Original ArticleBraz J Oral Sci. April/June 2009 - Volume 8, Number 2 Freshwater and salt-water influence in human identification by analysis of DNA: an epidemiologic and laboratory study Jamilly de Oliveira Musse1, Amanda da Costa Nardis2, Evelyn K. Anzai3, Mário H. Hirata4, Regina Maria Barreto Cicarelli5, Rogério Nogueira de Oliveira6 1 DDS, MSc, PhD Student in Community Dentistry, Faculdade de Odontologia, Universidade de São Paulo (USP), São Paulo (SP), Brazil 2 Undergraduate dental student, Faculdade de Odontologia, USP, São Paulo (SP), Brazil 3 PhD in Clinical Analysis, Faculdade de Ciências Farmacêuticas, USP, São Paulo (SP), Brazil 4 DDS, MSc, PhD, Associate Professor, Faculdade de Ciências Farmacêuticas, USP, São Paulo (SP), Brazil 5 DDS, MSc, PhD, Full Professor, Faculdade de Ciências Farmacêuticas, USP, São Paulo (SP), Brazil 6 DDS, MSc, PhD, Professor, Faculdade de Odontologia, USP, São Paulo (SP), Brazil Received for publication: December 22, 2008 Accepted: May 18, 2009 Correspondence to: Jamilly de Oliveira Musse Avenida Prof. Mello de Moraes, 1.235 – bloco C – apto. 205 – Cidade Universitária – Butantã CEP 05508000 – São Paulo (SP), Brazil E-mail: jamillymusse@usp.br; musse_jo@hotmail.com Abstract Aim: To investigate the casuistry of drowning cases by reviewing the records from the Forensic Medicine Institute Nina Rodrigues in the city of Salvador, BA, Brazil, and to verify the potential of DNA recovery in human teeth im- mersed in water. Methods: An epidemiological survey was conducted followed by a laboratorial phase, in which 40 teeth were immersed in fresh and salt-water, the DNA was extracted by the organic method and amplified by polymerase chain reaction, using the amelogenin as initiator. The electrophoresis initially occurred in agarose gel and later in polyacrylamide gel. Results: In the present survey, 346 deaths from drowning were observed, most of them in salt-water (51.73%), with a predominance of male victims (86.13%) aged from 18 to 35 years-old (37.94%). Dentists identified 14.74% of the victims. DNA was recovered in 37.5% from the samples, most from teeth immersed in freshwater. Polyacrylamide gel analysis in samples that were amplified in agarose gel allowed correct gender identification in 83.3% of the cases. However, allele loss was observed in samples of two victims, jeopardizing gender determination. Conclusions: Dental exposure to water interfered in DNA recovery. The gen- der investigation using the amelogenin as initiator was effective. Keywords: human identification, forensic dentistry, teeth, DNA, drowning. Introduction Forensic Dentistry has contributed to human identification for a long time. Dentists, enforced by law #5.081/66, are able to perform reports involving biological materials derived from hu- man body under several conditions (quartered, dilacerated, carbonized, macerated, decom- posed, in skeletonization and skeletonized), with the goal of establishing human identity1. Historically, fingerprints have been used in identification. However, in certain situations, in which the body is found in advanced stage of putrefaction, fingerprints are easily destroyed. Moreover, the technical experts frequently need to use comparative antemortem elements, such as dental records, in human body identification, but they are not always available. Before the development of molecular biology techniques, these situations would make the experts es- tablish the victim’s identification without the comparative elements. Whenever the documen- 72 Musse JO, Nardis AC, Anzai EK, Hirata MH, Cicarelli RMB, Oliveira RN Braz J Oral Sci. 8(2): 71-5 tation was available, but the corpses were degraded, the exams would try to recover the individual’s profile by establishing the species, gen- der, built, age, phenotype, skin color, among other characteristics2. Nowadays, biomolecular resources have been employed in hu- man identification whenever the use of traditional methods is not viable. Applying these resources allows identifying victims even without antemortem information or with deteriorated biological material in insignificant amounts, which are relatively frequent con- ditions in forensic