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JOSS:

Journal of Social Science

THE EFFECT OF STREPTOMYCIN AND CLINDAMYCIN ON DNASE

I ACTIVITY ON THE QUALITY OF DNA OF SALIVARY ORIGIN

Hamimatur Rohmah

, Zulham

Fakultas Kedokteran Universitas Muhammadiyah Sumatera Utara

, Departemen Histologi,

Fakultas Kedokteran, Universitas Sumatera Utara

Email : [email protected]

, [email protected]

KEYWORDS

Clindamycin,

DNase, salivary

DNA,

Streptomycin,

Preservation

ABSTRACT

Deoxyribonucleic acid (DNA) contains genetic material with all an

individual’s information. Saliva has the potential to be a good source

of human DNA. When compared to blood sampling, saliva is easier

to collect and the collection process is non-invasive. However, human

DNA collected from saliva has the potential to be degraded due to

deoxyribonuclease (DNase) activity in saliva. DNase activity can be

inhibited by antibiotics, such as aminoglycoside antibiotics and

lincomycin derivatives. This study aimed to determine the effect of

streptomycin and clindamycin on DNAse I activity against the quality

of human genomic DNA from saliva. The sampling technique was

that samples were collected from 9 subjects with each subject flowing

into a saliva pot. Before saliva collection, subjects were asked to rinse

their mouth with a solution of chlorhexidine gargle (Listerin™) for

30 seconds, then the samples were divided into four groups. The

average concentration of DNA extracted from the spin-column

method was 32.91 g/mL (7.10-99.45 g/mL) while the average purity

was 1.813 g/mL (1.639-2.043 g/mL). With clindamycin treatment,

PCR was able to amplify the human NOTCH2 gene (~704 bp) while

various concentrations of streptomycin produced multiple bands of

~100 bp. The concentration of 3.2 mM clindamycin effectively

inhibits DNAse I activity and can amplify the human NOTCH2 gene

while streptomycin cannot protect.

INTRODUCTION

Deoxyribonucleic acid (DNA) contains genetic material that contains all the identifying

information of an individual (Syaifiatul, 2017). DNA resides in the nucleus of eukaryote cells

and is found in almost all individual cells. Human body parts such as saliva, blood, sperm, skin

cells, hair, urine, sweat, etc. can be a source of DNA. (Putri & Yudianto, 2016), (Wahab et al.,

2017).

Saliva has the potential to be a good source of human DNA. When compared to blood

sampling, saliva is easy to collect and its collection is non-invasive. (Takeshita et al., 2016),

(Garbieri et al., 2017) Saliva is produced and secreted by the salivary glands as a complex fluid

with 99% saliva consisting of water, organic, and inorganic substances. (Wahab et al., 2017)

In addition, saliva also contains normal flora, enzymes, cells, electrolyte fluids,

microorganisms, epithelial cells, hormones, immunoglobulins and so on. (Tiwari, 2011), (Bibi

et al., 2021), (Aas et al., 2005) Epithelial cells in the oral mucosa that are released along with

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saliva have the potential to be a source of DNA for diagnostic purposes. However,

contaminants in saliva can damage DNA (Garbieri et al., 2017).

Human DNA collected from saliva is potentially damaged due to deoxyribonuclease

(DNase) activity in saliva (Garbieri et al., 2017). DNase is an enzyme capable of breaking

down nucleic acids (Rodwell et al., 2015). Belonging to a heterogeneous class of enzymes,

DNase catalyzes the hydrolysis of DNA and eventually degrades it. The two main types of

DNase are DNase I and DNase II (Kolarevic et al., 2014). DNase II is known as acidic DNase

because its optimal activity is in the low pH environment of lysosomes (Varela-Ramirez et al.,

2017).

Activity DNase can be inhibited by antibiotics, such as aminoglycoside antibiotics and

lincomycin derivatives (lincosamides) (Kolarevic et al., 2014), (Spížek & Řezanka, 2017).

Aminoglycosides are antibacterial produced by Streptomyces or other fungi. Since 1943,

various aminoglycoside derivatives have been developed. Examples of aminoglycoside drugs

are streptomycin, neomycin, kanamycin, gentamicin, amikacin, and tobramycin (Rohmah et

al., 2019).

Aminoglycosides with the suffix "mycin" (streptomycin, neomycin, kanamycin,

paromomycin, and tobramycin) are derived from Streptomyces; aminoglycosides ending in

"main" (gentamicin, netilmicin, and amikacin) are derived from Micromonospores (Fransiska,

2019).

