The Effect of Goat Milk Yoghurt Casein Antioxidant Activity

on KidneyTumor Necrosis Factor Alpha (TNF-Α) and Interleukin 1

Beta (IL-1β) Levels in Rattus norvegicus Exposed by 2,3,7,8

tetrachlorodibenzo-p-dioxin (TCDD)

C. Mahdi

1,*

, A. E. P. Haskito

, M. C. Padaga

, A. Roosdiana

Faculty of Mathematics and Natural Sciences, Brawijaya University, Veteran Street, Malang 65145

Faculty of Veterinary Medicine, Brawijaya University, Puncak Dieng Eksklusif, Dau, Malang 65145

Keywords: Antioxidant, IL-1β, goat milk yoghurt casein, TCDD, TNF-α

Abstract: Dioxins are highly toxic and carcinogenic compounds. The most dangerous dioxin is2,3,7,8-tetrachloro-

dibenzo-p-diozin (TCDD). TCDD can cause health problem accumulates overtime. Goat milk yoghurtcasein

can reduce free radicals caused by TCDD. Goat milk yoghurt casein contains bioactive peptide that acts as

antioxidant. The aim of the current research was to determine the effect of goat milk yoghurt towards TNF-

αand IL1-β level after TCDD exposure. This research was an experimental study using a completely

randomized design (CRD). Rattus norvegicus were divided into 6 groups: K- (negative control), K+ (positive

control induced by TCDD 100 mg/kgBW), KP (placebo control given goat milk yoghurt casein 300

mg/kgBB), K1 (given TCDD 100 mg/kgBW and goat milk yoghurt casein 300 mg/kgBB), K2 (given TCDD

100 mg/kgBW and goat milk yoghurt casein 600 mg/kgBB), and K3 (given TCDD 100 mg/kgBW and goat

milk yoghurt casein 900 mg/kgBB). Quantitative analysis withone-way ANOVA and post hoc Tukey test

showed that goat milk yoghurt could prevent TNF-α increase significantly (p<0,05) from control groups, and

also prevent IL1-β increase although not significantly different from control groups (p>0,05). It can be

concluded that goat milk yoghurt casein 900 mg/kgBW can prevent the increase of TNF-αand IL1-β. Thus,

goat milk yoghurt casein could be used as antioxidant source against TCDD exposure.

1 INTRODUCTION

Earth ecosystem is continuously contaminated by

various pollutant. Several pollutants have better

durability against environmental degradation be it

chemically, biologically, or even to photolytic

reaction and may persist for a long time in the

environment (Ritter et al., 2007),(Gupta and Ali.,

2012). Pollutants able to persist for a long time in the

environment is called persistent organic pollutants

(POPs). There are several persistent environmental

pollutants, one of them is dioxin (Alharbi et al.,

2018).

Dioxinis produced out of combustion of

household and industrial waste, especially chlorine

compounds such as chemical industry, pesticide,

plastic, pulp, paper, and so on (Winarti and Munarso,

2005). The most toxic dioxin is 2,3,7,8-tetrachloro-

dibenzo-p-dioxin (TCDD). According to

International Agency for Research on Cancer

(IARC), TCDD is included in group 1, which means

it is carcinogenic to human (World Health

Organization. 1997). Toxicity effects caused by

TCDD are cancer, reproductive and growth disorder,

immune system disorder, and affecting immune

system (Patrizi and de Cumis, 2018).

TCDD toxicity is mediated byArylhydrocarbon

Receptor (AhR). Upon TCDD exposure, activated

AhR would move towards nucleus and form dimer

compound with Ah Receptor Nuclear Translocator

(ARNT) (Harvey et al., 2015). TCDD-AhR-ARNT

compound complex will then form Reactive Oxygen

Species (ROS) and is toxic to various organ,

including kidney 1 Oxidative stress by TCDD will

induce NF-ĸB which is a proinflammation

transcription factor. Active NF-ĸB will increase

COX-2 expression (Shen et al., 2005; Wan et al.,

2014).

Goat milk is known as natural ingredient with the

most complete component and can neutralize acid and

122

Mahdi, C., Haskito, A., Padaga, M. and Roosdiana, A.

The Effect of Goat Milk Yoghurt Casein Antioxidant Activity on KidneyTumor Necrosis Factor Alpha (TNF-) and Interleukin 1 Beta (IL-1) Levels in RattusnorvegicusExposed by

2,3,7,8-tetrachlorodibenzo-p- dioxin (TCDD).

DOI: 10.5220/0009586701220125

In Proceedings of the 6th International Conference on Advanced Molecular Bioscience and Biomedical Engineering (ICAMBBE 2019) - Bio-Prospecting Natural Biological Compounds for

Seeds Vaccine and Drug Discovery, pages 122-125

ISBN: 978-989-758-483-1

toxin in the body (Getaneh et al., 2016). Protein in

goat milk consists of casein as much as 74% of total

protein, whey protein as much as 17%, and non-

protein nitrogen as much as 9% (Al-Saadi et al.,

2014). Casein is a source of peptide and has

antioxidant properties (Khan et al., 2019). Yoghurt is

produced from fermentation process of milk lactose

by lactic acid bacteria (LAB)

1. Yoghurt is proven to have antioxidant

activity higher than fresh milk

2. Administration of casein from goat milk

yoghurt is hoped to be an alternative antioxidant

source to lower free radicals out of TCDD exposure

as observed based on the level of TNF-α and IL-1β.