analysis, especially in mass accidents3. In this context, some authors have emphasized the importance of gender investigation in victims’ identification and the need of the joint action among several healthcare workers (physicians, dentists, psychol- ogists, geneticists, radiologists) and professionals from related sciences (anthropologists, technical examiners, among others) in this process4,5. Recently reported in the media, events such as the tsunami, in Thailand, in 2004 and in Indonesia, in 2006, the hurricanes “Katrina” in New Orleans and “Rita” in Texas, both in 20056, reinforce the need of mastering DNA extraction techniques in the process of identify- ing skeletonized bodies or bodies in advanced state of putrefaction, under the influence of aqueous environment7. The practical applicability of these methods can be observed in several studies that used DNA-based techniques in the identification of victims, whose bodies had undergone the action of water8,3. The in- fluence of environmental factors on DNA stability has been empha- sized, since corpses or their pieces are frequently found carbonized, submersed or buried. According to Schwartz et al.9, those factors can interfere in the amount of recovered DNA and influence in the iden- tification process9. According to Bender et al.10, this is due to the fact that the rate of DNA degradation varies according to light conditions, water content and temperature, and bacterial and fungal contamina- tion might occur followed by microbial growth, and resulting in the physical, chemical and biological degradation of the genomic DNA10. Although mass accidents, including natural disasters, have been poorly documented in Brazil, records from the Ministry of Health re- port a significant increase in mortality rate due to external causes, among which drowning is mentioned. In this sense, the official re- cords report the occurrence of 57,595 deaths by drowning and ac- cidental submersion between 1996 and 2004, which represents 0.67% from the total number of deaths that occurred in the country in this period11. Concern is expressed when the Brazilian coastal line exten- sion and its population concentration are considered, especially in Bahia, where, according to the Brazilian Institute of Geography and Statistics12, there is a prevalence of coastal cities. The present study aimed at understanding the casuistry in situ- ations involving drowning cases, and verifying the potential of DNA recovery from teeth immersed in water. Material and methods An epidemiological survey was conducted with records from the Forensic Medicine Institute Nina Rodrigues in Salvador, BA, Brazil, between 2003 and 2005. Initially, information such as number of deaths by drowning, gender, age, type of water in which the body was submerged (freshwater or salt-water) and the contribution or not of Forensic Dentistry in the victims’ identification was collected. Later, a research laboratorial phase was carried out. The sample was com- posed by 40 teeth from 20 individuals (two from each victim). Sa- liva in MGM® cards was collected from oral mucosa swabs. The teeth were submitted to a physicochemical process for tooth-cleaning, through scaling with curettes and 70% alcohol-wash and then im- mersed in fresh and salt-water for one month, according to Schwartz et al.9. After the immersion, the tooth crown was sectioned13 and pul- verization was done according to the method proposed by Sweet and Hildebrand14, adapted to the use of a mortar and pestle, and manual storage by immersion in liquid nitrogen. DNA extraction from the saliva stored on cards was performed with those fragments using a FTA kit. The protocol to extract DNA from the teeth was based on the organic method described by Hoch- meister et al.15. The region for amplification by polymerase chain re- action (PCR) was the amelogenin. The sequences of initiators were sense: ACCTCATCCTgggCACCCTgg (21BP) and anti-sense: AggCT- TgAggCCA ACCATCAg (21BP). The following cycling protocol was used: denaturation at 96° C for two minutes, followed by ten dena- turation cycles at 94 °C for one minute, hybridization at 64 °C for one minute, elongation at 