Environ Sci Pollut Res DOI 10.1007/s11356-013-2447-6
RESEARCH ARTICLE
Cytogenetic biomonitoring of oral mucosa cells of crack cocaine users Maria das Graças Alonso de Oliveira & Jean Nunes dos Santos & Patrícia Ramos Cury & Victor Hugo Pereira da Silva & Nara Rejane Cruz Oliveira & Ricardo da Costa Padovani & Adriana Marcassa Tucci & Daniel Araki Ribeiro
Received: 28 September 2013 / Accepted: 9 December 2013 # Springer-Verlag Berlin Heidelberg 2014
Abstract The aim of the present study was to comparatively evaluate genomic damage (micronucleus) and cellular death (pyknosis, karyolysis, and karyorrhexis) in exfoliated oral mucosa cells from crack cocaine users by micronucleus test. A total of 30 crack cocaine users and 30 health controls (nonexposed individuals) were included in this setting. Individuals had epithelial cells from cheek mechanically exfoliated, placed in fixative, and dropped in clean slides, which were checked for the above nuclear phenotypes. The results pointed out significant statistical differences (p< 0.05) of micronucleated oral mucosa cells from crack cocaine users. Exposure to crack cocaine caused an increase of other nuclear Responsible editor: Philippe Garrigues M. das Graças Alonso de Oliveira Department of Propedeutics and Integrated Clinic, School of Dentistry, Federal University of Bahia, Salvador, Bahia, Brazil J. N. dos Santos Department of Oral Pathology, School of Dentistry, Federal University of Bahia, Salvador, Bahia, Brazil P. R. Cury Department of Periodontics, School of Dentistry, Federal University of Bahia, Salvador, Bahia, Brazil V. H. P. da Silva : D. A. Ribeiro (*) Department of Biosciences, Federal University of Sao Paulo, Av. Ana Costa, 95 Vila Mathias, Santos, Sao Paulo 11060-001, Brazil e-mail: [email protected] D. A. Ribeiro e-mail: [email protected] N. R. C. Oliveira Department of Human Movement Sciences, Federal University of Sao Paulo, Santos, Sao Paulo, Brazil R. da Costa Padovani : A. M. Tucci Department of Health, Education and Society, Federal University of Sao Paulo, Santos, Sao Paulo, Brazil
alterations closely related to cytotoxicity such as karyolysis in oral cells as well. In summary, these data indicate that crack cocaine is able to induce chromosomal breakage and cellular death in oral mucosa cells of users. Keywords Buccal mucosa cells . Crack cocaine users . Micronucleus test
Introduction Crack cocaine is a complex substance insoluble in water and thermostable that vaporizes at temperatures above 93 °C. This is considered a smokable form of cocaine being inhaled, smoked, or used intravenously (Thieli et al. 2013). However, this by-product of cocaine is usually smoked in small homemade pipes or by inhalation of its vapor. Its outcomes include intense euphoria and excitement, and a stone produces no more than two hours of euphoria (Cruz et al. 2013). Illicit drug use has increased dramatically in the past decades in developing countries of South America (Jhonson et al. 2013). Particularly in Brazil, crack cocaine use has widely disseminated since the early 1990s. In 1999, the Brazilian Center for Psychotropic Drug Information (CEBRID) found a 0.4 % lifetime rate of crack cocaine use (Gálduroz et al. 2000). The most recent Brazilian national population-based survey was conducted in 2012 (Laranjeira et al. 2012). Researchers interviewed 4,607 subjects over the age of 14 in 149 cities. The results showed that two million Brazilians were using crack cocaine. The lifetime rate of crack cocaine use was 1.4 % for adults. These data show that the use of crack cocaine in Brazil is continuing to increase. Today, the crack cocaine use might be considered as an “epidemic”, mainly in metropolitan regions (Inciardi et al. 2006). In this scenario, the images of “cracolandias”, urban neighborhoods devastated by public crack cocaine use and its
Environ Sci Pollut Res
related violence and exchanging sex for drugs, has been associated with popular image of crack cocaine use in Brazil (Raupp and Adorno 2011). Ongoing debates of public health interventions, so called war on crack, have been conducted in Brazil. The dependence of crack cocaine can develop rapidly and has been linked to cardiovascular, respiratory, neurological, and psychiatric problems, and to other psychoactive substance use (Falck et al. 2008). Crack cocaine is usually smoked in pipes, but users in Brazil have adopted other techniques, among which the use of aluminum cans with holes and the addition of cigarette ash to improve combustion stands out (Almeida et al. 2012). Such use causes the increase in aluminum rates in the blood, causing more damage to the central nervous system and also burning the users' lips (Almeida et al. 2012). Crack cocaine is rapidly absorbed by mucous membranes when smoked, reaching cerebral circulation within 6 to 8 s (Alvarez et al. 2008). The effect is similar to intravenous administration, due to the large lung surface, but its duration is less than 20 