A study of the presence of Aflatoxin B1 on patients with liver disease and effect some immunological parameters
Haider F. AL-Kelabi1, Atheer B. AL-Obaidi2
1Department of Biology, College of Science, University of Kufa, Najaf, Iraq, Email:
2Department of Biology, College of Science, University of Kufa, Najaf, Iraq, Email:
[email protected] Email: [email protected]
Abstract
The study included the relationship between the Aflatoxin B1 and liver disease in the Province of Al- Najaf Al Ashraf city and estimating the amount of AFB1 in the blood of patients with the use high-performance liquid chromatography (HPLC) technique in addition to studying the extent of the effect of Aflatoxin B1 on IL10 for patients liver disease using Enzyme-linked Immunosorbent Assay( ELISA ).
In patients suffering from hepatitis disease (60) person, the (36) with aflatoxin B1and ( 24) without aflatoxin B1 was recognized after the examinations of the serum samples by using the TLC plate,
the results showed that hepatitis disease patients with Aflatoxin B1 was 36 (60%) , while hepatitis disease patients without Aflatoxin B1 was 24 (40%).
In patients suffering from hepatitis disease with AFB1 (36) person. AFB1 was recognized by TLC method to 19 male (52.8%) and female were 17 (47.2%).theincidence of males' injury increases because the males are more susceptible to contact with the cause through continuous exit from their homes for the purpose of working and eating from shops and streets that lack cleanliness or play sports.
The HPLC results have confirmed that the highest concentration of Aflatoxin B1 for liver disease patients (3.586ng / ml) and the lowest concentration (0.686ng / ml) with a significant difference with the control group (0.3096ng / ml).
As for interleukin-10, the toxicity of Aflatoxin B1 leads to a marked decrease in their levels significantly in liver disease patients , as reached value IL10 (3.785ng / l) compared to the control group (3.493ng / l) respectively.
The results of the study have revealed that the concentrations of (IL10) in the total number of patients and the control group have a significant increase (P>0.05)to (3.785 ng/l) and (3.493 ng/l) respectively, Also they have revealed that the concentration of (IL10) in male and female patients suffering from hepatitis disease have significantly increase (P>0.05)to male (4.163 pg/ml) as compared with the control group (3.659 pg/ml) and female (3.711pg/ml)) compared with control group (3.307pg/ml) .
Keywords: Hepatitis disease , Afllatoxin B1(AFB1) ,Interlukin10 (IL10)
1-Introduction
Hepatocellular carcinoma (HCC) is the most common type of liver cancer worldwide, The incidence of HCC is on the rise in Thailand, where it has become the most common malignancy in males and the third most common in females.Hepatitis B virus (HBV) and hepatitis C virus (HCV) associated molecules are viral factors that may play a role in the development of HCC [1,2].Patients with underlying chronic liver disease, especially that caused by hepatitis C virus infection, have an increased risk of death from acute viral infection, suggesting that such patients, who lack naturally acquired immunity to type A virus, might benefit most from targeted vaccination.[3,4].
Since both AFB1 exposure and cirrhosis are major risk factors for HCC, it's unknown if AFB1
plays a role in the early stages of the disease, such as cirrhosis [5]. Low doses of aflatoxins are repeatedly exposed to over a lifetime, resulting in chronic diseases, the most common and serious of which is cancer [6].Aflatoxinis the most toxic and carcinogenic among the known mycotoxins [7].Acute exposure to AFB1 in humans has been reported in outbreaks of aflatoxicosis, in which clinical symptoms have been linked to the ingestion of AFB1
contaminated food [8].
1 1
dominant type of liver cancer. In Guatemala, the estimated incidence of HCC is the highest in the Western hemisphere [9].
Among the major aflatoxins (B1, B2, G1 and G2) of Aspergillusflavus or Aspergillusparasiticus, AFB1 is the most potent one. It has hepatotoxic, immunosuppressive, carcinogenic, teratogenic and mutagenic activities[10].Following a review of the AFB1
biosynthetic pathway and its internal gene regulation, we go through other genes that have been found to interact with AFB1 synthesis and their roles in fungal metabolism.[11,12]
Chemical structure of aflatoxin B1 (AFB1 ).The C8 -C9 double bond is highlighted in blue, while the lactone bond is indicated in red.[13].
Interleukin10(IL-10) a cytokine with anti-inflammatory and immunomodulatoryfunctions [14].plays an important role in normal renal physiology[15].Effects of aflatoxins on immune cells that play key roles in the innate immunity, such as monocytes, dendritic cells,macrophages and natural killer cells to restrict their viability, function, or genetic expression of chemokines and cytokines is well documented.[16,17].
High-performance liquid chromatographic method( HPLC):
An HPLC instrument connected to a post-column Kobra cell derivatizer for aflatoxin B1 was used to qualitatively and quantitatively determine the amount of AFB1 in various peanuts and peanut butter samples (Dickson, 2019). This thesis examines a variety of analytical techniques for analyzing AFB1 in feed or feedstuffs, beginning with sampling, procedure/technique, and related pros and cons associated with those analytical methods.
This study also summarized the risk assessment of AFB1 [18,19].
