Introduction

One of the most important longstanding problems in the field of forensic medicine is the determination of the time of death upon the discovery of a possible homicide victim. With a majority of homicide victims discovered within the first 48h, it is critically important to be able to determine time of death quickly, and with accuracy and precision. Current methods of determining postmortem interval (PMI) vary, but none can provide better than an 8-h window time estimate (Johnson and Ferris, 2002).

The time of death of an individual can easily be determined if the postmortem interval can be assessed. Although livor mortis, rigor mortis, and, to a lesser degree, algor mortis have been used to estimate the postmortem interval, most experienced forensic pathologists agree that these characteristics provide, at best, “postmortem windows” (Cina, 1994).

During postmortem autolysis, cellular organelles and nuclear DNA break down into their constituent parts. DNA analysis was applied as a possible method for postmortem interval determination (Boy et al., 2003). Determining the quantity of DNA should be an objective and exact way to estimate the PMI (Liu et al., 2001). So, it is important to know which organ is most reliable for DNA extraction and also to know the effect of PMI on DNA degradation.

Several methods have been developed to quantify DNA, from basic UV spectrometry, through gel-based techniques, to dye staining, blotting techniques, and, very recently, DNA amplification methods (polymerase chain reaction, PCR) (Nicklas and Buel, 2003). The present search used a simple, easy, applicable and highly informative electrophoresis method that make it an ideal for the busy forensic laboratory. Liu et al. (2007) suggest that computerized image analysis technique CIAT is a useful and promising tool for the estimation of early PMI with good objectivity and reproducibility as quantitative indicator for the estimation of PMI within the first 36 h after death in rats.

Animals And Methods

Animals: The study included forty Albino rats that were classified into 5 groups; first group rats were sacrificed immediately after drowning(as a method of inducing death), while the 2nd, 3rd , 4th , 5th groups were sacrificed at 3, 6, 12, 24 hours postmortem respectively. Animals were dissected to obtain organs (lung, liver, spleen, muscle and brain). DNA of rats' viscera were detected by gel electrophoresis and the amount of DNA were measured by detecting its optical density (OD) employing an image analysis program.

Chemicals: 1-Lysing buffer: NaCl, tris (Camresco), Na2edita (Alpha), SDS (sodium diodo sulphate) PH 8.5. (Fisher Biotech). 2-Running buffer: tris (Camresco), boric acid, EDTA (Alpha). 3-Loading buffer: bromonphenol,glycerol (Adwic). 4-Agarose (Gibcobr). 5-NaCl 50mm (Adwic). 6-Isopropanol (Adwic). 7-Ethyl alcohol (Chemaget). 8-Ethidium bromide (Sigma). 9-200 bp ladder (Sigma).

Equipments: 1-Eppendorf tube (Naser company). 2-Blue tips. 3-Deepfreezer-20ºC (Ideal company). 4-Microcentrifuge (Beckman). 5-UV trasilluminator (Biometra). 6-Horizontal electrophoresis (Biometra standard power PACKP25). 7-Polaroid camera (Gelcam electrophoresis HOOD 0,7x). 8- Micropipette (Finn pipette 40-200 ul). 9-Incubator (Bst 5010).

Method of DNA Studying:

Results

Liver (Plate 1 and gel Proanalyzer curve 1); there is intact DNA at zerotime , mild DNA damage at 3h and 6h PM, moderate DNA damage at 12h PM, severe DNA damage at 24h PM. Table (1) show comparison of optical density of liver DNA at different PMI.

Spleen (Plate 2 and gel Proanalyzer curve 2); intact DNA at zerotime , mild DNA damage at 3h PM, moderate DNA damage at 6h and 12h PM and severe DNA damage at 24h PM. Table (2) show comparison of optical density of spleen DNA at different PMI.

Lung (Plate 3 and gel Proanalyzer curve 3); intact DNA at zerotime , moderate DNA damage at 3h and 6h PM, severe DNA damage at 12h and 24h PM. Table (3) show comparison of optical density of lung DNA at different PMI.

Brain (Plate 4 and gel Proanalyzer curve 4); intact DNA at zerotime , 3h and 6h PM with mild DNA damage at 12h and moderate DNA damage at 24h PM. Table (4) show comparison of optical density of brain DNA at different PMI.

Muscle (Plate 5 and gel Proanalyzer curve 5); there is intact DNA at zerotime, mild DNA damage at 3h, moderate DNA damage at 6h PM and severe DNA damage at 12h and 24h PM. Table (5) show comparison of optical density of muscle DNA at different PMI.

Figure 1

Plate (1): Gel electrophoresis of extracted DNA from rat liver at different PMI after drowning.

Figure 2

Plate (2): Gel electrophoresis of extracted DNA from rat spleen at different PMI after drowning.