The term lincomycin originated in Lincoln, Nebraska (United States), the area where this

type of antibiotic was first isolated from Streptomyces lincolnensis in soil samples (Spížek &

Řezanka, 2017).

Lincosamide is a relatively small class of antibiotics with a chemical structure

consisting of amino acids and sugars. Members of the group of antibiotics that form naturally

from lincosamide are lincomycin and celesticetin. Many semisynthetic lincomycin derivatives

have been synthesized. Of these types, only clindamycin is effective. Lincosamide is produced

by several species of Streptomyces especially S. lincolnensis, S. Roseland, S. caelestetis, and

Micromonospora halophytica. Lincosamide is widely active against anaerobic bacteria (Spížek

& Řezanka, 2017).

The potential of aminoglycoside antibiotics on DNase activity appears to vary.

Clindamycin, a derivative of lincomycin produced by Streptomyces lincolnensis, shows

activity against bacteria, especially Gram-positive bacteria, and protozoa. These antibiotics are

bacteriostatic, inhibit protein synthesis in sensitive bacteria, and generally at higher

concentrations can be bactericidal. Clindamycin is more effective than lincomycin in the

treatment of bacterial infections, in particular those caused by anaerobic species. In addition,

clindamycin effectively inhibited DNase activity in mouse lesions by necrotizing fasciitis at a

high dose of 100 μg/rat twice daily intraperitoneally, although its role in inhibiting DNA

degradation is unclear (Andreoni et al., 2017), (Bertram, 1998), (Spížek & Řezanka, 2017).

Preservation of human DNA from saliva can be done using aminoglycoside antibiotics

and lincomycin derivatsives. Streptomycin and clindamycin, in addition to inhibiting DNase

activity, are also easily obtained at low prices. The assessment of the successful preservation

of human DNA of salivary origin rests on the inhibition of DNase activity and can be assessed

in quality and quantity. This study aims to assess the effect of streptomycin and clindamycin

on DNAse I activity on the DNA quality of the human genome of salivary origin.

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METHOD RESEARCH

This type of research is using purely experimental research with a pretest-posttest design.

This research was carried out at the Integrated Laboratory of the Faculty of Medicine,

University of North Sumatra (USU). This study took place in December 2021-January 2022.

In this study, saliva was collected from each subject. Before saliva collection, subjects were

asked to gargle with a solution of chlorhexidine gargle (Listerin™) for 30 seconds and sterile

equates. Inclusion and exclusion criteria are not required. A total of 15 mL of clean saliva was

collected from each subject with each subject draining into a clean saliva pot.

Saliva samples from each subject were immediately divided into 4 groups: negative

control (K1), positive control (K2), Streptomycin (K3), and Clindamycin (K4). Data analysis

was carried out with univariate mean and standard deviation for the concentration and purity

of extracted DNA. DNase I activity on DNA extraction from saliva was qualitatively assessed

through observation of the electrophoresis result of 1% agarose gel. Assessment of PCR results

for human NOTCH2 genes from DNase I inhibition results by antibiotics, was carried out using

a descriptive approach with observations on the results of 1% agarose gel electrophoresis. The

expected result on 1% agarose gel electrophoresis is the presence of an expected amplicon

length (~704 bp).

RESULT AND DISCUSSION

After conducting research, the data that has been collected is that nine DNA samples

obtained have been successfully extracted by the spin column method. The concentration and

purity of DNA obtained varied from sample to sample (Table 4.1). The mean DNA

concentration was 32.91 μg/mL in the range of 7.10-99.45 μg/mL. The average purity of the

extraction yield was 1.813 μg/mL in the range of 1.639-2.043 μg/mL.

Table 1

Purity and Concentration of DNA of Salivary Origin

Sample

Concentration

(ng/uL)

Purity

13,25

1,791

7,10

2,043

20,05

1,759

20,10

1,803

7,95

1,639

10,60

1,927

33,45

1,848

99,45

1,742

84,25

1,761

Average

32,91

1,813

Range

(Minimum-

Maximum)

7,10-99,45

1,639-2,043

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DNA degradation testing was performed to determine the activity of DNAse I against

DNA extracts of salivary origin and visualized by electrophoresis on 1% agarose gel. DNase I

degrades a sample of salivary-origin DNA (Figure 1).