min (He et al. 2006). Before reaching the lungs, crack cocaine comes into contact with the oral mucosa. To the best of our knowledge, there are few studies regarding the outcomes induced by crack cocaine on the oral mucosa (Woyceichoski et al. 2008; Almeida et al. 2012). There are some cases of oral, oropharynx, and laryngopharynx lesions induced by cocaine, such as caused by crack cocaine use (Fava et al. 2008; Nastro et al. 2013). Biomarkers are considered useful indicators of environmental exposure and play a key role in the prevention of the effects of carcinogen exposure by early detection, because they are not diagnostic assays but rather early damage indicators (Bartolotta et al. 2011). The strategies for risk assessment are more effective with screening for potential mutagenicity in crack cocaine users by the use of some assays, for example, the bacterial gene mutation assay, the mammalian cell gene mutation assay, and mouse lymphoma assays or chromosomal aberration test. However, these methods are typically laborious and time-consuming or require highly trained technicians to accurately read and interpret slides. For this purpose, a great deal of enthusiasm was raised by the application of the micronucleus test to uncultured exfoliated cells (Stich et al. 1992). Micronucleus arises from acentric fragments or whole chromosomes which are not included into the main nuclei of the daughter cells. The formation of micronuclei can be induced by substances that cause chromosome breakage (clastogens) as well as by agents that affect the spindle apparatus (aneugens) (Belien 1995). According to Tolbert et al. (1992), the specificity of the test to detect genotoxic and cytotoxic effects is increased by recording other degenerative nuclear alterations indicative of cell death, besides the micronucleus. Among them, pyknosis, karyolysis, and
karyorrhexis are suitable for this purpose. Recently, we have applied this methodology with success in individuals exposed to dental X-ray, petrol station attendants, and those with malignant tumors undergoing chemotherapy (Minicucci et al. 2008; Ribeiro et al. 2008; Martins et al. 2009). In the present study, we investigated the frequencies of micronucleated cells in oral mucosa from crack cocaine users and control subjects. To monitor the cytotoxic effects, pyknosis, karyolysis, and karyorrhexis were also evaluated in this setting.
Material and methods Subjects The subjects of this study comprised a total of 30 crack cocaine users with a mean age of 31.4±9.3 years and living on the streets in Salvador city, previously identified as key areas for street drug use. The study's eligibility criteria included (1) crack cocaine use in the last 1 month, (2) 18–52 years of age, and (3) consent to participate to the study. The control group consisted of 30 healthy men, 33.6±11.6 years, and not exposed to crack cocaine. Each person was interviewed concerning possible confounding factors and was excluded from this study when there was lesion on the oral mucosa visible at clinical examination, a history of cancer, previous radio- or chemotherapy, use of therapeutic drugs, and exposure to diagnostic X-rays during the last 6 months. Exposure to known genotoxic agents such as smoking habits, alcohol consumption, and the time of exposure to crack cocaine was not recorded because recall bias phenomenon has been reached. The study was approved by the Ethics Committee of UFBA, Federal University of Bahia. Informed consent was obtained from the individuals included in the study. Micronucleus test in oral mucosa cells Exfoliated oral mucosa cells were collected from buccal mucosa of all individuals. After rinsing the mouth with tap water, the cells were obtained by scraping the right/left cheek mucosa with a disposable plastic spatula. The cells were transferred to a tube containing saline solution (NaCl at 0.9 % concentration in distilled water), centrifuged (800 rpm) for 5 min, fixed in 3:1 methanol/acetic acid, and dropped onto pre-cleaned slides. Later, the air-dried slides were stained using the Feulgen/Fast Green method (Belien 1995) and examined under a light microscope at×400 magnification. A total of 1,000 cells were scored from each person from the control and exposed groups as described elsewhere (Buajeeb et al. 2007).
Environ Sci Pollut Res
Data analysis Micronuclei were scored according to the criteria described by Sarto et al. (1990) as a parameter of DNA damage (mutagenicity). For cytotoxicity, the following nuclear alterations were considered: pyknosis, karyolysis, and karyorrhexis. The results were expressed in percentage (%). Such analysis was established in a previous study group (Sarto et al. 1990). Statistical methods Differences were statistically analyzed using the conditional test for comparing proportions in situations in which events are rare using SigmaStat software, version 1.0 (Jadel Scientific, USA). The level of statistical significance was set at 5 %.