72 °C for one minute and 30 seconds and final elongation at 72 oC for ten minutes. The PCR products were initially analyzed in agarose gel at a 1.5% concentration. Electrophoretic separation occurred in 100 V and 60 mA for 40 minutes. Later, the samples that amplified in the agarose gel were submitted to electrophoresis in silver-stained 8% polyacrylamide gel. Results The review of the records referring to the total of deaths occurred between 2003 and 2005 showed a prevalence of only 2.7% deaths re- sulting from drowning. The records also revealed a higher prevalence of death in male individuals and young adults (37.94%), as seen in Figures 1 and 2. Regarding the type of water in which the bodies were submerged, salt-water prevailed, followed by freshwater and swimming pool water, except for 2003, when the first two categories were inversed (Figure 3). When evaluating Forensic Dentistry contribution to vic- tim identification, little participation of the forensic dentist was ob- served (Figure 4). During the laboratorial phase, the DNA was initially by electro- phoresis in 1.5% agarose gel (Figure 5). In this phase, genetic ma- terial recovery was observed in 37.5% of the dental samples, with a predominance of teeth immersed in freshwater (45%) over teeth im- mersed in salt-water (30%). All saliva samples used as controls pre- sented positive amplification. The analysis of polyacr ylamide gel in 27 samples (15 teeth and 12 saliva samples), from 12 v ictims w ith positive amplif ica- 73Freshwater and salt-water influence in human identification by analysis of DNA: an epidemiologic and laboratory study Braz J Oral Sci. 8(2): 71-5 87.50% 86.90% 88.89% 12.50% 13.10% 11.11% 0.00% 10.00% 20.00% 30.00% 40.00% 50.00% 60.00% 70.00% 80.00% 90.00% 100.00% 2003 2004 2005 Male Female Figure 1. Percent distribution of drowning victims according to gender, Salvador, 2003 to 2005. 15% 17.64% 37.94% 21.77% 1.76% 5.89% 0% 5% 10% 15% 20% 25% 30% 35% 40% 0 to 11 years 12 to 17 years 18 to 35 years 36 to 59 years 60 or more Not informed Figure 2. Percent distribution of drowning victims according to age, Salvador, 2003 to 2005. 46% 28,70% 36.68% 43.00% 59.00% 57.77% 3.90% 2.46% 1.11% 7% 9.84% 4.44% 0% 10% 20% 30% 40% 50% 60% 70% 2003 2004 2005 Freshwater Salt-water Pool Not informed Figure 3. Percent distribution of drowning victims according to the place where the body was found, Salvador, 2003 to 2005. 33% 4.00% 4.44% 67.20% 96.00% 95.56% 0% 20% 40% 60% 80% 100% 120% 2003 2004 2005 Yes No Figure 4. Percent distribution of drowning cases according to the contribution of forensic dentistry in victim identification, Salvador, 2003 to 2005. 1 2 3 4 5 6 Figure 5. Photograph in 1.5% agarose gel stained with ethidium bromide, after AMEL locus amplification. Samples: 1 = 100 pb marker; 2 = saliva; 3 = tooth submerged in salt-water; 4 = tooth submerged in freshwater; 5 = PCR negative control and 6 = PCR positive control. 1 2 3 4 5 6 7 8 9 10 A B C Figure 6. Photograph in 8% polyacrylamide gel stained with silver. Samples: 1, 5 and 8 = tooth submerged in salt-water; 2 and 6 = tooth submerged in freshwater; 3, 7 and 9 = saliva; 4 and 10 = 10 pb standard. A: male; B: female; C: no gender identification. tion in agarose gel, allowed the v isualization of the amelogenin different alleles, enabling the correct identif ication of gender in 83.3% of the cases (ten indiv iduals). Moreover, an adequate correlation was obser ved between band intensit y and the t y pe of material used in the exam (tooth or saliva) (Fig ure 6). How- ever, loss of alleles in the polyacr ylamide gel was obser ved in the samples of two indiv iduals, jeopardizing gender identif ication in these cases. 