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 Table of Contents  
ORIGINAL ARTICLE
Year : 2022  |  Volume : 11  |  Issue : 1  |  Page : 37-41

Role of 4-methylimidazole in liver toxicity: A histomorphometric study in albino rats


1 Assistant Professor, Department of Anatomy, Postgraduate Institute of Medical Sciences, Rohtak, Haryana, India
2 Professor, Department of Anatomy, University College of Medical Sciences, Delhi, India
3 Associate Professor, Department of Anatomy, Maulana Azad Medical College, Delhi, India
4 Associate Professor, Department of Anatomy, Postgraduate Institute of Medical Sciences, Rohtak, Haryana, India

Date of Submission21-Sep-2021
Date of Decision05-Nov-2021
Date of Acceptance11-Dec-2021
Date of Web Publication01-Feb-2022

Correspondence Address:
Suman Yadav
Department of Anatomy, Postgraduate Institute of Medical Sciences, Rohtak - 124 001, Haryana
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/NJCA.NJCA_126_21

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  Abstract 


Background: These days, food products industry involves the commercial production and addition of food colors, which comprises many food dyes, including caramel colors. 4-methylimidazole (4-MEI) is a component of various caramel-colored food products such as bakery, beer, soft drinks, coffee and sauces, which are consumed daily. The most common route of exposure is ingestion and liver is the organ of detoxification. The histomorphometric observations in liver after 4-MEI consumption have not been reported to the best of our knowledge and so the present study have been designed to evaluate the same. Methodology: Adult male Wistar albino rats weighing 150–200 g were procured for the study and divided into the control and experimental groups. The experimental animals were given 4-MEI orally by gavage tube at a dose of 250 mg/kg body weight daily for 1 month, while the control animals received distilled water. At the end of experiment, all the animals were euthanized under ether anesthesia by perfusion with Formal saline. Dissection and histopathological processing of liver was done for the examination. Results: The histomorphometric observations of the liver in experimental rats revealed a significant increase in sinusoidal width indicative of sinusoidal dilatation and congestion. There was a significant increase in the size of hepatocytes while a decrease in the size of their nuclei was noted, suggestive of ballooning degeneration. Conclusion: The findings in the present study strongly suggest hepatocellular damage caused by ingestion of 4-MEI found in caramel color dyes used routinely in the food industry.

Keywords: 4-Methylimidazole, caramel, hepatic damage


How to cite this article:
Yadav S, Kalra S, Wadhwa S, Rani P. Role of 4-methylimidazole in liver toxicity: A histomorphometric study in albino rats. Natl J Clin Anat 2022;11:37-41

How to cite this URL:
Yadav S, Kalra S, Wadhwa S, Rani P. Role of 4-methylimidazole in liver toxicity: A histomorphometric study in albino rats. Natl J Clin Anat [serial online] 2022 [cited 2022 May 22];11:37-41. Available from: http://www.njca.info/text.asp?2022/11/1/37/337041




  Introduction Top


Nowadays, the food industry involves large-scale production and addition of food colors. Food color additives are substances that may enhance, restore or impart color to food products when added to them.[1] They can be in the form of a dye, pigment, or any other substance and are found in diverse states such as liquid, powder, gel, or paste.[2] Many food dyes such as caramel colors, turmeric, cochineal, chlorophyllin, saffron, annatto, betanin, paprika, lycopene, etc., are used on a very large scale.[3] Besides commercial food production, domestic cooking also includes use of food colors. We consume these color additives in one form or the other as part of many foods we eat daily. Caramel is one of the extensively used food color which imparts color to colas, soy sauces, seasonings, bread, grilled meat, coffee, roasted foods, pet foods, cereals, and beverages.[3],[4],[5] United Nations Food and Agriculture Organization and the World Health Organization Joint Expert Committee on Food Additives has divided caramel color into four classes depending on the nature of food-grade reactants used in its manufacturing.[4],[5],[6],[7] Among these, the Class 1 and 2 of caramel colors are formed by the controlled heat treatment of carbohydrates in the absence of ammonium compounds.