Figure 1

DNA degradation assay

Lane 2 and lane 4 (10μL and 5μL salivary DNA extract) as positive control DNA

degradation assays, showing intact salivary origin DNA. The addition of DNAse I to salivary

origin DNA samples (lanes 3 and 5 (10μL and 5μL salivary DNA extract) showed empty

lanes 3 and 5 and showed no presence of DNA. There is no DNA smear or DNA

fragmentation in lanes 3 and 5.

Electrophoresis of 1% agarose gel was performed for DNA visualization after the

addition of various concentrations of streptomycin as an inhibitor of DNAse I. Although at a

concentration of 0.1 M streptomycin was not able to inhibit, DNAse I activity appeared to be

inhibited with the addition of streptomycin concentrations (Figure 4.2). Streptomycin

concentration of 0.2 mM can maintain the integrity of 1 μg DNA from 2.5 mg/mL DNase I

activity (Figure 2).

The PCR approach could not show the amplicon length of the NOTCH2 gene as expected

(~704 bp). Instead, the amplicon length formed measures about 100 bp (Figure 3).

Figure 2

Hasil electrophoresis Streptomycin-DNase I protection assay.

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There is protection from DNA degradation by DNase I in lanes 3, 4, 5, 6, 7, and 8. At a

streptomycin concentration of 0.1 mM (lane 2), there is a DNA smear. Increased streptom

concentrations to 0.2 mM (lane 3), 0.4 mM (lane 4), 0.8 mM (lane 5), 1.6 mM (lane 6), 2.0 mM (lane

7), 3.2 mM (lane 8) showed streptomycin could maintain whole DNA bands of DNase I activity.

Figure 3

Electrophoresis results from human NOTCH2 gene PCR

PCR results of the human NOTCH2 gene from salivary DNA samples on agarose gel 1% after

administration of streptomycin, 2.5 mg/mL DNase I added to salivary DNA showed the presence of

bands measuring 100 bp in all lanes with streptomycin concentrations of 0.1-3.2 mM.

Clindamycin with different concentrations, affects the preservation of salivary origin DNA

(Figure 4.4). The higher the concentration of clindamycin, the higher its preservation ability to salivary

DNA that undergoes DNA degradation assay. DNA bands appear more pronounced at concentrations

of 3.2 mM (Figure 4.4). DNA smears still appear at 0.8 mM clindamycin, showing the partial ability of

clindamycin to prevent salivary DNA degradation by 2.5 mg/mL DNase I.

Figure 4

The effect of clindamycin on DNAse I activity.

Clindamycin inhibits DNase I activity at different concentrations with an effect that gets

stronger with increasing concentration. Clindamycin concentrations are 0.1 mM (lane 2), 0.2 mM

(lane 3), 0.4 mM (lane 4), 0.8 mM (lane 5), 1.6 mM (lane 6), 2.0 mM (lane 7), and 3.2 mM (lane 8).

DNA smear (lanes 2, 3, 4, 5) becomes DNA without it (lanes 6, 7, 8). Lane 8 shows very clear and

thick intact DNA with the highest concentration of clindamycin (3.2 mM).

PCR electrophoresis results from the human NOTCH2 gene that had been given varying

concentrations of clindamycin (0.1-3.8 mM) and 2.5 mg/dL showed a single band at ~704 bp.

Expected bands (~704 bp) of the human NOTCH2 gene were seen at all concentrations of

clindamycin (Figure 5).

Figure 5

Human NOTCH2 gene PCR Electrophoresis Results

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The expected band of the human NOTCH2 gene (~704 bp) is in the lane (2, 3, 4, 5, 6, 7, 8).

PCR can amplify human NOTCH2 genes from DNA given clindamycin in concentrations of 0.1-3.2

mM and 2.5 mg/dL DNase I. Clindamycin concentrations are 0.1 mM (lane 2), 0.2 mM (lane 3), 0.4

mM (lane 4), 0.8 mM (lane 5), 1.6 mM (lane 6), 2.0 mM (lane 7), 3.2 mM (lane 8).

Salivary DNA obtained from extraction needs to be observed in terms of quantity and quality.

DNA quantity analysis is performed to determine the purity and concentration of DNA. DNA purity

can be determined through spectrophotometric rules with a ratio of A260 nm and A280 nm.

The results of this study are by previous studies, where the results of DNA extraction are said

to be pure if the ratio of A260 nm and A280 nm reaches 1.8-2.027 or the extraction results are also

considered pure if the absorbance ratio is between 1.6 and 2.0.5 The range of values indicates that the

amount of DNA in the sample is more than that of proteins. If the ratio of A260/A280 is < 1.8 or >

2.0, it is indicated that DNA is still contaminated with RNA and proteins.