Figure 3

Plate (3): Gel electrophoresis of extracted DNA from rat lung at different PMI after drowning

Figure 4

Plate (4): Gel electrophoresis of extracted DNA from rat brain at different PMI after drowning.

Figure 5

Plate (5): Gel electrophoresis of extracted DNA from rat muscle at different PMI after drowning.

Figure 6

Chart (1): Computer chart by gel Pro-analyzer shows the intensity of DNA against the molecular weight of each specified base pairs in rat liver at the studied PMI after drowning

Figure 7

Chart (2): Computer chart by gel Pro-analyzer shows the intensity of DNA against the molecular weight of each specified base pairs in rat spleen at the studied PMI after drowning

Figure 8

Chart (3): Computer chart by gel Pro-analyzer shows the intensity of DNA against the molecular weight of each specified base pairs in rat lung at the studied PMI after drowning

Figure 9

Chart (4): Computer chart by gel Pro-analyzer shows the intensity of DNA against the molecular weight of each specified base pairs in rat brain at the studied PMI after drowning

Figure 10

Chart (5): Computer chart by gel Pro-analyzer shows the intensity of DNA against the molecular weight of each specified base pairs in rat muscle at the studied PMI after drowning

Figure 11

Table (1): Comparison of optical density of liver DNA at different PMI

Figure 12

Table (2): Comparison of optical density of spleen DNA at different PMI

Figure 13

Table (3): Comparison of optical density of lung DNA at different PMI

Figure 14

Table (4): Comparison of optical density of brain DNA at different PMI

Figure 15

Table (5): Comparison of optical density of muscle DNA at different PMI

Discussion

Determination of the PMI is one of the most valuable subjects in forensic practice. However, it is often very difficult to accurately determine the PMI in daily practice. Forensic DNA technology has recently been used to estimate the PMI (Hao et al., 2007). DNA decays after death, in biological samples, and the ensuing damage is manifested in many forms (Gilbert et al., 2003). So, this study aimed to profile postmortem degradation of DNA in relation to PMI. DNA was extracted from the brain, lungs, spleen, liver and skeletal muscles of drowned rats at different PMI (0, 3, 6, 12 and 24 hours postmortem). Total genomic damage of DNA was determined by gel electrophoresis and its intensity was measured by software Gel Pro analyzer computer program as maximum optical density.

In drowning cases, no previous researches studied the relation between PMI and DNA amount in these organs by using this modified electrophoresis method.

Generally, results of this study revealed gradual degradation of intact nuclear DNA in the studied organs with increasing PMI. These findings coincide with those of Luo et al. (2006) who showed gradual decrease of bone marrow DNA with prolongation of PMI. Concerning DNA maximal optical density, it showed a significant lower mean values in the studied organs with increasing the PMI than control group at zero time at intact DNA which was prominent in the lungs beginning from 3 hours PM and in the spleen beginning from 6 hours PM as seen in their computer charts. While, there is a significant higher mean value of maximum optical density than control group at 600, 400 and 200 base pairs which is prominent in the liver. In agreement with these findings, Johnson and Ferris (2002) reported that in tissues such as liver and kidneys, enzymes tend to be more active and accelerate DNA decomposition. In the present study, the used method was useful in detection of fragmented DNA in the liver up to 24 hours PM. Also, Lin et al. (2000) observed that the DNA degeneration rate of liver cells had a linear relationship to early postmortem period in rats.

Regarding spleen, there was descendent trend of the amount of intact DNA at the different PMI. This was similar to conclusion of Liu et al. (2004) by using flow cytometry while the method used in this study is much easier in application. Chen et al. (2005) showed also a good relationship between spleenic DNA degradation and PMI. The fragmentation in DNA had begun in lungs and skeletal muscles at 3 and 6 hours respectively; this may be also attributed to the presence of many enzymes in these organs. Also, postmortem skeletal muscles up regulate proteolysis related genes (Sanodou et al., 2004). Considering brain DNA degradation, it occurred at slower rate than other organs and become prominent at 24 hours PM. Leonard et al. (1993) concluded that human postmortem brain collections will continue to be valuable resources for the study of gene expression and isolation of nucleotide sequences.

According to the previous results of this study, it can be concluded that the degradation of DNA shows a well relationship with early PMI (up to 24 hours) in the studied organs. This degradation revealed sequential time dependent process with the potential for use as a predictor of PMI. The slower degradation of brain DNA invites more research use of molecular genetic techniques for the study of PMI from this organ.

The present study used a simple, easy, applicable and highly informative electrophoresis method that make it an ideal for the busy forensic laboratory. So, this method can be used for a reliable and sensitive analysis of PMI and future human studies should be considered with more prolonged PMI. It is also recommended to study DNA degradation and PMI in different causes of death for revealing if there is any effect of the cause of death on DNA degradation rate.