In contrast, the Class 3 and 4 are prepared in the presence of ammonium compounds. Class 3 caramel colors are also called ammonia caramel and are used in bakery products, beer, soy sauce, gravy, and other products. Class 4 caramel colors are also known as sulfite ammonia caramel and is used in pet foods, soups, colas, and some other soft drinks.[8] These class 3 and 4 caramel colors contain the chemical 4-methylimidazole (4-MEI) which has been labeled as potentially toxic on different body organs.[8] 4-MEI is a nitrogen-containing heterocyclic compound formed through the mallard reaction between carbohydrates and ammonia-containing compounds lead to production of this compound.[4] 4-MEI is used in the manufacturing of a variety of pharmaceuticals, agricultural chemicals, rubber, photo thermographic chemicals, dyes, and pigments.[9] It is even found in ammoniated hay forage used for livestock.[6],[7] The United States Center for Science in the Public Interest submitted a petition on February 16, 2011, to bar caramel colorings produced with ammonia or ammonia sulfite process containing 4-MEI and related chemicals.[8] 4-MEI has been labeled as possible carcinogen by the International Agency for Research on Cancer in 2012.[10],[11],[12]

4-MEI exposure is a subject of concern in human as well as veterinary toxicology. Humans can be exposed to 4-MEI through ingestion, inhalation, and dermal contact; the most common route of exposure is ingestion. Liver is the organ of metabolism and detoxification of exogenously administered chemicals in our body. Despite the extensive human exposure and controversy regarding its carcinogenic potential, literature is scarce on its histopathological effects on the liver. Hence, the current study evaluated the histomorphometric changes on the liver in albino rats.


  Materials and Methods Top


The study was conducted in the Department of Anatomy Research Laboratory Collaborating with Pathology at University College of Medical Sciences Delhi. The primary objective of the present study was to compare histomorphometric characteristics of the liver (size of hepatocytes and their nuclei, sinusoidal width) in 4-MEI treated and control albino rats. Adult male Wistar albino rats weighing 150–200 g were procured from the Animal House of the Institute after obtaining approval from the Animal Ethics Committee. As there is a lack of literature on histomorphometry of liver after 4-MEI consumption, the present study was planned as a pilot project. It was proposed to procure 10 rats as a convenient sample for this pilot study in each group but the institutional ethics committee gave approval for 6 animals in each group due to ethical issues.

Experimental animals were given 4-MEI by oral route because ingestion is the most common route for human exposure. The chemical (4-MEI) was obtained from sigma pharmaceuticals (99% pure) in powder form and was kept away from light and moisture in the refrigerator. Fresh solution was prepared every day by dissolving the compound in 2 ml distilled water. All the animals were group-housed in cages in the animal house (12 h light/dark cycle) with adequate access to food and water.

Animals were assigned two groups

Experimental group

Comprise of six animals and received 250 mg/kg body weight of 4-MEI daily, dissolved in distilled water orally using a gavage tube for continuous 30 days.

Control group

Comprise of six animals in which an equal amount of distilled water was given orally for the same time period by the same route.

At the end of the experiment, animals were euthanized using ether anesthesia within 24 h of the last dose of 4-MEI by the process of perfusion with formal saline till the animals turned out to be pale and stiff. The perfused rats were kept in formalin for 7 days. The liver was then dissected and observed for gross changes. Small pieces of 5 mm were cut for further histopathological processing. Tissue was then embedded in paraffin wax, and blocks were prepared. Seven-micron thick sections were cut using a rotary microtome. Sections were picked up by the floatation method on a glass slide smeared with egg albumin, glycerin, and thymol mixture and kept for incubation. Hundred slides from each rat liver were prepared. The sections were stained with Hematoxylin and eosin. Twenty five slides were chosen among 100 slides/ per animal via systematic randomization for measurement (to avoid selection bias and for adequate representation of all segments of the liver). Then, 4 hepatocytes/slide were measured and a total of 100 hepatocytes were measured in 25 slides of each animal.The variables studied and compared were as follows:

  • Long diameters of hepatocytes
  • Short diameters of hepatocytes
  • Long diameters of nuclei of hepatocytes
  • Short diameters of nuclei of hepatocytes
  • Sinusoidal width.


The measurements were done on Hematoxylin and Eosin-stained slides under a Zeiss light microscope. For all linear measurements, Abercrombie's (1946) technique was used in which an ocular micrometer was calibrated with a standard stage micrometer.[13]

Measurement of hepatocytes [Figure 1]a, [Figure 2] and [Figure 3]: The sizes of the mononuclear hepatocytes with a clearly visible nucleus with nucleolus were measured from randomly selected sections of the liver. Two diameters of hepatocytes were measured at right angles to each other, passing through the center of each cell. The longest diameter was recorded as the long diameter, and the other diameter at right angle to the maximum diameter was recorded as the short diameter.
Figure 1: (a and b) Diagrammatic representation of individual hepatocyte to show the long and short diameters measured in an hepatocyte (a) and its nucleus (b). Long diameter of hepatocyte, (short diameter of hepatocyte), (long diameter of nucleus of hepatocyte), (short diameter of nucleus of hepatocyte)