Results Table 1 shows the frequencies of micronucleated cells in crack cocaine users and control individuals. In the control group, the mean frequency of micronucleated cells (MNC) was approximately 0.05 % for buccal mucosa. In the group exposed to crack cocaine, the mean frequency of MNC was 0.06 % in the buccal mucosa cells, being significantly statistically different (p
RESEARCH ARTICLE
Cytogenetic biomonitoring of oral mucosa cells of crack cocaine users Maria das Graças Alonso de Oliveira & Jean Nunes dos Santos & Patrícia Ramos Cury & Victor Hugo Pereira da Silva & Nara Rejane Cruz Oliveira & Ricardo da Costa Padovani & Adriana Marcassa Tucci & Daniel Araki Ribeiro
Received: 28 September 2013 / Accepted: 9 December 2013 # Springer-Verlag Berlin Heidelberg 2014
Abstract The aim of the present study was to comparatively evaluate genomic damage (micronucleus) and cellular death (pyknosis, karyolysis, and karyorrhexis) in exfoliated oral mucosa cells from crack cocaine users by micronucleus test. A total of 30 crack cocaine users and 30 health controls (nonexposed individuals) were included in this setting. Individuals had epithelial cells from cheek mechanically exfoliated, placed in fixative, and dropped in clean slides, which were checked for the above nuclear phenotypes. The results pointed out significant statistical differences (p< 0.05) of micronucleated oral mucosa cells from crack cocaine users. Exposure to crack cocaine caused an increase of other nuclear Responsible editor: Philippe Garrigues M. das Graças Alonso de Oliveira Department of Propedeutics and Integrated Clinic, School of Dentistry, Federal University of Bahia, Salvador, Bahia, Brazil J. N. dos Santos Department of Oral Pathology, School of Dentistry, Federal University of Bahia, Salvador, Bahia, Brazil P. R. Cury Department of Periodontics, School of Dentistry, Federal University of Bahia, Salvador, Bahia, Brazil V. H. P. da Silva : D. A. Ribeiro (*) Department of Biosciences, Federal University of Sao Paulo, Av. Ana Costa, 95 Vila Mathias, Santos, Sao Paulo 11060-001, Brazil e-mail: [email protected] D. A. Ribeiro e-mail: [email protected] N. R. C. Oliveira Department of Human Movement Sciences, Federal University of Sao Paulo, Santos, Sao Paulo, Brazil R. da Costa Padovani : A. M. Tucci Department of Health, Education and Society, Federal University of Sao Paulo, Santos, Sao Paulo, Brazil
alterations closely related to cytotoxicity such as karyolysis in oral cells as well. In summary, these data indicate that crack cocaine is able to induce chromosomal breakage and cellular death in oral mucosa cells of users. Keywords Buccal mucosa cells . Crack cocaine users . Micronucleus test
Introduction Crack cocaine is a complex substance insoluble in water and thermostable that vaporizes at temperatures above 93 °C. This is considered a smokable form of cocaine being inhaled, smoked, or used intravenously (Thieli et al. 2013). However, this by-product of cocaine is usually smoked in small homemade pipes or by inhalation of its vapor. Its outcomes include intense euphoria and excitement, and a stone produces no more than two hours of euphoria (Cruz et al. 2013). Illicit drug use has increased dramatically in the past decades in developing countries of South America (Jhonson et al. 2013). Particularly in Brazil, crack cocaine use has widely disseminated since the early 1990s. In 1999, the Brazilian Center for Psychotropic Drug Information (CEBRID) found a 0.4 % lifetime rate of crack cocaine use (Gálduroz et al. 2000). The most recent Brazilian national population-based survey was conducted in 2012 (Laranjeira et al. 2012). Researchers interviewed 4,607 subjects over the age of 14 in 149 cities. The results showed that two million Brazilians were using crack cocaine. The lifetime rate of crack cocaine use was 1.4 % for adults. These data show that the use of crack cocaine in Brazil is continuing to increase. Today, the crack cocaine use might be considered as an “epidemic”, mainly in metropolitan regions (Inciardi et al. 2006). In this scenario, the images of “cracolandias”, urban neighborhoods devastated by public crack cocaine use and its
Environ Sci Pollut Res
related violence and exchanging sex for drugs, has been associated with popular image of crack cocaine use in Brazil (Raupp and Adorno 2011). Ongoing debates of public health interventions, so called war on crack, have been conducted in Brazil. The dependence of crack cocaine can develop rapidly and has been linked to cardiovascular, respiratory, neurological, and psychiatric problems, and to other psychoactive substance use (Falck et al. 2008). Crack cocaine is usually smoked in pipes, but users in Brazil have adopted other techniques, among which the use of aluminum cans with holes and the addition of cigarette ash to improve combustion stands out (Almeida et al. 2012). Such use causes the increase in aluminum rates in the blood, causing more damage to the central nervous system and also burning the users' lips (Almeida et al. 2012). Crack cocaine is rapidly absorbed by mucous membranes when smoked, reaching cerebral circulation within 6 to 8 s (Alvarez et al. 2008). The effect is similar to intravenous administration, due to the large lung surface, but its duration is less than 20 min (He et al. 2006). Before