Sleep loss and acute drug abuse can induce DNA damage in multiple organs of mice

Abstract

The purpose of the present study was to characterize the genetic damage induced by paradoxical sleep deprivation (PSD) in combination with cocaine or ecstasy (3,4-methylenedioxymethamphetamine; MDMA) in multiple organs of male mice using the single cell gel (comet) assay. C57BL/6J mice were submitted to PSD by the platform technique for 72 hours, followed by drug administration and evaluation of DNA damage in peripheral blood, liver and brain tissues. Cocaine was able to induce genetic damage in the blood, brain and liver cells of sleep-deprived mice at the majority of the doses evaluated. Ecstasy also induced increased DNA migration in peripheral blood cells for all concentrations tested. Analysis of damaged cells by the tail moment data suggests that ecstasy is a genotoxic chemical at the highest concentrations tested, inducing damage in liver or brain cells after sleep deprivation in mice. Taken together, our results suggest that cocaine and ecstasy/MDMA act as potent genotoxins in multiple organs of mice when associated with sleep loss.

Introduction

The World Drug Report produced by the United Nations Office on Drugs and Crime estimated that between 172 and 250 million people used illicit drugs at least once during 2007. ¹ In particular, over 12 million individuals worldwide used ecstasy (the full chemical name is 3,4-methylenedioxymethamphetamine, MDMA) or cocaine. ¹

Complex interactions between genetic and environmental factors have been implicated in the transition from drug abuse to addiction as well as in the propensity to relapse after many drug-free years. ^{2 –5} Pre-clinical studies have shown a role for gene expression in cocaine-related behavioral processes, such as long-term adaptation in response to drugs of abuse. ^6,7 Recently, Romieu and colleagues ⁷ demonstrated the regulation of gene transcription in neurons by chromatin remodeling, suggesting that post-translational modifications of histones play a major role in this process. Taken together, these findings demonstrate a close relationship between drugs of abuse and genetic factors. However, the genetic mechanisms involved in addiction remain unknown.

Drugs like cocaine and ecstasy are known to lead to modifications of sleep architecture, ^{8 –11} difficulty in initiating and maintaining sleep, lowered sleep efficiency and significant delay in sleep onset. ¹² These facts suggest a close association between sleep deprivation and psychostimulant drug use. Indeed, Smith and colleagues ¹³ demonstrated that ecstasy users show impairments in tracking performance and divided attention that were more pronounced when the individuals were sleep deprived. Furthermore, in cocaine-dependent individuals, alterations in objective sleep quality accompany their characteristic binge-abstinence cycle. ¹²

Both sleep deprivation ^14,15 and drugs of abuse such as cocaine and MDMA ¹⁶ can induce genetic damage in multiple organs of rodents, and sleep loss can potentiate the risk caused by psychostimulant drugs. ¹⁷ Thus, the aim of this study was to evaluate the influence of paradoxical sleep deprivation (PSD) on genotoxicity in mice challenged with acute exposure to cocaine and MDMA.

Materials and methods

Animals

Male C57BL/6J inbred mice were bred and raised in the animal facility of the Centro de Desenvolvimento de Modelos Experimentais para Medicina e Biologia (CEDEME). The animals were housed in a colony that was maintained at 22°C with a 12:12 hour light-dark cycle (lights on at 07:00 a.m.) and allowed free access to food and water inside standard polypropylene cages. All procedures used in the present study complied with the Guide for the Care and Use of Laboratory Animals. All animal procedures were approved by the Ethical Committee of Universidade Federal de São Paulo (# 866/09).