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Figure 2: Photomicrograph of transverse section of the liver of control rat at ×200 magnification showing measurements of hepatocytes and their nuclei along with sinusoidal width (SD: short diameter of hepatocyte; LD: long diameter of hepatocyte; SD: sinusoidal width; CV: central vein; SW: sinusoid width; N: nuclei of hepatocytes)

Click here to view
Figure 3: Photomicrograph of transverse section of the liver of experimental rat at ×200 magnification showing measurements of hepatocytes and their nuclei along with sinusoidal width (SD: short diameter of hepatocyte; LD: long diameter of hepatocyte; SD: sinusoidal width; CV: central vein; SW: sinusoid width; S: sinusoid; N: nuclei of hepatocytes)

Click here to view


Measurement of the nuclei of hepatocytes [Figure 1]b, [Figure 2] and [Figure 3]: The long and short diameters of the nuclei of hepatocytes with a clearly visible nucleolus were measured similarly to that of hepatocytes and were recorded as long and short diameters.

Sinusoidal width

The sinusoidal width was determined by measuring the gap between two plates of hepatocytes along the line of measurement of short diameter of hepatocytes. The measurements from both groups were compared and statistically analyzed.

Image Pro-Express Analyzer System and a (charged couple device) camera connected to a frame grabber in a Compaq P.C. interfaced with the microscope was used to capture images.

All the measurements for liver parenchymal histomorphometry were recorded, tabulated, and statistically analyzed by using the unpaired Student's t-test.


  Results Top


Size of the hepatocytes

The mean long and short diameters of hepatocytes in control rats were 17.77 ± 2.39 μ and 14.45 ± 2.36 μ, respectively, while mean long and short diameters of hepatocytes in experimental rats were 20.00 ± 3.07 μ and 15 ± 2.55 μ, respectively. The increase in mean long diameter was of statistical significance with a P < 0.001, while the increase was nonsignificant for short diameter (P = 0.590) [Table 1].
Table 1: Comparison of mean diameters (microns) of the hepatocytes in both groups of rats

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Size of the nuclei of hepatocytes

The mean long and short diameters of nuclei of control rats were 6.24 ± 1.23 μ and 5.29 ± 0.85 μ, respectively, while the mean long and short diameters of nuclei of experimental rats were 5.78 ± 1.10 μ and 4.97 ± 0.70 μ, respectively. Both diameters of nuclei of hepatocytes were decreased. Decrease in the long diameter of the nucleus was statistically significant, with a P < 0.001, while the decrease in short diameter was not significant (P = 0.533) [Table 2].
Table 2: Comparison of mean diameters (microns) of the nuclei of hepatocytes of the two groups of rats

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Sinusoidal width

The mean sinusoidal width was 8.89 ± 2.76 μ in control rats while it was 15.276 ± 3.57 μ in experimental rats, respectively. The mean sinusoidal width of both groups was compared using the unpaired Student's t-test, and the P value was significant (<0.001) [Table 3].
Table 3: Comparison of mean sinusoidal width (microns) of the sinusoids in control and experimental rats

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  Discussion Top


Consumption of food products in daily life includes many caramel-colored foods which contain the chemical 4-MEI. The usual route of exposure to 4-MEI is ingestion, and liver is the target organ of toxicity.[14] In the current study, significant histopathological findings were observed in the liver of 4-MEI treated rats at daily oral dose of 250 mg/kg body weight for continuous 30 days.

4-MEI treated rats in the present study showed a significant increase in the width of sinusoids as compared to control. The mean sinusoidal width was 15.276 ± 3.57 μ in experimental rats, while 8.89 ± 2.76 μ in control rats. The effect of 4-MEI consumption on liver sinusoids has not been documented in literature as far as we are aware. But many other chemicals and drugs such as cyclophosphamide and oxaliplatin-based chemotherapy has been reported to be associated with sinusoidal ectasia in the liver.[15],[16] Various authors have observed liver sinusoidal dilatation in association with prothrombotic disorders also.[17] They explained this change as a result of activation of perisinusoidal cells, which can modulate the caliber of sinusoids and also direct injury to endothelial cells of sinusoids. Centrilobular hepatic congestion and a significant increase in the volume of sinusoids in the liver has been reported in rats exposed to Glycyrrhiza glabra extract and in conditions of hepatic venous outflow impairment, nodular regenerative hyperplasia, sickle cell anemia along with other systemic inflammatory disorders such as Castleman disease, sarcoidosis, Crohn's disease, rheumatoid arthritis, and Still's disease.[18],[19],[20] Moreover, in one of the studies, the authors reported a definite relationship of sinusoidal ectasia to the presence of a tumor or granulomatous disease in the liver or elsewhere in the body.[21] Chan et al. observed significant increase in the incidences of chronic inflammatory changes in liver which included hepatic histiocytosis, focal fatty changes, hepatocellular eosinophilic and mixed cell foci in rats exposed to 4-MEI for 106 weeks. In the same study, 4-MEI was found carcinogenic to lungs in mice while induced mononuclear cell leukemias in female rats.[14]