74 Musse JO, Nardis AC, Anzai EK, Hirata MH, Cicarelli RMB, Oliveira RN Braz J Oral Sci. 8(2): 71-5 Discussion The mortality rates due to external causes in Brazil, among which drowning, have increased significantly in the last few years, reaching the second place in Brazilian mortality statistics16. Thus, it is neces- sary to know the population profile that have mostly contributed to this statistics, in such a way that planners and executors of govern- ment policies are able to define, in concrete basis, actions that should be a priority, in order to contemplate prevention and attention to those victims. In this sense, the findings of the present epidemiological survey showed prevalence of 2.7% of deaths by drowning in the city of Sal- vador. Baptista et al.17 and Martins and Andrade18 have found 26 and 19.5% of deaths by drowning in Portugal and in Paraná/Brazil, re- spectively. These discrepant percent values can be attributed to the methodological differences in the studies and their duration. Regarding the distribution of deaths by drowning, the findings of the present study agree with the literature19,20 regarding the most prevalent gender (male) and age group (adults). The findings of a 12-year survey of deaths by drowning stated that 63% of the cases occurred in freshwater18, while another study19 found a predominance of death cases in salt-water. In the present research, most cases occurred in salt-water, which is probably due to the fact that 95% of the water in the Brazilian territory is formed by salt-water20. There is a low prevalence of Forensic Dentistry contribution in the identification of victims by drowning in the present study. Fo- rensic dentists participated in only 14.74% of the cases within three years. This result is probably due to the lack of Forensic experts in Bahia during the period of this study, since the first public selection for these positions occurred in 200621. The results of the review of the records referring to the total of deaths in the Forensic Medicine Institute served as basis for the next phase of the present study: the laboratorial. Considering the recent world casuistry of mass accidents involving the aquatic environ- ment, and the consequent need of mastering the DNA-based tech- nique in the process of identifying victims that were under the influ- ence of water, teeth were immersed in fresh and salt-water in order to simulate cases of forensic investigation. In this sense, one of the first steps to proceed with the victim identification is gender investigation4, usually performed accord- ing to anatomical characteristics from male and female genitals. However, when forensic examination has to be done in skeletonized corpses or bodies in advanced state of putrefaction, bones and teeth are, many times, the only materials available and identification can be performed by molecular biology techniques, using amelogenin analysis22. In the present paper, the analysis by electrophoresis in agarose gel showed only one DNA region for gender investigation (amelogenin). Genetic material recovery was possible in only 37.5% of the teeth, demonstrating that the water interfered directly in DNA preserva- tion. On the other hand, DNA from all saliva samples used as controls was successfully amplified. There was greater DNA degradation in salt-water (70%) compared to freshwater (55%), probably due to the chemical composition of both aqueous conditions. Both types of wa- ter present ions such as: calcium, magnesium, sodium, potassium, bicarbonate, chloride, sulfate, nitrate, among others. Traces of lead, copper, arsenic, manganese and a large spectrum of organic com- pounds from decomposition of organic matter of animal and vegetal origin can also be found. In addition, in some cases, residues from agricultural fields and disposal of effluents from domestic and in- dustrial origin are present, varying from humic acids to synthetic organic compounds such as detergents, pesticides and solvents20. It was not possible to determine the mechanism by which the water affects DNA recovery. However, some components present in aquatic ecosystems have already been reported as being able to degrade genetic material (microbial growth, humidity)10 or inhibit the Taq DNA polymerase enzyme (humic acid). This enzyme is re- sponsible for the incorporation of free nucleotides, which will form a new fragment of the DNA molecule. Thus, its inhibition is a hin- drance to PCR 23. The analysis of the agarose gel does not allow gender identifica- tion due to a slight difference in base pairs6 present in men and wom- en, when the amelogenin is used as a region. In this sense, Mukherjee