2 MATERIALS AND METHOD

2.1 Materials

Yoghurt was made by yoghurt starter containing 3

LAB strains: L. bulgaricus, S.thermophilus, andL.

acidophilus (Yogourmet

, Lyo-SAN INC: 500

Aeroparc, C.P. 589, and Lachute, QC. Canada, J8H,

464). Chemical used was TCDDwith more than 99%

purity (Supelco Analytical Bellefonte, PA, Cat

No:48599). Stock solution of 10 µg/mL TCDDwas

diluted into 100 ml of corn syrup as solution dosage.

Animal model used were 24 male Rattus

norvegicusofWistar strain, aged 8-12 weeks,

weighing 150-200 gramsobtained from animal house

D’wistar Bandung. Rattus norvegicuswere kept in

Bioscience Institute, Brawijaya University.

Laboratory condition was kept in below normal

temperature of 24±2

C and given 12 hours light/dark

lighting durations. Acclimatization was conducted for

14 days before the experiment with animal general

condition observation occurred every day. Feed and

drink were givenad libitum.

3 METHOD

3.1 Making of Goat Milk Yoghurt and

Goat Milk Yoghurt Casein

Preparation

Initially mother working culture was made by adding

0,35 grams of starter powder

(Yogourmet

containingL. bulgaricus, S.

thermophilus, andL. acidophilus ) into70 ml of

pasteurized goat milk and incubated in 45◦C for 4

hours until it reached pH 4,4-4,5. Yoghurt making

followed with the addition of working mother culture

in 3% concentration into 480 ml into pasteurized goat

milk and incubated in 45◦C for 4 hours until it reached

pH 4,5-5. Goat milk yoghurt was centrifuged in 5◦C

with1200 rpm for 5 minutes and then meshed to

separate casein and whey. Afterwards, casein was

freeze dried to stabilize casein pH. Goat milk yoghurt

casein was stored in -20◦C until used.

3.2 Goat Milk Yoghurt Casein

Antioxidant Activity Test:

Goat milk yoghurt casein with 50 µg/mL. 75 µg/mL,

100 µg/mL, 125 µg/mL, and 150 µg/mL

concentration were added into 2 mL DPPH 0,1 mM

respectively. The solutions were mixed and incubated

in room temperature for 30 minutes in dark room.

These solutions were then measured for absorbance

with spectrophotometer in λmax 516 nm. The same

treatment was done to blank solution containing 2 mL

DPPH 0,1 mM and 1 mL methanol p.a.

3.3 Research Design

This research has been ethically approved with

certificate from Research Ethical Committee of

Brawijaya University (Komisi

EtikPenelitianUniversitasBrawijaya—KEP).

Rattusnovergicus were randomly divided into 6

groups. Each group contained 4 Rattusnovergicus as

repetition for every treatment. Negative control group

(K-) was given standard feed and drink without

treatment. Place of control group (KP) was given goat

milk yoghurt casein 600 mg/kgBW/day. Positive

control group (K+) was exposed to TCDD 100

ng/kgBW/day. Treatment group 1 (P1) was given

goat milk yoghurt casein 300 mg/kgBW/dayand

exposed to TCDD 100 ng/kgBW/day. Treatment

group 2 (P2) was given goat milk yoghurt casein600

mg/kgBW/day and exposed toTCDD 100

ng/kgBW/day. Treatment group 3 (P3) was given

goat milk yoghurt casein900 mg/kgBW/dayand

exposed toTCDD 100 ng/kgBW/day. Goat milk

yoghurt casein was given orally by being diluted in

reverse osmosis water, while TCDD exposure was

given orally diluted with corn syrup. The volume of

casein solution and TCDD given was 1 ml for each.

Treatment was conducted for 21 days and ended with

Rattus novergicus euthanized with cervical

dislocation.

The Effect of Goat Milk Yoghurt Casein Antioxidant Activity on KidneyTumor Necrosis Factor Alpha (TNF-) and Interleukin 1 Beta (IL-1)

Levels in RattusnorvegicusExposed by 2,3,7,8-tetrachlorodibenzo-p- dioxin (TCDD)

123

3.4 Measurement of TNF-α and IL-1β

Level in Kidney

Rattusnorvegicuswas dissected and their kidneys

taken and put into 10% formalin. Kidney samples

were made into histopathological preparates with

immunohistochemistry stain (IHC) of TNF-α andIL1-

β antibody, and followed by preparates reading under

light microscope with 400x magnification. The

measurement of TNF-α and IL-1β was calculated by

Immunoratio software

3.5 Data Analysis

Obtained TNF-α,and IL1-β data were analyzed by

one-way Analysis of Variants (ANOVA) statistical

analysis followed by Tukey test with the help of SPSS

for Windows software.