Alterations in the sizes of hepatocytes and their nuclei were observed in the current study. There was an increase in the mean long and short diameters of hepatocytes in experimental rats, which were 20.00 ± 3.07 μ and 15 ± 2.55 μ, respectively. In contrast, the mean long and short diameters of nuclei of hepatocytes were decreased, which were 5.78 ± 1.10 μ and 4.97 ± 0.70 μ, respectively. This kind of association is found in the ballooning degeneration of cells wherein the cell size increases while the nucleus becomes smaller or pyknotic. The decrease in nuclear size is an indicator of decreased metabolic activity of cells.[18] Hepatocellular ballooning has been reported to occur in phosphorous poisoning and after exposure to toxins like petrochemicals, amanita phalloides mushrooms, and bacillus cereus toxin;[19] however, there is a paucity of literature on histomorphometric observations of liver after exposure to 4-MEI and caramel colors. The increase in hepatocytes' size and their nuclei were reported by few authors in carbaryl and Glycyrrhiza glabra extract treated animals.[22],[23],[24] In one of the studies, the decrease in nuclear diameter of hepatocytes was also observed in offsprings of protein deficient female rats.[25] The increase in size of hepatocytes with a simultaneous decrease in size of nuclei in the present study indicates the ballooning degeneration of hepatocytes.

The present study revealed distinct histomorphometric observations in the liver of 4-MEI treated rats. The degenerative and congestive findings in the liver of experimental rats indicate toxic and harmful effects of 4-MEI exposure.

Limitations

The small sample size and shorter duration of the study are possible limitations of the study.


  Conclusion Top


4-MEI is consumed daily as a component of various food products containing caramel colors. Human exposure to 4-MEI occurs via ingestion, inhalation, and dermal contact. Most common route of exposure is ingestion. Liver is the site of first-pass metabolism and detoxification of various exogenously administered chemicals and xenobiotics. Significant histomorphometric findings of an increase in sinusoidal width and size of hepatocytes along with a decrease in the dimensions of their nuclei are suggestive of congestive and degenerative changes in liver of 4-MEI treated rats. Therefore, rational consumption of caramel-colored foods should be considered in daily life.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Fierens T, Van Holderbeke M, Cornelis C, Jacobs G, Sioen I, De Maeyer M, et al. Caramel colour and process contaminants in foods and beverages: Part II – Occurrence data and exposure assessment of 2-acetyl-4-(1,2,3,4-tetrahydroxybutyl) imidazole (THI) and 4-methylimidazole (4-MEI) in Belgium. Food Chem 2018;255:372-9.  Back to cited text no. 1
    
2.
Food and Health Survey. Food Insight; 2020. Available from: https://foodinsight.org/2020-food-and-health-survey. [Last accessed on 2021 Oct 09].  Back to cited text no. 2
    
3.
Petruci JF, Pereira EA, Cardoso AA. Determination of 2-methylimidazole and 4-methylimidazole in caramel colors by capillary electrophoresis. J Agric Food Chem 2013;61:2263-7.  Back to cited text no. 3
    
4.
Kamuf W, Nixon A, Parker O, Barnum GC. Overview of caramel colors. Cereal Foods World 2003;48:64-9.  Back to cited text no. 4
    
5.
Chan PC; National Toxicology Program; US Department of Health and Human Services; Public Health Service; National Institutes of Health. NTP technical report on the toxicity studies of 2- and 4-Methylimidazole (CAS No. 693-98-1 and 822-36-6) administered in feed to F344/N rats and B6C3F1 mice. Toxic Rep Ser 2004;67:1-G12.  Back to cited text no. 5
    
6.
Weiss WP, Conrad HR, Martin CM, Cross RF, Shockey WL. Etiology of ammoniated hay toxicosis. J Anim Sci 1986;63:525-32.  Back to cited text no. 6
    
7.
Perdok HB, Leng RA. Hyperexcitability in cattle fed ammoniated roughages. Anim Feed Sci Technol 1987;17:121-43.  Back to cited text no. 7
    