In other studies, the

average purity of salivary DNA ranged from 1.8 to 2.0.3 If the sample has a ratio of about 1.8 or close

to 1.8, then the DNA sample can be said to be relatively pure (Garbieri et al., 2017).

The DNA concentration in this study has a range of 7.10–99.45 μg/mL with a salivary volume

of 2 mL and the spin column method. Another study with a volume of 1 mL using the Oragene™ kit

showed a lower DNA concentration of 10 ng/μL.5 This can occur due to factors that affect DNA

concentration. Factors that can affect DNA concentration such as incubation temperature factors,

types of chemicals, and technical factors during DNA extraction work (Khare et al., 2014).

The results of this study, the administration of streptomycin did show the ability of

streptomycin to maintain DNA measuring > 1000 bp (Figure 2.). However, this does not guarantee

that the DNA is intact. PCR on NOTCH2 showed the ~100 bp band was not as expected ~704 bp.

The NOTCH2 gene may have also been degraded because streptomycin is unable to inhibit DNase

activity (WOEGERBAUER et al., 2000). Indeed, forward and reverse primers work in PCR reactions

to multiply the remaining (pieces) of the NOTCH2 gene while producing complementary sequences

from the reverse and forward directions of the primer.

While clindamycin as a DNase inhibitor, can preserve whole DNA (Figure 4). The results of

this study were that 3.2 mM levels effectively inhibited DNAse I activity in human genomic DNA of

salivary origin and were able to maintain genes by amplification of human NOTCH2 genes such as

the estimated length of amplicon (~704 bp) (Figure 5), while streptomycin was not able to do the

same (Figure 3). The results of this study are in line with previous studies that clindamycin can inhibit

DNase activity in vivo 100 μg/mL.15 In human tissue, DNase activity is completely lost after two

days of additional treatment with clindamycin although high bacterial concentrations remain

(Andreoni et al., 2017).

The results of this study are also by Liu Yawen and colleagues' research on DNA preservation

that salivary DNA can be damaged due to DNase activity in saliva, causing a decrease in DNA quality

and quantity.5,29 Therefore DNA preservation or protection is necessary to maintain DNA quality

and integrity.29 As a type of enzyme, DNase activity can be affected or even eliminated by regulating

, cofactor30a, and temperature (Huque et al., 2020). In another study, it was said that DNAse

activity decreased drastically when it was at temperatures above or below the optimum temperature

range (20-70 °C) (Huque et al., 2020).

One strategy of DNA preservation from saliva is to protect DNA from DNase activity by

administering DNase inhibitors. Aminoglycoside antibiotics such as clindamycin, gentamycin, and

neomycin can work to inhibit DNase activity.10,15,31 Other types of DNase inhibitors are available

as synthetic and natural ingredients (Kolarevic et al., 2014).

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CONCLUSION

Clindamycin can inhibit DNAse I activity so that it can maintain the quality of human

genome DNA from saliva. Streptomycin cannot inhibit DNAse I activity so it cannot maintain

the DNA quality of the human genome of salivary origin. Clindamycin 3.2 mM effectively

inhibits DNAse I activity in human genome DNA of salivary origin while streptomycin does

not inhibit The quality of human genes of salivary origin and can be protected with clindamycin

0.1 mM with PCR can amplify human NOTCH2 genes while streptomycin cannot protect.

REFERENCES

Aas, J. A., Paster, B. J., Stokes, L. N., Olsen, I., & Dewhirst, F. E. (2005). Defining The Normal

Bacterial Flora Of The Oral Cavity. Journal Of Clinical Microbiology, 43(11), 5721–

5732. Google Scholar

Andreoni, F., Zürcher, C., Tarnutzer, A., Schilcher, K., Neff, A., Keller, N., Marques Maggio,

E., Poyart, C., Schuepbach, R. A., & Zinkernagel, A. S. (2017). Clindamycin Affects

Group A Streptococcus Virulence Factors And Improves Clinical Outcome. The Journal

Of Infectious Diseases, 215(2), 269–277. Google Scholar

Bertram, K. G. (1998). Basic And Clinical Pharmacology. Editions Appelton And Lange.

Stanford, Conn, 14, 16. Google Scholar

Bibi, T., Khurshid, Z., Rehman, A., Imran, E., Srivastava, K. C., & Shrivastava, D. (2021).