4 RESULTS

4.1 Kidney TNF-α Level

The result of P1 treatment group (20,10007,554), P2

(17,8750 ± 9,141),and P3 (12,9550 ± 4,826) did not

show significantly different results with positive

control (K+), but were significantly different with

negative control (K-). This provides information that

goat milk yoghurt casein in 300, 600, and 900

mg/kgBW dosage managed to protect kidney from

TCDD exposure injury, indicated by the inhibition of

TNF-α increase, even if the inhibition is not

significantly different when compared to TNF-α level

from positive control (K+). The average level of

TNF-αfor every treatment can be see in Table 1.

Table 1. The Influence of Goat Milk Casein on

KidneyTNF-α Level on Average

Group

TNF-α Level (pg/mL)

K-

23,50 ± 0,000

K+

23,7467

± 2,555

21,0300

± 8,327

20,1000

± 7,554

17,8750

± 9,141

12,9550

± 4,826

Annotation: Different notation

showed

significant difference

between treatment groups

(p<0,05)

4.2 Kidney IL-1β Level

The value from treatment group P2 (31,495 ± 2,7554)

and P3 (21,615 ± 25,502) showed IL-1β increase

inhibition compared to positive control (K+),

however, there were no statistically significant

difference. This provides information that goat milk

yoghurt casein was able to protect against TCDD

exposure in 600 and 900 mg/kgBW dosage, indicated

by inhibition of IL-1β level increase. The average

value of IL-1βlevel for every treatment group can be

seen on Table 2.

Table 2. Goat Milk Yoghurt Casein Influence on Kidney

IL1-βLevel on Average

Group IL-1βLevel (pg/mL)

K- 31,070

± 33,267

K+ 35,700 ± 0,000

KP 22,310

± 20,79

P1 43,140

± 7,318

P2 31,495 ± 2,7554

P3 21,615

±25,502

Annotation: Different notation showed

significant difference between treatment groups

(p,0,05)

5 DISCUSSIONS

Administration of TCDD to positive control caused

increase of TNF-α level (23,7467 ± 2,555) which is

higher compared to negative control (23,50 ±

0,000)and increase of IL 1-βlevel (35,700(World

Health Organization. 1997)0,000) which is higher

compared to negative control (31,070 ± 33,267). The

increase of TNF-α and IL 1-β was caused by TCDD

exposure being accumulated in the body. TCDD will

bind with AhR in cytosol and disturbed lysosomal

enzymatic activity. TCDD-AhR complex would

trigger CYP1A1 and CYP1B1 protein expression and

induce intracellular ROS increase. This caused lipid

peroxidation and lipid acid conversion through

lipoxygenase pathway which will then disturbed

glomerulus function (Jigyasi and Kundu, 2013). ROS

is able to bind H atom from lipid, protein, and

nucleotide existing within cell organelles or DNA and

thus caused disfunction and DNA structure injury as

well as cell organelles damage. AhR activity by

TCDD also induce inflammatory cell response within

kidney tubules, signified by the increase of TNF-α

and IL 1-β (Manabe, 2011).

ICAMBBE 2019 - 6th ICAMBBE (International Conference on Advance Molecular Bioscience Biomedical Engineering) 2019

124

Goat milk yoghurt casein exhibited strong

antioxidant activity (4,52 µg/mL), and thus able to

prevent inflammation in the kidney. Administration

of goat milk yoghurt casein has the most effective

effect in 900 mg/kgBW/day dosage which could

lower oxidative stress level, indicated by the

inhibition of TNF-α level (12,9550 ± 4,826) increase

and IL-1β (21,615 ± 25,502) level increase. This

treatment showed highest difference compared to

positive control(K+), which showedTNF-αlevel

being 23,7467 ± 2,555and IL-1βlevel being 35,700 ±

0,000.The kidney protective effect exhibited by goat

milk yoghurt casein against TCDD was possible for

casein’s function as antioxidant. Milk fermentation

process by LABs produces bioactive peptides (Wan

et al., 2014). Goat milk yoghurt bioactive peptides

would stabilize superoxide radicals by donating

hydrogen atom (H). Superoxide radicals were

captured by bioactive peptides from goat milk

yoghurt casein and this prevented unstable radical

lipid from forming out of lipid molecules losing one

hydrogen atom (H). Moreover, bioactive peptides

also prevent oxygen molecule from transferring

electron to peroxyl radicals and thus prevents

propagation process, stopping free radicals from

reacting with oxygen (Kullisaar et al., 2003).

6 CONCLUSION

Goat milk yoghurt casein may prevent the rise of

proinflammatory cytokine TNF-α andIL-1β level in

animal modelRattus norvegicusexposed to TCDD

and thus can be used as alternative nutrition with

antioxidant that can prevent cell damage caused by

toxic environmental pollutants.

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The Effect of Goat Milk Yoghurt Casein Antioxidant Activity on KidneyTumor Necrosis Factor Alpha (TNF-) and Interleukin 1 Beta (IL-1)

Levels in RattusnorvegicusExposed by 2,3,7,8-tetrachlorodibenzo-p- dioxin (TCDD)

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