8.
Petition to Bar the Use of Caramel Colorings Produced with Ammonia and Containing Certain Carcinogens. Center for Science in the Public Interest. Available from: https://cspinet.org/resource/petition-bar-use-caramel-colorings-produced-ammonia-and-containing-certain-carcinogens. [Last accessed on 2021 Oct 09].  Back to cited text no. 8
    
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Yuan JH, Burka LT. Toxicokinetics of 4-methylimidazole in the male F344 rat. Xenobiotica 1995;25:885-94.  Back to cited text no. 9
    
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IARC Working Group on the Evaluation of Carcinogenic Risks to Humans. Some chemicals present in industrial and consumer products, food and drinking-water. IARC Monogr Eval Carcinog Risks Hum 2013;101:9-549.  Back to cited text no. 10
    
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Borghoff SJ, Fitch SE, Black MB, McMullen PD, Andersen ME, Chappell GA. A systematic approach to evaluate plausible modes of actions for mouse lung tumors in mice exposed to 4-methylimidozole. Regul Toxicol Pharmacol 2021;124:104977.  Back to cited text no. 11
    
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Hong SM, Park M, Lee KG. Development of caramel colour with improved colour stability and reduced 4-methylimidazole. Food Addit Contam Part A Chem Anal Control Expo Risk Assess 2020;37:1110-7.  Back to cited text no. 12
    
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Abercrombie M. Estimation of nuclear population from microtome sections. Anat Rec 1946;94:239-47.  Back to cited text no. 13
    
14.
Chan PC, Hill GD, Kissling GE, Nyska A. Toxicity and carcinogenicity studies of 4-methylimidazole in F344/N rats and B6C3F1 mice. Arch Toxicol 2008;82:45-53.  Back to cited text no. 14
    
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Shokrzadeh M, Chabra A, Ahmadi A, Naghshvar F, Habibi E, Salehi F, et al. Hepatoprotective effects of zataria multiflora ethanolic extract on liver toxicity induced by cyclophosphamide in mice. Drug Res (Stuttg) 2015;65:169-75.  Back to cited text no. 15
    
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Rubbia-Brandt L, Audard V, Sartoretti P, Roth AD, Brezault C, Le Charpentier M, et al. Severe hepatic sinusoidal obstruction associated with oxaliplatin-based chemotherapy in patients with metastatic colorectal cancer. Ann Oncol 2004;15:460-6.  Back to cited text no. 16
    
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Saadoun D, Cazals-Hatem D, Denninger MH, Boudaoud L, Pham BN, Mallet V, et al. Association of idiopathic hepatic sinusoidal dilatation with the immunological features of the antiphospholipid syndrome. Gut 2004;53:1516-9.  Back to cited text no. 17
    
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Monfared AL. Biochemical and histomorphometric studies on the liver rats administrated with Glycyrrhiza glabra extracts. Adv Biol Res 2013;7:67-71.  Back to cited text no. 18
    
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Patel JM, Bahadur A. Histopathological manifestations of sub lethal toxicity of copper ions in Catla catla. Am Eurasian J Toxicol Sci 2011;3:1-5.  Back to cited text no. 19
    
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Kakar S, Kamath PS, Burgart LJ. Sinusoidal dilatation and congestion in liver biopsy: Is it always due to venous outflow impairment? Arch Pathol Lab Med 2004;128:901-4.  Back to cited text no. 20
    
21.
Bruguera M, Aranguibel F, Ros E, Rodés J. Incidence and clinical significance of sinusoidal dilatation in liver biopsies. Gastroenterology 1978;75:474-8.  Back to cited text no. 21
    
22.
Kneeman JM, Misdraji J, Corey KE. Secondary causes of nonalcoholic fatty liver disease. Therap Adv Gastroenterol 2012;5:199-207.  Back to cited text no. 22
    
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Munglang M, Nagar M, Prakash R. Liver in carbaryl treated rats – A morphological and morphometric study. J Anat Soc India 2009;58:6-9.  Back to cited text no. 23
    
24.
Doycheva I, Loomba R. Effect of metformin on ballooning degeneration in Nonalcoholic Steatohepatitis (NASH): When to use metformin in Nonalcoholic Fatty Liver Disease (NAFLD). Adv Ther 2014;31:30-43.  Back to cited text no. 24
    
25.
Almeida FR, Silva GA, Fiúza AT, Chianca DA Jr., Ferreira AJ, Chiarini-Garcia H. Gestational and postnatal protein deficiency affects postnatal development and histomorphometry of liver, kidneys, and ovaries of female rats' offspring. Appl Physiol Nutr Metab 2012;37:293-300.  Back to cited text no. 25
    


    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

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