and Biwas24 have suggested the use of polyacrylamide gel in those cases because it promotes a better band separation and, consequent- ly, allele visualization. Running polyacrylamide gel was performed in samples that amplified in agarose gel, enabling the accurate vi- sualization of the amelogenin alleles, and identifying correctly the gender of ten victims. However, although gender determination us- ing amelogenin is a recognized and scientifically accredited meth- odology, loss of alleles in the polyacrylamide gel was observed in the samples of two individuals. Pinheiro1 comments on this possibility when he states that sometimes DNA degradation, instead of imped- ing the results, can cause the visualization of one allele, instead of two, and the disappearing of the allele with larger dimension is more frequent. Hence, when old vestiges are analyzed and homozygous profile is obtained by some systems, one should be careful using the results because they might be from a heterozygous profile. An alter- native to solve this problem was proposed by Steinlechner et al.25, while studying the genetic profile of 29,432 men stored in an Aus- tralian database, they verified the absence of PCR product specific of the amelogenin related to chromosome Y in six individuals. After changing the methodology and analyzing eight short tander repeat (STR) from the same chromosome, the complete genetic profile of five victims was obtained, confirming their male genotype. The error rate in gender investigation using the amelogenin test in the present work was 0.018%. A factor that may lead to DNA non-amplification is the quality of the forensic biological sample. The insignificant amount of biologi- cal material to DNA extraction may result in absence of the target sequence in the fraction used for the reaction or the same can be degraded, not allowing DNA amplification by PCR24. In an attempt to minimize the effects of this degradation and even as legal endorse- 75Freshwater and salt-water influence in human identification by analysis of DNA: an epidemiologic and laboratory study Braz J Oral Sci. 8(2): 71-5 ment to avoid the exam result to be questioned, it is necessary that forensic laboratories keep the chain-of-custody of the samples. The records must be readily accessible and followed since data collection until the final disposition, in such a way to warrant that every pre- caution has been taken to avoid falsification, break, loss or contami- nation of the samples. In addition, inadequate procedures that may lead to sample contamination, by the lab investigators and forensic professionals, may result in erroneous interpretation of the genetic profile26. Therefore, the maintenance of good laboratorial practices is essential and specific training on DNA-based identification tech- niques is mandatory. The following conclusions may be draw from the obtained re- sults: the epidemiological survey allowed outlining the population profile that most contributed to the statistics surveyed, during a three-year period in the Forensic Medicine Institute of Salvador; there was a predominance of adult male victims exposed to the action of salt-water; tooth exposure to water interfered directly in DNA recovery; despite its well-established efficacy, recognition and credibility, gender investigation by amelogenin, as every procedure that uses biological molecular resources, requires a careful inter- pretation of the results by the researcher, therefore, the observation of all the criteria related to the maintenance of the chain-of-cus- tody and DNA analysis are important tools in the processes of hu- man identification and must be used with caution and considered within a set of varied evidences. References 1. Pinheiro MFP. Genética e biologia forense e criminalística. In: Noções Gerais sobre outras ciências forenses. In: Noções Gerais sobre outras ciências forenses. [2004 fev. 26]. Disponível em: http://medicina.med.up.pt/legal/ NocoesGeraisCF.pdf 2. Oliveira RN, Nunes FD, Anzai EK, Daruge E, Mesquita RA, Ozaki AN, et al. Population studies os the Y-chromosome of loci DYS390, DYS391 and DYS393 in Brazilian subjects and its use in human identification. J Forensic Odontostomatol. 