Aflatoxins were detected in all chloroform extracts from strains showing fluorescence at the third day by an HPLC method with a detection limit of lower than 5 ppb for each aflatoxin[20].
ELISA Assays:
Enzyme immunoassays make use of enzymes attached to one of the reactants in an immunoassay to allow quantification through the development of color after the addition of a suitable substrate/chromoge, In humans and domestic animals, many traditional serological approaches, including ELISA, have been used to diagnose forinterlukin 10 [21,22].
2-1 Materials and Methods
Thisstudy has been conducted in the laboratories of the Department Biology / Faculty of Science / University of Kufa. Standard Aflatoxin B1 was obtained from the Sigma - Aldrich Corporation.
2-2 Samples Collection
sixty samples of patients with hepatitis disease and twenty four samples from individuals without hepatitis disease as controls were collected from Al-Sader hospital and central
laboratory in the Najaf city province, it was taken up taking 5 ml of blood from each individual and placed in gel tube left at room temperature for 30 minutes. After that, is separated the serum from the blood sample by centrifugation process represented 3,000 RPM for (5) minutes and then the serum is collected in sterile tubes (Eppendorf tube) and each sample of serum is divided into 4 parts (each part 0.5 ml) and saved at a temperature of -20
0C till being used for the determination of concentration of (TLC), (HPLC) and Immunological Markers , this samples taken from both gender (male and female) and installed information for each patient in data sheet included all the ages and for both gender.
2-3 Samples preparation of aflatoxin B1
0.5ml of sample were dissolved in 30 ml 0.1% trichloro acetic acid ( TCA ),then agitated in ultra sonic bath for 10 minutes ,then the extract were filtered on disposable sepak filtered paper 0.25um to remove the fibres and un-dissolved sample, then prepared aflatoxin standards solution by serial dilution of (1.5,3,4,5.5,7 ng\ml, from 100 ng\ml stock solution authentic standard obtained from sigma co, ), then 50 ul of the aqueous filtrate were injected to HPLC column under the optimum separation condition ,analysed occurred on HPLC column as in above separated condition for quantitatively identify the AFB1 in each sample either by comparison the peak area of sample with peak area of authentic standard or from the calibration curve between peak area of sample and standard.
2-4 Analysis of Aflatoxin B1 in serum of hepatitis diseases by (HPLC)
Mobile phase : 0.1% trichloro acetic acid ( TCA )in deionized water pH 3.0: 0.1% tricolor acetic acid ( TCA ) in acetonitrile linear gradient program from 0% B to 100% B for 10 minutes
2-5Assay procedure(Interleukin 10) ELISA
Add 100 μL standard or sample to each well. Incubate for 90 min at 37°C. Remove the liquid.
Add 100 μLBiotinylated Detection Ab. Incubate for 1 hour at 37°C. Aspirate and wash 3 times and Add 100 μL HRP Conjugate put Incubate for 30 min at 37°C. Aspirate and wash 5 times. 6. Add 90 μL Substrate Reagent. Incubate for 15 min at 37°C and Add 50 μL Stop Solution. Read at 450 nm immediately.
3-Results and Discussion
3.1.Detection of AFB1 with hepatitis disease patients according to gender:
In patients suffering from hepatitis disease with AFB1 (36) person. AFB1 was recognized by TLC method to 19 male (52.8%) and female were 17 (47.2%).theincidence of males' injury increases because the males are more susceptible to contact with the cause through continuous exit from their homes for the purpose of working and eating from shops and streets that lack cleanliness or play sports.as seen in Table (3-1).this result is agree withgovernment revealed that comprised 207 HCC patients with 189 (91.3%) males and 18 (8.7%) females. The mean age was 46.9 ± 11.5 years[23].
Table (3.1): The number and percentage of patients borne the AFB1 according to the gender.
Gender NO. %
Male 19 52.78
Female 17 47.22
Total 36 100
3.2.Detection of AFB1 in the blood serum by HPLC:
The result indicated that a significant difference in the serum level of AFB1 (male and female) patients and control group. Higher concentration of AFB1 inmale was (3.568 ng/ml) then (2.684 ng/ml) in famale patients at (29) and (58) years old respectively, The results of the study have revealed a significant increase (P>0.05) to (1.569 ng/ml) and (0.3096 ng/ml) respectively in the concentrations of (AFB1) in the total number patientssuffering from hepatitis disease with AFB1 and the control group , Also they revealed that the concentration of (AFB1) in male and female patients suffering from liver disease have significantly increased to (2.984 ng/ml) respectively compared with the control group (0.531 ng/ml) . it also revealed a variation depending on ages (18-34),(35-54) and(55-71) yearsold was the concentration of AFB1
(1.508 ng/ml),(1.41 ng/ml),(1.796 ng/ml) respectively compard with the control group (0.2345 ng/ml), (0.3284 ng/ml) and (0.4556 ng/m),as seen in ta Figures (3.1),(3.2), (3.3),(3.4) and table (3.2),(3.3).In the current study a (HPLC) is used to detect the presence of AFB1 in serum of patients suffering from hepatitis disease and measure the concentration of it in the province Al-Najafcity.
