Food and medicine are vital to human survival. Hybrid food products are produced to feed humans and livestock. Recently nucleic acid treatment through parenteral or GIT administration has been reported. The nucleic acid fate in the GIT tract has been unclear. A mouse model was developed to study the fate of DNA in the digestive tract in mice.
The advancement rate was found to be 20.27±2.60% at 10 min post gavage. The advancement in the movement of bowel/chyme was observed in the GIT in mice. At 180 to 360 min post-gavage, the mouse stomach was completely emptied, and the contents of the stomach had fully entered the small intestine. This finding is consistent to those reported by Chul-Hyun Lim, who used a 13C octanoic acid breath test to measure gastric emptying times in mice. After 510 to 570 min of digestion, all contents had entered the cecum. This would provide understanding of the advancement of bowel movement.
Currently, the most commonly used DNA-based detection techniques are PCR and qPCR. qPCR, the most commonly used DNA quantification method, has the advantages of low pollution and high specificity. As shown in Figures 5 and 6, clear bands in the mouse stomach were observed pre-gavage (0 min) and post-gavage (i.e., during 40–120 min). These results confirmed the presence of human leukocyte DNA in the stomach in mice after 120 min of digestion (>200 bp). There were no observed DNA bands for the four target genes in the contents of the small intestine in mice at 40, 80 and 120 min after gavage. The DNA had degraded into fragments of <77 bp in the small intestine in mice. Nawaz found that food DNA can survive in the digestion process, and DNA fragments up to several hundred bp can be detected in the GIT. Same as the results of other scholars[9–13], DNA fragments can clearly be detected in the GIT of animals; however, whether they can be detected in the blood and other tissues requires further experiments.
Anatomically, the mouse stomach consists of two regions—the non-glandular/fore-stomach and glandular stomach—which are separated by a limiting ridge. Gastric juice consists of pepsin and gastric acid. Pepsin's main function is to digest protein, but in recent years, Liu found that the pepsin in gastric juice can digest not only protein but also nucleic acid. The digestion of nucleic acid starts in the stomach, and various animal pepsins have different abilities to digest nucleic acid. Most dietary DNA is in the form of histones, which form nucleosomes. The complex components of the diet may affect the digestion of DNA by pepsin. Zhang demonstrated that common food components, including proteins, carbohydrates, metal cations and polycationic compounds, are closely associated with the digestion of DNA through in vitro simulation studies.
Ct values in the gastric contents of mice at 0, 40, 80 and 120 min respectively after gavage were measured through qPCR as shown in Figure 7. The DNA concentration decreased consistently from 0 to 120 min post gavage, and at 120 min, 85.62±8.10% decrease was noted as compared with 0 min (Table 3). The half-life of DNA degradation in mouse stomach was 70.50±5.46 min (Figure 9). This indicated that the DNA concentration in the mice stomach decreased significantly. Wiedemann S  analyze rubisco and cry1Ab gene through real-time PCR and reported that the degradation was 20% of the initial value at 2 hours as compare with 0 hour. The degradation was 0.5% of the initial value (0 hour) after incubated for 48 hours in the rumen.
DNA degradation may be associated with mechanical aspects, gastric juice and microorganisms in the mice stomach. The DNA was not completely degraded in the stomach of mice, and remained >200 bp of DNA fragments. Protein and carbohydrate, the main components of food, do not affect DNA digestion at the concentrations recommended by the WHO (40:1 and 80:1). When the ratio of protein to DNA is >80:1, DNA digestion is inhibited. Divalent cations (Ca2+ and Mg2+) can result in greater DNA digestion than monovalent cations (Na+ and K+). The gavage included nutritive semi-solid paste and human white blood cells. The sodium carboxymethyl cellulose, starch and milk powder in the nutritive semi-solid paste resembled normal dietary components, thus potentially inhibiting DNA digestion. In addition, the structure of human leukocytes includes a cell membrane and nucleus, which may protect against DNA digestion. According to Zhang, pepsin has a digestive effect toward nucleic acid, on the basis of in vitro simulation: pepsin can digest specific sequences nucleic acids, such as 5´-AAG↓AA-3´ and CGA↓T. The target genes TH01, TPOX and D7S820 have repetitive sequences rich in TCAT, GAAT and GATA, respectively. Mouse pepsin may have a restriction enzymatic effect on these sequences, thus resulting in DNA degradation.
In qPCR method, when the Ct value is >35, the target gene is considered absent. In Table 4, from the amplification curve, the Ct value of the mouse gastric contents at 40 min was approximately 26 (<35). The Ct values of the four target genes in the upper part of the small intestine of the mice at 40 min were all less than 35, thus indicating that the four target genes were present in very low amounts; in the lower part of the mouse small intestine at 40 min, only the GAPDH gene had a Ct value less than 35. These results indicated the presence of a small amount of GAPDH, whereas the Ct values of the other three target genes were >35, indicating the absence of the target genes. Similarly, the target genes were not detected in the DNA of the small intestine contents in mice at other times after gavage (Figure 10). These results were consistent with the PCR results, indicating that DNA was further degraded into small fragments <77 bp in the small intestine by digestive enzymes and intestinal microorganisms. The DNA in human white blood cells was more easily degraded digested by the gastric juices in mice when it entered the intestines.
The STR technique was used to amplify small fragments of DNA. The average peak area (38 alleles; 77–446 bp) of 21 gene loci amplified by STR provides a good representation of DNA degradation. The capillary zone electrophoresis-laser induced fluorescence method can be used to determine the DNA concentrations in serum and plasma, and is as accurate and sensitive as the widely used real-time PCR method[31, 32].
The results obtained from plotting the average peak area and gavage time were consistent with the results of qPCR (Figure 11A). According to the natural logarithm of the average peak area versus the digestion time, the half-life of DNA degradation in the mouse stomach was 63.13 min. This result was consistent with the half-life of DNA degradation calculated by qPCR method. After food enters the stomach, through mechanical digestion and chemical digestion, part of the food bolus enters the intestines in the form of chyme. Our results showed that the rate of cellular DNA degradation in the mouse stomach was slow. STR map analysis of DNA in the small intestine in mice revealed that as the chyme advances in the small intestine, the number of human DNA alleles decreased (Table 5). qPCR and STR both clearly showed that human genomic DNA was markedly more degraded in the mouse intestine than the mouse stomach, whereas human target genes were degraded gradually in the mouse intestine with chyme advancement. Gene degradation times were also predicted through the STR method (Figure 11B).
Liu et al. reported that nucleic acids are digested in the stomach in blackhead fish and banded grouper, whereas the digestion of nucleic acids by bovine gastric enzymes was not observed. Different animals have varying ability to digest nucleic acids with pepsin. According to the experimental results of the current study, the mouse pepsin can be assumed to have a digestive effect toward human genomic DNA, thus, providing a potential reference for future experiments.
In recent years, no detailed research has performed a quantitative analysis of the degradation of DNA in the digestive tract in mice. Our findings should contribute to future food and drug research, and risk assessment of genetically modified foods.