2002;20(1):6-9. 3. de la Grandmaison GL, Leterreux M, Lasseuguette K, Alvarez JC, de Mazancourt P, Durigon M. Study of the diagnostic value of iron in fresh water drowning. Forensic Sci Int. 2006;157:117-20. 4. Lau G, Tan WF, Tan PH. After the Indian Ocean tsunami: Singapore’s contribution to the international disaster victim identification effort in Thailand. Ann Acad Med Singapore. 2005;34:341-51. 5. Budowle B, Bieber FR, Eisenberg AJ. Forensic aspects of mass disasters: strategic considerations for DNA-based human identification. Leg Med. 2005;7:230-43. 6. Mccarthy M. Desastres naturais crescem com aquecimento global, diz especialista britânico. Folha de São Paulo. 2006;8:13. 7. Alonso A, Martin P, Albarrán C, Garcia P, Fernandez de Simon L, Jesús Iturralde M, et al. Challenges of DNA profiling in mass disaster investigations. Croat Med J. 2005;46:540-8. 8. Mannucci A, Casarino L, Bruni G, Lomi A, De Stefano F. Individual identification of flood victims by DNA polymorphisms and autopsy findings. Int J Legal Med. 1995;107:213-5. 9. Schwartz TR, Schwartz EA, Mieszerski L, McNally L, Kobilinsky L. Characterization of deoxyribonucleic acid (DNA) obtained from teeth subjected to various environmental conditions. J Forensic Sci. 1991;36:979-90. 10. Bender K, Farfán MJ, Schneider PM. Preparation of degraded human DNA under controlled conditions. Forensic Sci Int. 2004;139:135-40. 11. Brasil. Ministério da Saúde. Departamento de Informação e Informática do SUS (DATASUS). Tabela CID BR – 10: óbitos por ocorrência por faixa etária segundo região - afogamento e submersões acidentais de 1996 à 2004. [2006 fev. 24]. Brasília: MS/DATASUS. Disponível em: http://www.datasus.gov.br.html 12. Instituto Brasileiro de Geografia e Estatística (IBGE). Distribuição territorial da população brasileira. [2006 fev. 26]. Disponível em: http://www.ibge.gov.br/ home/. 13. Gaytmenn R, Sweet D. Quantification of forensic DNA from various regions of human teeth. J Forensic Sci. 2003;48:622-5. 14. Sweet D, Hildebrand D. Recovery of DNA from human teeth by cryogenic grinding. J Forensic Sci. 1998;43:1199-202. 15. Hochmeister MN, Rudin O, Ambach E. PCR analysis from cigarette butts, postage stamps, envelope sealing flaps, and other saliva-stained material. In: Lincoln PJ, Thompson J. Methods in molecular biology. v.48. Forensic DNA profiling protocols. Totowa: Humana press; 1998. p. 27-32. 16. Reis JC, Fradique FS. Significações sobre causas e prevenção das doenças em jovens adultos, adultos de meia-idade e idosos. Psicol Teor Pesqui. 2003;19:47-57. 17. Baptista JP, Casanova PC, Sousa JP, Martins PJ, Simões A, Fernandes V, et al. Afogamento: revisão temática a propósito de análise casuística do Serviço de Medicina intensiva dos Hospitais da Universidade de Coimbra (1989-2002). Rev Port Pneumol. 2003;9:311-25. 18. Martins CBG, Andrade SM. Causas Externas entre menores de 15 anos em cidade do Sul do Brasil: atendimentos em pronto-socorro, internações e óbitos. Rev Bras Epidemiol. 2005;8:194-204. 19. Suomionem PB, Korpela R. Impact of age, submersion time and water temperature on outcome in near-drowning. Ressuscitation. 2002;52(3):247-54. 20. Bernardo DL, Dantas ADB. Métodos e técnicas de tratamento de água. 2a ed. São Carlos: Rima; 2005. 21. Bahia. Diário Oficial da Bahia. [2006 jan. 12]. Disponível em: URL: http://www. egba.ba.gov.br/diario/_DODia/DO_frm0.html. 22. Murakami H, Yamamoto Y, Yoshitome K, Ono T, Okamoto O, Shigeta Y, et al. Forensic study of sex determination using PCR on teeth simples. Acta Med Okayama. 2000;54:21-32. 23. Burger J, Hummel S, Hermann B, Henke W. DNA preservation: a microsatellite- DNA study on ancient skeletal remains. Electrophoresis. 1999;20:1722-8. 24. Mukherjee KK, Biswas R. Short tandem repeat (STRs) and sex specific Amelogenin analysis of blood samples from neurosurgical female transfused patients. J Clin Forensic Med. 2005;12:10-3. 25. Steinlechner M, Berger M, Niederstätter H, Parson W. Rare failures in the amelogenin sex test. Int J Legal Med. 2002;116:117-20. 26. Bonaccorso NS. Aplicação do exame de DNA na elucidação de crimes. São Paulo: Faculdade de Direito da USP; 2005.