Drugs

Systemic injections were administered intraperitoneally (i.p.) in a concentration of 1 mL/kg of cocaine (1.75, 3.5 and 7 mg/kg b.w.; Sigma, St. Louis, Missouri, USA) and MDMA (1.25, 2.5 and 5 mg/kg b.w.). The drug dosages were chosen based on previous studies, ^18,19 as well as our recent investigation using the same doses and protocol design. ¹⁶ The drugs were prepared immediately prior to administration by dissolving them in saline. The MDMA that we used in this experiment was donated to us by the Criminalistic Institute (São Paulo). The ecstasy that we used was prepared from ecstasy pills and was quantified using a validated high performance liquid chromatography/fluorescence detection (HPLC/FD) method. To confirm the absence of other active substances, the material was analyzed using the liquid chromatography/tandem mass spectrometry (LC/MS/MS) screening method.

Groups

The mice were randomly distributed into 7 independent groups (n = 5/group). The experimental groups were given cocaine (1.75, 3.5 and 7 mg/kg) or MDMA (1.25, 2.5 and 5 mg/kg) and the control group was given saline. All animals were submitted to 72 hours of PSD. Immediately after this period, mice were subjected to the respective drug or saline injections. One hour after administration, all mice were euthanized for blood, liver and brain (prefrontal cortex) sample collection. The CTRL-drugs groups were not included since our previous study reported the isolated effects of a single exposure to cocaine or ecstasy in multiple organs in mice. ¹⁶

Paradoxical sleep deprivation

The experimental groups were submitted to PSD for 72 hours using the modified multiple platform method, which consists of placing 5 mice in a conventional cage (38 × 31 × 17 cm) containing 12 circular platforms (3.5 cm in diameter) with water 1 cm below the platform surface. At the onset of each paradoxical sleep episode, the animal experiences a loss of muscle tonus and falls into the water, thereby awakening the animal. Food and water were available ad libitum. This procedure for inducing PSD in mice has been previously published by our group. ^20,21

Sample collection

Mice were decapitated using a rapid procedure that was carried out less than 1 minute after removal from the home cage. This time-sensitive procedure was used because DNA damage due to death can occur within 1 hour postmortem. ²² Blood was collected in sterile tubes containing liquid EDTA. An aliquot of blood was removed, and cellular suspensions (~10 μL) were used for the single cell gel (comet) assay. In addition, central fragments from the liver and brain (prefrontal cortex) were collected and minced in 0.9% NaCl. The supernatant was removed, and the cellular suspensions (~10 μL) were used for the single cell gel (comet) assay as well.

Single cell gel (comet) assay

The protocol used for peripheral blood, liver and brain (prefrontal cortex) cells followed the guidelines outlined by Tice et al. ²³ with some modifications. Briefly, 5 μL of peripheral blood or cellular suspensions from liver or brain were added to 120 μL of 0.5% low-melting-point agarose at 37°C. Cells were then layered onto a pre-coated slide with 1.5% regular agarose and covered with a coverslip. Prior to electrophoresis, the slides were left in alkaline buffer (pH > 13) for 20 minutes and then electrophoresed for another 20 minutes at 0.7 V/cm and 300 mA. After electrophoresis, the slides were neutralized in 0.4 M Tris-HCl (pH 7.5), fixed in absolute ethanol and stored until the time of analysis on a fluorescent microscope using a 400× magnification. Independent positive controls using cells from peripheral blood that were treated in vitro with 10 μM H₂O₂ (hydrogen peroxide) for 10 min at 4°C were used to ensure the reproducibility and sensitivity of the assay.

Genotoxicity data analysis

A total of 50 randomly captured comets per animal (25 cells from each slide) ²⁴ were examined blindly by one expert observer at 400× magnification using a fluorescent microscope (Olympus). The microscope was connected through a black and white camera to an image analysis system (Comet Assay II, Perceptive Instruments, Suffolk, Haverhill, UK) that had been previously calibrated according to the manufacturer’s instructions. The computerized image analysis system acquires images, computes the integrated intensity profiles for each cell, estimates the comet cell components and ultimately evaluates the range of derived parameters. Undamaged cells have an intact nucleus without a tail, whereas damaged cells have the appearance of a comet. To measure DNA damage, 2 image analysis system parameters were considered: tail intensity (% migrated DNA) and tail moment (the product of the tail length and the fraction of DNA in the comet tail). ²⁴ Given that none of the groups we analyzed showed significant differences between these parameters, we chose to present our results in terms of tail moment.

