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JOSS:

Journal of Social Science

EFFECT OF GENTAMICIN AND NEOMYCIN+BACITRACIN ON

DEOXYRIBONUCLEASE ACTIVITY I ON THE QUALITY OF

HUMAN DNA OF SALIVARY ORIGIN

Siti Chairani

, Zulham

Faculty of Medicine, Muhammadiyah University of Sumatera Utara

, Department of

Histology, Departemen Histologi, Fakultas Kedokteran, Universitas Sumatera Utara

Email : [email protected]

, [email protected]2

KEYWORDS

deoxyribonucleic

acid;

deoxyribonuclease

;gentamicin,

neomycin, saliva

ABSTRACT

Deoxyribonucleic acid (DNA) can be found in saliva and various

other sources. Saliva contains epithelial cells and leukocytes that are

released into the oral cavity. Cells in saliva are potential sources of

DNA for diagnostic purposes. DNA in saliva can be degraded due to

the activity of deoxyribonuclease (DNase), an enzyme that catalyzes

the hydrolysis of DNA. DNase I is expressed in exocrine cells while

DNase II is expressed in macrophages. Inhibition of DNase activity

will preserve DNA. Besides being able to kill bacteria,

aminoglycosides (i.e. gentamicin and neomycin) can inhibit DNase.

Methods: Saliva was collected from 8 subjects. Furthermore, saliva

samples from each subject were divided into 4 groups, namely

negative control (K1), positive control (K2), gentamicin (K3), and

neomycin + bacitracin (K4). DNA was extracted from saliva using

the spin column method. DNA from groups K3 and K4 were added

to 1 mg/mL gentamicin and 20 mM neomycin + bacitracin,

respectively, before adding 2.5 g/mL DNase I (DNA degradation

assay). The quality of human genomic DNA from saliva was

determined by the presence of the NOTCH2 gene amplicons (~704

bp). DNA was separated by 1% agarose gel electrophoresis and

recorded using the gel documentation system. Results: The DNA

extracted from saliva using the spin column method had an average

concentration of 26.49 + 30.70 ng/mL and an average purity of 1.819

+ 0.122. The administration of distilled water as a negative control in

the DNA degradation assay showed that no DNA was digested.

Gentamicin inhibited the activity of 2.5 mg/mL of DNase I at a

concentration of 35 g/mL while neomycin + bacitracin inhibited it at

a concentration of 0.8 mM. Conclusion: Certain levels of gentamicin

and neomycin+ bacitracin inhibited the activity of 2.5 mg/mL DNase

I and maintained the quality of human genomic DNA from saliva.

INTRODUCTION

Deoxyribonucleic acid (DNA) is a macromolecule in the form of a linear polymer

composed of nucleotide monomers. The process of transcription and translation of DNA

produces products in the form of proteins. DNA can be found in saliva, skin, sweat, blood,

teeth, hair, mucus, sperm, and vaginal fluids (Syaifiatul, 2017).

Saliva is an acidic oral liquid (pH 6-7). Three major glands and thousands of minor

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salivary glands in the oral cavity are the main components of salivary development (Kaczor-

Urbanowicz et al., 2017). Saliva consists of 99% water and the rest is protein, inorganic, and

organic substances. Gingival crevicular fluid, cell debris, plaque, bacteria, nasal secretions,

epithelial cells, blood, and exogenous substances are also present in saliva. Some oral

microbiota are present in saliva and consist of bacteria, archaea, fungi

(Valentijn-Benz et al.,

2015), protozoa

(Khairnar & Parija, 2008), and viruses

(Samaranayake & Matsubara, 2017).

Most of the organisms in the salivary microbiota can be found in almost all individuals as

normal flora. This normal flora consists of Streptococcus, Neisseria, Rothia, Prevotella,

Actinomyces, Granulicatella, Porphyromonia, Haemophilus, and Porphyromonas species

(Yamashita & Takeshita, 2017).

In saliva can be found epithelial cells that are released mucosa

of the oral cavity (Garbieri et al., 2017).

Epithelial cells released into saliva could potentially be used as a source of DNA for

diagnostic purposes using nucleic acid sources. Saliva is easy to collect because it is non-

invasive and there are many (Garbieri et al., 2017).

Unfortunately, efforts to obtain human

DNA from saliva are not easy because contaminants in saliva have the potential to damage

human DNA. Normal flora, enzymes, hormones, immunoglobulins and other biomolecules

secreted in saliva have the potential to immediately damage human DNA in saliva (Garbieri et

al., 2017).

Deoxyribonuclease (DNase) is an enzyme capable of catalyzing DNA (Kolarevic et al.,

2014). There are 2 types of DNase. DNase I is expressed by exocrine cells in the

gastrointestinal tract. DNase II is expressed by macrophage cells located throughout the tissue

(Keyel, 2017).

