. Hence, apart from
in nasopharyngeal region or trachea. Furthermore, most DFE isoflurane and DFE groups, 2 other experimental groups were
absorption occurs through alveoli, where it equilibrates with used in our study. Epinephrine + isoflurane and epinephrine + DFE
the blood of the pulmonary capillaries. Thus, DFE diffuses into were 2 additional groups, which relate to clinical conditions dur-
the blood and can be well distributed throughout the different ing such abuse cases.
organs in the body.2 Two important mechanisms behind arrhythmias are ectopic
The present study deals with arrhythmogenic effects and the foci and re-entry phenomena. As discussed earlier, changes in
possible mechanism behind DFE toxicity. Electrocardiographic electrolyte levels play an important role in the genesis of these ar-
monitoring was performed to see changes in cardiac rhythms, rhythmias. Many studies have shown an increase in extracellular
and several arrhythmias were observed ranging from low to high potassium and a decrease in extracellular calcium (or increase in
severity. After multiple DFE doses in rats, arrhythmias such as intracellular calcium) can cause arrhythmias.17,18
skipped beat, PVCs, coupled beats, ventricular tachycardia, and We checked electrolyte levels for sodium, calcium, potas-
ventricular fibrillation were observed. This confirms previous sium, and magnesium in rat plasma. Results showed no significant
data in which 42-year-old man was found dead after repeated ex- change in plasma sodium and plasma calcium levels. Plasma
posure to DFE.29
Studies have shown cardiac arrhythmias to be the most com-
mon cause of deaths due to volatile solvent abuse.30 The evidence TABLE 1. Histopathology Grading for Treatment and Control
for this has been shown during the early 1900s when some pa- Groups
tients under chloroform anesthesia suddenly die because of car-
diac arrhythmias.31 In that study, the group showed that when Treatment Groups Grading
myocardium was exposed to chloroform, it was sensitive to epi-
nephrine. Epinephrine and sympathetic nervous system stimula- Isoflurane 0.8
tion later resulted in abnormal cardiac muscle contraction.31 Later, DFE 2.8+*
many such studies were reported with sensitization of myocardium EPI + isoflurane 0.8
to epinephrine by volatile solvents during their use in hospitals, EPI + DFE 2.7+*
factories, industrial environment, or abuse.30 Hence, it is impor-
Data are expressed as mean SEM, N = 6 rats per group analyzed by
tant to know the amount of epinephrine released endogenously
Mann Whitney U test, P 0.05 was considered statistically significant
during abuse cases. Thus, as shown in Figure 2, the release of en- and was denoted as * for EPI + isoflurane and + for isoflurane group.
dogenous epinephrine in plasma was checked for isoflurane- and
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10. Dubin D. Rapid Interpretation of EKG's: An Interactive Course. 6th ed. 25. Cai H, Dikalov S, Griendling KK, et al. Detection of reactive oxygen
Tampa, FL: Cover Pub. Co.; 2000:368. species and nitric oxide in vascular cells and tissues: comparison of
11. Thaler MS. The Only EKG Book You'll Ever Need. 7th ed. Philadelphia: sensitivity and specificity. Methods Mol Med. 2007;139:293311.
Wolters Kluwer Health/Lippincott Williams & Wilkins; 2012:333. 26. Aebi H. Catalase in vitro. Methods Enzymol. 1984;105:121126.
12. Nattel S. New ideas about atrial fibrillation 50 years on. Nature. 2002;415 27. Paglia DE, Valentine WN. Studies on the quantitative and qualitative
(6868):219226. characterization of erythrocyte glutathione peroxidase. J Lab Clin Med.
1967;70(1):158169.
13. Klabunde RE. Cardiovascular Physiology Concepts. Philadelphia:
Lippincott Williams & Wilkins; 2005:235. 28. Greenwald RA. CRC handbook of methods for oxygen radical research.
Boca Raton, FL: CRC Press; 1985:447.
14. Gaztaaga L, Marchlinski FE, Betensky BP. Mechanisms of cardiac
arrhythmias. Rev Esp Cardiol (Engl Ed). 2012;65(2):174185. 29. Avella J, Wilson JC, Lehrer M. Fatal cardiac arrhythmia after repeated
exposure to 1,1-difluoroethane (DFE). Am J Forensic Med Pathol.
15. Hoffman BF, Rosen MR. Cellular mechanisms for cardiac arrhythmias.
2006;27(1):5860.
Circ Res. 1981;49(1):115.
30. Shepherd RT. Mechanism of sudden death associated with volatile
16. Fisch C. Relation of electrolyte disturbances to cardiac arrhythmias. substance abuse. Hum Toxicol. 1989;8(4):287291.
Circulation. 1973;47(2):408419.
31. Levy AG. Sudden death under light chloroform anaesthesia. Proc R Soc
17. Niemann JT, Cairns CB. Hyperkalemia and ionized hypocalcemia during Med. 1914;7(Sect Anaesth):5784.
cardiac arrest and resuscitation: possible culprits for postcountershock
arrhythmias? Ann Emerg Med. 1999;34(1):17. 32. Bellet S. The electrocardiogram in electrolyte imbalance. AMA Arch Intern
Med. 1955;96(5):618638.
18. El-Sherif N, Turitto G. Electrolyte disorders and arrhythmogenesis. Cardiol
J. 2011;18(3):233245. 33. Cieslik-Guerra UI, Rechciski T, Trzos E, et al. Cardiotoxic effect due to
accidental ingestion of an organic solvent. Int J Occup Med Environ
19. Singh V, Martinezclark P, Pascual M, et al. Cardiac biomarkers - the old and Health. 2015;28(1):174179.
the new: a review. Coron Artery Dis. 2010;21(4):244256.
34. Eshaghi M, Zare S, Banihabib N, et al. Cardioprotective effect of Cornus
20. Adams JE 3rd, Miracle VA. Cardiac biomarkers: past, present, and future. mas fruit extract against carbon tetrachloride induced-cardiotoxicity in
Am J Crit Care. 1998;7(6):418423. albino rats. J Basic Appl Sci Res 2012;2(11):1110611114.
21. Jeong EM, Liu M, Sturdy M, et al. Metabolic stress, reactive oxygen 35. Barth AS, Tomaselli GF. Cardiac metabolism and arrhythmias.
species, and arrhythmia. J Mol Cell Cardiol. 2012;52(2):454463. Circ Arrhythm Electrophysiol. 2009;2(3):327335.
22. Singal PK, Khaper N, Palace V, et al. The role of oxidative stress in the 36. Murphy E, Steenbergen C. Ion transport and energetics during cell death
genesis of heart disease. Cardiovasc Res. 1998;40(3):426432. and protection. Physiology (Bethesda). 2008;23:115123.
23. Lefer DJ, Granger DN. Oxidative stress and cardiac disease. Am J Med. 37. Dndarz MR, Trkbay T, Akay C, et al. Antioxidant enzymes and lipid
2000;109(4):315323. peroxidation in adolescents with inhalant abuse. Turk J Pediatr.
24. Bardales RH, Hailey LS, Xie SS, et al. In situ apoptosis assay for the 2003;45(1):4345.
detection of early acute myocardial infarction. Am J Pathol. 1996;149(3): 38. Mee AS, Wright PL. Congestive (dilated) cardiomyopathy in association
821829. with solvent abuse. J R Soc Med. 1980;73(9):671672.
2017 Wolters Kluwer Health, Inc. All rights reserved. www.amjforensicmedicine.com 125