Gingival Crevicular Fluid (Gcf): A Diagnostic Tool For The Detection Of Periodontal

Health And Diseases. Molecules, 26(5), 1–16. Google Scholar

Fransiska, F. (2019). Ototoksisitas Aminoglikosida. Keluwih: Jurnal Kesehatan Dan

Kedokteran, 1(1), 36–46. Google Scholar

Garbieri, T. F., Brozoki, D. T., Dionisio, Thiago Jose, Santos, C. F., & Neves, L. T. Das. (2017).

Human Dna Extraction From Whole Saliva That Was Fresh Or Stored For 3,6 Or 12

Month Using Five Different Protocols. 25(2), 147–148. Google Scholar

Huque, A. K. M. M., So, W. M., You, M. K., & Shin, J. S. (2020). Phylogenetic Analysis And

In Vitro Bifunctional Nuclease Assay Of Arabidopsis Bbd1 And Bbd2. Molecules, 25(9),

2169. Google Scholar

Khare, P., Raj, V., Chandra, S., & Agarwal, S. (2014). Quantitative And Qualitative

Assessment Of Dna Extracted From Saliva For Its Use In Forensic Identification. Journal

Of Forensic Dental Sciences, 6(2), 81. Google Scholar

Kolarevic, A., Yancheva, D., Kocic, G., & Smelcerovic, A. (2014). Deoxyribonuclease

Inhibitors. European Journal Of Medicinal Chemistry, 88, 101–111. Google Scholar

Putri, N. P. P. E., & Yudianto, A. (2016). Pengaruh Tanah Dan Air Laut Terhadap Kualitas

Dna Dari Otot Psoas Jenazah Melalui Metode Str. Jurnal Biosains Pascasarjana Vol. 18,

18(3), 205. Google Scholar

Rodwell, V. W., Bender, D. A., Botham, K. M., Kennelly, P. J., & Weil, A. P. (2015).

Illustrated Biochemistry. New York: Mcgraw-Hill. Google Scholar

Rohmah, J., Cholifah, S., & Rezania, V. (2019). Pelatihan Higiene Dan Sanitasi Makanan Pada

Pedagang Makanan Di Kantin Sd. Loyalitas, Jurnal Pengabdian Kepada Masyarakat,

2(2), 170. Https://Doi.Org/10.30739/Loyal.V2i2.473 .Google Scholar

Spížek, J., & Řezanka, T. (2017). Lincosamides: Chemical Structure, Biosynthesis, Mechanism

Of Action, Resistance, And Applications. Biochemical Pharmacology, 133, 20–28.

Google Scholar

Syaifiatul, H. (2017). Dna Sebagai Bukti Untuk Memecahkan Tindakan Kriminal. Semin Nas

Humanira Apl Teknelogi Inf, 2017. Google Scholar

Takeshita, T., Kageyama, S., Furuta, M., Tsuboi, H., Takeuchi, K., Shibata, Y., Shimazaki, Y.,

Akifusa, S., Ninomiya, T., Kiyohara, Y., & Yamashita, Y. (2016). Bacterial Diversity In

Saliva And Oral Health-Related Conditions: The Hisayama Study. Scientific Reports, 6,

Vol 2, No 4 April 2023

The Effect Of Streptomycin And Clindamycin On Dnase I

Activity On The Quality Of Dna Of Salivary Origin

410

https://joss.al-makkipublisher.com/index.php/js

1. Google Scholar

Tiwari, M. (2011). Science Behind Human Saliva. Journal Of Natural Science, Biology And

Medicine, 1–3. Google Scholar

Varela-Ramirez, A., Abendroth, J., Mejia, A. A., Phan, I. Q., Lorimer, D. D., Edwards, T. E.,

& Aguilera, R. J. (2017). Structure Of Acid Deoxyribonuclease. Nucleic Acids Research,

45(10), 6217–6227. Google Scholar

Wahab, R. M. A., Nasri, F. A. M., & Abidin, I. Z. Z. (2017). Kesan Penyimpanan Sampel Air

Liur Terhadap Kualiti Dna Genom. Sains Malaysiana, 46(6), 909–910. Google Scholar

Woegerbauer, M., Burgmann, H., Davies, J., & Graninger, W. (2000). Dnase I Induced Dna

Degradation Is Inhibited By Neomycin. The Journal Of Antibiotics, 53(3), 276–285.

Google Scholar

Hamimatur Rohmah, Zulham (2023)

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