Inhibition of DNase activity can use aminoglycosides (Krause et al., 2016).

The use of aminoglycosides to preserve human DNA from saliva will be beneficial

because aminoglycosides inhibit DNase activity as well as bactericidal properties that kill

bacteria in saliva (Kolarevic et al., 2014), (Krause et al., 2016). Aminoglycosides inhibit

bacterial protein synthesis by blocking the initiation process or by changing the conformation

of amino acids (Krause et al., 2016). In addition, aminoglycosides can enter bacterial cells and

increase membrane permeability by involving the electrostatic binding of polycationic

aminoglycosides to negatively charged components, such as phospholipids, Gram-positive

bacterial teichoic acid, and Gram-negative bacterial phospholipids and lipopolysaccharides

(Krause et al., 2016).

The aminoglycosides used in the form of gentamicin and neomycin + bacitracin are types

of aminoglycosides that can be purchased cheaply and are also easy to obtain. In addition,

gentamicin works as an antibiotic and DNase inhibitor (McGuire et al., 2015).

Preservation of human DNA of salivary origin is possible using aminoglycosides. The

assessment of the success of preservation can be assessed in quantity and quality. This study

aims to see the effect of aminoglycosides on DNase activity on the quality of human DNA of

salivary origin (Alipour et al., 2009).

METHOD RESEARCH

This study used experimental methods. The study was conducted from December 2021

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Of Salivary Origin

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to January 2022. This research was conducted at the Integrated Laboratory of the Faculty of

Medicine, University of North Sumatra (Kolarevic et al., 2014). This study used saliva and

salivary DNA from 8 subjects. Saliva was divided into four groups, each consisting of

Negative control (K1) given Aquades, positive control (K2) given DNAse I, treatment group

1 given gentamicin (K3), and treatment group 2 given neomycin + bacitracin (K4)

(Woegerbauer Et Al., 2000).

Research procedure

a. Saliva collection from 10 subjects was collected in one room at 07.00 WIB and was not

allowed to brush their teeth and gargle with oral cleaning liquid. Subjects were instructed

not to eat and drink for 90 minutes before saliva collection. Next, the subject was given 10

minutes to spit in a salivary pot.

b. DNA isolation preparations. Each saliva of as much as 2 mL is inserted in a microtube then

DNA isolation using the spin column method by looking at DNA purity with UV-Vis

spectrophotometry and calculated absorbance value of 260 nm divided by absorbance value

of 280 nm (A260 / A280), and DNA purity value ranges from 1.8-2.0.

c. Negative control (K1). A total of 1 μg of salivary DNA was given the addition of aqua dest.

d. Positive control (K2). A total of 1 μg of salivary DNA was treated with DNA degradation

assay with the addition of 2.5 μg/mL DNase I (DN25, Merck).

e. Treatment group (K3). A total of 1 μg of salivary DNA will be added with 1 mg / mL of

gentamicin antibiotics and 2.5 μg / mL DNase I (DNA degradation assay).

f. Treatment group (K4). A total of 1 μg of salivary DNA will be added with 20 mM of

neomycin + bacitracin antibiotics and 2.5 μg / mL DNase I (DNA degradation assay)

g. DNA testing with 1% agarose gel. A total of 1.3 g of 1% agarose was included in the

Erlenmeyer flask and given 130 mL of 1X TAE buffer.

h. PCR preparation. The PCR method was run using 25 μL of PCR reaction mixture containing

1 ng of salivary DNA as a template, 1 μM for each primer, and 200 μM for each dNTP, 2.5

mM MgCl2, 5 μL 5X PCR buffer and also 5 U/μL Taq DNA polymerase in PCR master

mix (2GFRMKB, Kapa2G Fast ReadyMix, Merck).

RESULT AND DISCUSSION

The following research data are data obtained from 4 test groups, namely the negative

control group (K1), positive control (K2), the treatment group given gentamicin (K3), and the

treatment group given neomycin + bacitracin (K4) which have each been given salivary DNA

of 1 μg.

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a. Negative Control (K1) and Positive Control (K2)

Figure 1

DNA degradation assay

Figure 1 is a treatment with a concentration of 10 μL. Lane 1 contains a DNA ladder of

100 bp. Lane 2 is a negative control that is 1 μg DNA + equates. Lane 3 was a positive control

containing 1 μg of DNA + 2.5 μg/mL DNase I.

Kelompok perlakuan 1 (K3)

Figure 2

Effect of gentamicin in DNA degradation assay

The presence of gentamicin has prevented DNA digestion by DNase I Lane 1 contains

a DNA ladder of 100 bp. Each lane 2-9 contains 1 μg of DNA that has been added with

gentamicin 35, 70, 140, 280, 560, 1120, 1500, and 2240 μg / mL, and received 2.5 mg / mL

DNase I. DNA electrophoresis was carried out 1% agarose gel with conditions of 80 mV,

400 mA, and 90 minutes

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Figure 3

PCR results of human NOTCH2 genes from DNA samples that received additional

concentrations of gentamicin and 2.5 mg / mL DNase I.