Statistical methods

Considering that tail moment data are expressed in arbitrary units, values were evaluated statistically using the Kruskal-Wallis non-parametric test followed by the post-hoc Dunn’s test using Sigma Stat for Windows (Jadel Scientific, San Rafael, CA, USA). Values are expressed as mean ± SD. The level of significance was set at p < 0.05.

Results

In this study, we were able to evaluate genetic damage in peripheral blood cells induced by cocaine or MDMA exposure when associated with sleep loss in vivo. Cocaine induced genetic damage in blood in all doses evaluated and in liver cells at the highest concentrations. Furthermore, cocaine was able to induce genetic damage in the brain of sleep-deprived mice at all of the concentrations that we tested. This data is summarized in Figure 1 .

Figure 1.

DNA damage (tail moment) induced by cocaine following sleep deprivation in multiple organs of mice. Results are expressed as mean ± SD.*p < 0.05 when compared to C0 − negative control; C1 − 1.75; C2 − 3.5 and C3 − 7 mg/kg.

In mice treated with MDMA, an increased DNA migration rate was detected in blood cells for all concentrations tested, suggesting extensive genotoxic effects of MDMA after sleep deprivation in mice. These results are displayed in Figure 2 . Interestingly, the analysis of the tail moment data suggests that high concentrations of MDMA are genotoxic to liver and brain cells when the animal is under the effects of sleep deprivation.

Figure 2.

DNA damage (tail moment) induced by 3,4-methylenedioxymethamphetamine (MDMA) following sleep deprivation in multiple organs of mice. Results are expressed as mean ± SD.*p < 0.05 when compared to M0 − negative control; M1 − 1.25; M2 − 2.5 and M3 − 5 mg/kg.

Mouse blood was also treated with H₂O₂ to ensure the sensitivity of the assay as a positive control. A clear sensitivity to DNA damage was observed in animals given drugs (p < 0.05) as compared to negative controls. No animal died unexpectedly during this experiment. Figure 3 shows an undamaged rat blood cell from a negative control, a blood cell exposed to cocaine and an H₂O₂-induced comet cell (positive control).

Figure 3.

Representative comet images of a blood cell from a negative control (A), a cell exposed to cocaine (B) and an H₂O₂-treated cell (positive control; C). DNA was stained with ethidium bromide; 400× magnification.

Discussion

The aim of the present study was to evaluate the genetic damage induced by experimental sleep loss associated with the use of 2 very common illicit drugs (cocaine and ecstasy) in mice. The investigation was conducted using a single cell gel (comet) assay. To the best of our knowledge, this approach has not been demonstrated previously.

The alkaline version of the single cell gel (comet) assay that was used here is sensitive to a wide variety of DNA lesions, including single- and double-strand breaks, oxidative DNA base damage, alkali-labile sites including a basic and incomplete repair sites and DNA-DNA/DNA-protein/DNA-drug cross-linking in eukaryotic cells. ²³ Tail moment is a virtual measure that is calculated with a computerized image analysis system that considers both the length of DNA migration in the comet tail and the tail intensity. This parameter is one of the best indices for induced DNA damage as compared to the various parameters calculated using this method.