reaching the lungs, crack cocaine comes into contact with the oral mucosa. To the best of our knowledge, there are few studies regarding the outcomes induced by crack cocaine on the oral mucosa (Woyceichoski et al. 2008; Almeida et al. 2012). There are some cases of oral, oropharynx, and laryngopharynx lesions induced by cocaine, such as caused by crack cocaine use (Fava et al. 2008; Nastro et al. 2013). Biomarkers are considered useful indicators of environmental exposure and play a key role in the prevention of the effects of carcinogen exposure by early detection, because they are not diagnostic assays but rather early damage indicators (Bartolotta et al. 2011). The strategies for risk assessment are more effective with screening for potential mutagenicity in crack cocaine users by the use of some assays, for example, the bacterial gene mutation assay, the mammalian cell gene mutation assay, and mouse lymphoma assays or chromosomal aberration test. However, these methods are typically laborious and time-consuming or require highly trained technicians to accurately read and interpret slides. For this purpose, a great deal of enthusiasm was raised by the application of the micronucleus test to uncultured exfoliated cells (Stich et al. 1992). Micronucleus arises from acentric fragments or whole chromosomes which are not included into the main nuclei of the daughter cells. The formation of micronuclei can be induced by substances that cause chromosome breakage (clastogens) as well as by agents that affect the spindle apparatus (aneugens) (Belien 1995). According to Tolbert et al. (1992), the specificity of the test to detect genotoxic and cytotoxic effects is increased by recording other degenerative nuclear alterations indicative of cell death, besides the micronucleus. Among them, pyknosis, karyolysis, and
karyorrhexis are suitable for this purpose. Recently, we have applied this methodology with success in individuals exposed to dental X-ray, petrol station attendants, and those with malignant tumors undergoing chemotherapy (Minicucci et al. 2008; Ribeiro et al. 2008; Martins et al. 2009). In the present study, we investigated the frequencies of micronucleated cells in oral mucosa from crack cocaine users and control subjects. To monitor the cytotoxic effects, pyknosis, karyolysis, and karyorrhexis were also evaluated in this setting.
Material and methods Subjects The subjects of this study comprised a total of 30 crack cocaine users with a mean age of 31.4±9.3 years and living on the streets in Salvador city, previously identified as key areas for street drug use. The study's eligibility criteria included (1) crack cocaine use in the last 1 month, (2) 18–52 years of age, and (3) consent to participate to the study. The control group consisted of 30 healthy men, 33.6±11.6 years, and not exposed to crack cocaine. Each person was interviewed concerning possible confounding factors and was excluded from this study when there was lesion on the oral mucosa visible at clinical examination, a history of cancer, previous radio- or chemotherapy, use of therapeutic drugs, and exposure to diagnostic X-rays during the last 6 months. Exposure to known genotoxic agents such as smoking habits, alcohol consumption, and the time of exposure to crack cocaine was not recorded because recall bias phenomenon has been reached. The study was approved by the Ethics Committee of UFBA, Federal University of Bahia. Informed consent was obtained from the individuals included in the study. Micronucleus test in oral mucosa cells Exfoliated oral mucosa cells were collected from buccal mucosa of all individuals. After rinsing the mouth with tap water, the cells were obtained by scraping the right/left cheek mucosa with a disposable plastic spatula. The cells were transferred to a tube containing saline solution (NaCl at 0.9 % concentration in distilled water), centrifuged (800 rpm) for 5 min, fixed in 3:1 methanol/acetic acid, and dropped onto pre-cleaned slides. Later, the air-dried slides were stained using the Feulgen/Fast Green method (Belien 1995) and examined under a light microscope at×400 magnification. A total of 1,000 cells were scored from each person from the control and exposed groups as described elsewhere (Buajeeb et al. 2007).
Environ Sci Pollut Res
Data analysis Micronuclei were scored according to the criteria described by Sarto et al. (1990) as a parameter of DNA damage (mutagenicity). For cytotoxicity, the following nuclear alterations were considered: pyknosis, karyolysis, and karyorrhexis. The results were expressed in percentage (%). Such analysis was established in a previous study group (Sarto et al. 1990). Statistical methods Differences were statistically analyzed using the conditional test for comparing proportions in situations in which events are rare using SigmaStat software, version 1.0 (Jadel Scientific, USA). The level of statistical significance was set at 5 %.
Results Table 1 shows the frequencies of micronucleated cells in crack cocaine users and control individuals. In the control group, the mean frequency of micronucleated cells (MNC) was approximately 0.05 % for buccal mucosa. In the group exposed to crack cocaine, the mean frequency of MNC was 0.06 % in the buccal mucosa cells, being significantly statistically different (p