The human NOTCH2 gene (~704 bp) was successfully amplified from all DNA

samples. Lane 1 contains a 100 bp DNA ladder. Each lane 2-9 contains 1 μg of DNA that

has been added with gentamicin 35, 70, 140, 280, 560, 1120, 1500, 2240 μg / mL, and

received 2.5 mg / mL DNase I. DNA electrophoresis was carried out 1% agarose gel with

conditions of 80 mV, 400 mA, and 90 minutes.

c. Treatment group 2 (K4)

Figure 4

Effect of neomycin in DNA degradation assay.

The concentration of neomycin determines the digestion of DNA by DNase I. A

concentration of 0.8 mM has provided minimal inhibition of the activity of 2.5 mg/mL

DNase I. Lane 1 contains a DNA ladder of 100 bp. Each lane 2-8 contains 1 μg of DNA

that has been added with Nebacetin® 0.1, 0.2, 0.4, 0.8, 1.6, 3.2 mM, and received 2.5 mg

/ mL DNase I. DNA electrophoresis was carried out 1% agarose gel with conditions of 80

mV, 400 mA, and 90 minutes.

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Figure 5

Human NOTCH2 gene with DNA preservation influence with the addition of neomycin in

DNA degradation assay.

Presence of human NOTCH2 gene (~704 bp) in agarose gel electrophoresis 1%. Lane

1 contains 100 bp DNA ladder, Lane 2 contains 0.1 mM nebacetin, Lane 3 contains 0.2 mM

nebacetin, Lane 4 contains 0.4 mM nebacetin, Lane 5 contains 0.8 mM nebacetin, Lane 6

contains 1.6 mM nebacetin, Lane 7 contains mM nebacetin. 2.0 mM, Lane 8 contains 3.2.

This study aims to determine the effect of aminoglycoside antibiotics on DNase I

activity on the quality of human DNA of salivary origin using the spin column method as a

DNA isolation method. As well as knowing the effect of gentamicin and neomycin +

bacitracin on DNase I activity in human NOTCH2 genes and determining the minimum dose

of gentamicin and neomycin + bacitracin in inhibition of DNase I activity in human genome

DNA of salivary origin assessed through 1% agarose gel electrophoresis (b / v) and followed

by visualization under UV light after ethidium bromide (EtBr) staining was carried out with

considering the presence of PCR results in the form of a band measuring ~704 bp.

Based on the results of the research presented above, DNA extraction carried out by

the spin column method is following the average purity rule of 1.8-2.0. Then the use of

gentamicin with a concentration of 35 μg/mL was sufficient to prevent the digestion of 1 μg

of DNA by 2.5 mg/mL DNase I and the use of neomycin + bacitracin with a concentration

of 0.8-2.0 mM was sufficient in preventing the digestion of 1 μg of DNA by 2.5 mg/mL

DNase I.

The results of this study are different from previous research conducted by McGuire

AL in 2015 using antibiotics in the form of gentamicin. McGuire Al stated at a concentration

of >35 μg / mL gentamicin can inhibit 2.5 mg / mL Dnase 1.13 Then the results of research

conducted by (Alipour et al., 2009) stated that the combination of DNAse and gentamicin

can increase the work of gentamicin with a minimum concentration of 64 μg / mL.14 The

use of antibiotics in the form of neomycin + bacitracin was also carried out by Ana Kolarevic

in 2014 and Markus Woegerbauer in 2000 who states that neomycin can inhibit DNAse I

activity at neomycin concentrations of 2 mM.15,16 While bacitracin does not have a major

effect on DNAse I. This is reinforced by research conducted by Jerzy Ciesiolka in 2014 which

states bacitracin can induce DNA degradation. However, the use of bacitracin in inducing

DNA degradation should be greater in concentration, even with a concentration of 23 mM

does not change the migration of plasmid DNA in agarose gel by 1%. So in this study, the

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use of neomycin was much more instrumental than bacitracin which did not affect 2.5 mg /

mL DNAse I.

CONCLUSION

Gentamicin and neomycin + bacitracin can inhibit the activity of 2.5 mg / mL DNase I

to maintain the quality of intact DNA of the human genome from saliva. Gentamicin and

neomycin + bacitracin inhibit the activity of 2.5 mg / mL DNase I to maintain the NOTCH2

gene (704 bp) Gentamicin can inhibit the activity of 2.5 mg / mL DNase I in the DNA quality

of the human genome of salivary origin at a concentration of 35 μg / mL while neomycin +

bacitracin inhibits at a concentration of 0.8 mM.

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Siti Chairani, Zulham

(2023)

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