The most sensitive and notable effect is liver disease, but the toxin may also have effects on fetal development and on the immune system. Contrary to the clear evidence of liver toxicity and liver cancer due to Aflatoxin B1 exposure in animals, this association in humans is unclear however effects on liver have been demonstrated this result agree with the resultswhich show that the p-value equal 0.869 which is greater than 0.05 , and the value of Fstat =0.141which is less than Fcritical =3.25, that’s means there is no statistically significant differences at the level of α ≤ 0.05 the percent of Aflatoxin B1 in patients group at significant level α ≤ 0.05 due to HBV Duration[24].
Table(3.2 ) :The concentration of AFB1in blood serum according to the male and ages Concentration of
AFB1(ng/ml) Age
Gender
0.867 18
* M
1.234 20
M
2.308 22
M
1.899 27
M
3.586 29
M
1.24 30
M
1.078 33
M
1.746 37
M
1.055 42
M
2.21 42
M
1.409 43
M
1.518 45
M
2.984 46
M
0.8001 48
M
1.386 50
M
0.816 52
M
1.467 61
M
2.325 62
M
1.095 70
M
Table(3.3 ) :The concentration of AFB1in blood serum according to the female and ages Concentration of
AFB1(ng/ml Age
Gender
1.576 23
**F
1.609 26
F
0.686 30
F
1.195 36
F
1.512 41
F
1.71 41
F
1.234 43
F
0.89 47
F
1.492 55
F
1.3 56
F
1.072 58
F
2.684 59
F
Figure(3-1): Concentration of AFB1(ng/ml) in total number of patients and total number of control group. This different letter refers to significant differences at p>0.05
according to T test.
Figure (3-2):Concentration of Aflatoxin B1 (ng/ml) in Patients andcontrol Group. This different letter refers to significant differences at p<0.05 according to T test.
Figure (3-3):Concentration of AFB1 in patients and control group. This different letter refers to significant differences at P<0.05 according to Tukey's multiple comparisons test.
Figure (3-4) : HPLC Chromatography analysis shown major peaks in Hepatitis disease patients with AFB1
3.3 Interleukin 10 (IL10) concentration in serum
The results of the study have revealed that the concentrations of (IL10) in the total number of patients and the control group have a significant increase (p>0.05) to (3.785 ng/l) and (3.493 ng/l) respectively, Also they have revealed that the concentration of (IL10) in male and female patients suffering from hepatitis disease have significantly increase (P>0.05) to male (4.163 ng/l) as compared with the control group (3.659 ng/l) and female (3.711) compared with control group (3.307),it also revealed a variation depending on ages non signinficant (18-34),(35-54) and (55-71) years old in patients suffering from hepatitis disease (p<0.05) to (4.4 ng/l),(3.853 ng/l) and (4.191 ng/l) respectively compard with the control group (3.125 ng/l),(3.612 ng/l) and (4.118 ng/l) respectively in the concentration of (IL10) the total numberpatients suffering from hepatitis disease and the control group, as seen in Figures:
(3.5),(3.6) and (3.7).
The results of study proves a significant increase in the serum level of IL-10 in male and female patients suffering from hepatitis disease compared with the control group also in total patients with kidney failurecompared to the total control group,Therefore, these previous studies demonstrated the important role of IL-10 in paraquat poisoning. IL-10 inhibits the activity of monocyte macrophages, T cells and natural killer cells [25]
Interleukin 10 are responsible for suppressing anti-tumor immune response, thus facilitating tumor escape. Other causes or risk factors for hepatocellular carcinoma include autoimmune hepatitis, severe alcohol consumption, aflatoxin B1, age, and gender, by which male is more vulnerable than female.Since we do not yet have samples from patients with hepatocellular carcinoma, we could not evaluate the extent of immune cells, particularly Treg, in these patients.To complete our understanding of the intrahepatic immune response during hepatitis, we will need to recruit more participants at various stages of the disease.This study agrees with the study[26,27].
Figure 3.5: Concentration of IL10 (pg/ml) in total number of patients and total number of control group. This different letter refers to significant differences at p<0.05 according to T test
Figure ( 3-6): Concentration of IL10 (pg/ml) in patients and control group. This different letter refers to significant differences at p>0.05 according to T test.
Figure( 3-7): Level of IL10 (pg/ml) in patients and control group. This different letter refers to significant differences at p>0.05 according to ANOVA with Tukey multiple comparisons test.ns=non signinficant.
Conclusions
1-The results have showed the presence of the Aflatoxin B1 in the blood of patients with liver disease, as they 36 patients (60)% and the males were the most infected with liver disease with AFB1 as they reached (52.8)% compared to the number of infected females and by (47.2)%
2-The HPLC results have confirmed that the highest concentration of Aflatoxin B1 for liver disease patients (2.984 ng / ml) and the lowest concentration (0.59 ng / ml) with a significant difference with the control group (0.3096 ng / ml).
3-The AFB1 in patients with liver disease causes increasing serum level of immunoglobulin increase interleukin 10 (IL10) .
4- The AFB1 has an effect on some immunological biomarkers.
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