In the present study, as in our previous investigations using this single cell gel (comet) assay, we have excluded comets without clearly identifiable heads (i.e., comets with most of their DNA in their tails after electrophoresis) during the image analysis. Although the meaning of what these ‘clouds' actually represent remains unknown, this type of comet was excluded based on the assumption that these cells have died from the putative cytotoxic effects of cocaine or MDMA exposure rather than due to a direct interaction between DNA and a genotoxic agent that causes them to display primary DNA damage. ²⁵ Other researchers have also used this method of analysis. ²⁶

In this study, we were able to demonstrate that DNA damage occurs in peripheral blood, liver and brain cells of mice that are sleep-deprived and have experienced acute exposure to cocaine. The effect is more pronounced at the highest concentration. Our findings are consistent with previous research that has found evidence for DNA mutagenesis after 1 hour, with both cocaine hydrochloride and freebase. ²⁷ A recent study conducted by our group has consistently demonstrated genotoxic damage in mice after a single exposure to cocaine. ¹⁶ Sub-acute cocaine exposure promotes toxicity in multiple organs, and at low doses, it can induce hepatic damage without gross pathological injury to the heart in a murine model. ²⁸ Other studies have similarly shown that, following 2 hours of treatment, a reduction in the DNA content of protozoa was also observed. ²⁷ Therefore, cocaine N-oxidative metabolism may be an indirect contributor to oxidative stress ²⁹ increased oxidative stress reported in the frontal cortex and the striatum of rats exposed to cocaine suggests that oxidative damage plays a significant role in cocaine-induced disruption of the central nervous system and is also relevant to apoptosis in cocaine-exposed models. ³⁰ Taken together, our results suggest that cocaine can increase the genetic damage in peripheral blood, liver and brain cells when combined with sleep loss.

When we evaluated MDMA in this study, our results demonstrated that this compound induced extensive genotoxic damage in blood cells for all doses tested when it was associated with sleep loss in mice. However, the genotoxic damage in the brain cells was observed only at the highest dose of MDMA. Thus, in contrast to the effects of cocaine, it seems that it is necessary a higher dose of MDMA is required to promote brain damage. Several studies have also demonstrated the noxious effects induced by ecstasy exposure. For example, previous research suggests that very low doses of ecstasy lead to acute oxidative stress, DNA single- and double-strand breaks and long-lasting metabolic changes in the hippocampus. ^31,32 Exposure to ecstasy generates selective hippocampal alterations that may underlie cognitive impairment and seizure susceptibility. ³² Others have found that the administration of a neurotoxic binge dose of ecstasy to a pubertal rat model that was previously treated with the specific MAO-A inhibitor, clorgyline, resulted in synergistic effects on serotonin (5-HT)-mediated behavior and body temperature, eventually leading to high mortality. ³³ Inhibition of MAO-A by clorgyline had no protective effect on ecstasy-induced alterations of brain mitochondrial function, such as increased lipid peroxidation and protein carbonylation and decreased expression of respiratory chain subunits. In line with our previous study that demonstrates DNA damage in several organs after a single MDMA injection, it can be stated that ecstasy may have a genotoxic effect when associated with sleep loss in mice.

There are several different approaches that may be taken to understand the functional properties of paradoxical sleep. ³⁴ A common procedure involves deprivation of paradoxical sleep in various organisms and subsequent observation of their behavioral and physiological changes. Thus, most studies on sleep deprivation focus on paradoxical sleep deprivation (PSD). In the present study, we utilized the PSD strategy to investigate the interaction between selective sleep loss and drug exposure. We recently identified a total of 78 unique transcripts that were differentially expressed in animals that experienced PSD relative to controls. ³⁵ These differentially expressed transcripts include genes from many functional classes, including the circadian sleep-wake cycle, the response to a stimulus, regulation of cell proliferation and signaling pathways. Intriguingly, DNA damage is an important step in the events leading to genomic instability. This current study represents a relevant contribution to the understanding of the potential health risks of single drug exposure that is associated with sleep deprivation.

In conclusion, the present findings shows the sleep loss potentiating the genotoxic effects of cocaine and MDMA in mice. Considering that DNA damage is an important step in events leading to genomic instability and tumorigenesis, this study provides further insights into the potential health risks of some illicit drugs when associated with the sleep loss that is typical in our modern society. Further investigation is needed to confirm and extend these results.