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ORIGINAL ARTICLE
Year : 2014  |  Volume : 5  |  Issue : 1  |  Page : 39-44  

Long-term ethanol diet (based on waste powder as vehicle) does not cause liver injury in C57BL/6 and A/J mice


Laboratório de Virologia Molecular, Instituto Carlos Chagas (ICC-FIOCRUZ/PR), Curitiba, Paraná, Brazil

Date of Web Publication16-Jul-2014

Correspondence Address:
Lorena Bavia
Instituto Carlos Chagas, Fundacao Oswaldo Cruz, Rua Prof. Algacyr Munhoz Mader, 3775 - CIC, Curitiba, CEP: 81350 010, Parana
Brazil
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0976-9234.136795

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   Abstract 

Background: Alcohol abuse is increasing worldwide in the human population and causes severe damage such as steatosis, hepatitis, and cirrhosis. Alcoholic individuals recurrently are more susceptible to infectious diseases since alcohol metabolites affect the innate and acquired immune responses. Hence, there is a need for animal models to study alcohol-induced pathologies. Materials and Methods: In order to establish an alternative and cheaper protocol to perform mice alcohol feeding, powder from regular chow CR-1 was mixture with water and agar generating a semi-solid vehicle-diet in which ethanol was added. As pair-fed control glucose was equicalorically added to the semi-solid vehicle-diet. Results: Four weeks later, both ethanol and glucose diets did not cause hematological, physiological and pathological changes in C57BL/6 and A/J mice. Conclusions: Our results contributed to development of a new chow approach as vehicle-diet for substances soluble in ethanol or water. In addition, our results propose an alternative to powder chow destination.

Keywords: Diet, ethanol feeding, mice, powder chow


How to cite this article:
Bavia L. Long-term ethanol diet (based on waste powder as vehicle) does not cause liver injury in C57BL/6 and A/J mice. J Pharm Negative Results 2014;5:39-44

How to cite this URL:
Bavia L. Long-term ethanol diet (based on waste powder as vehicle) does not cause liver injury in C57BL/6 and A/J mice. J Pharm Negative Results [serial online] 2014 [cited 2019 Dec 12];5:39-44. Available from: http://www.pnrjournal.com/text.asp?2014/5/1/39/136795


   Introduction Top


Alcoholism is a worldwide public health problem and its consequences lead to serious pathological issues such as steatosis, hepatitis and cirrhosis. [1],[2] The World Health Organization [3] estimates that there are 140 million alcoholics in the world. In Brazil, until 2004, alcohol abuse and alcoholism affected approximately 620/100,000 people, who suffered alcohol-related disorders. According to the Brazilian Observatory of Drug Information in 2007 [4] 11% of adult men drink ethanol every day and 28% drink it 1-4 times a week. Regarding to alcohol consumption intensity (amount) in Brazil, 24% of the population drinks heavily and frequently, characterized by an intake of at least five or more drinks once a week. In any of these cases, chemical and psychological interventions are needed, nevertheless numerous patients fail to quit. [3]

Murine model is an alternative to observe experimentally the same human pathologies induced by alcoholism, but administer ethanol to mice is not a trivial task. Therefore, many studies have been focused to developed approaches to induce ethanol consumption features in animals and to monitor the volume of ethanol consumed as follow: (a) Intragastric ethanol administration, [5] (b) oral gavage, [6] (c) ethanol diluted in drinking water, [7] (d) voluntary intake [8] and (e) ethanol as part of the diet. [9] The technique of ethanol feeding as part of a totally liquid diet was devised almost 40 (>50) years ago [10] in response to the need of developing an animal model in which the alcohol consumption was clinically relevant, while dietary control was maintained. In addition, ethanol administration through the diet is less aggressive and less stressful to the mice.

As described before, alcoholism is a major problem worldwide [3] and there is a need for animal models to study alcohol-induced pathologies. However, the means of ethanol administration to animals are still an issue and therefore there is a need to establish an alternative and cheaper protocol to alcohol administration.

The standard chow CR-1 (Nuvilab CR-1 from Nuvital, Curitiba, Paranα, Brazil) is widely used in Brazilian laboratory animal facilities that maintain colonies of mice and rats. This pellet chow is kept in plastic bags resistant to moisture which does not allow light passage. During chow transportation (which is performed largely by land in Brazil) the mechanical abrasion, due to bag stacking, causes shatter in chow pellets generating the powder chow. The powder chow produced is around of 10% of the total weight of the bag feed (~2 kg/bag). Animal facilities use dozens of bags of chow per week to feed animals, but the powder waste generated cannot be used to feed the animals since they do not present both shape and hardness necessary for the dental wear (attrition) of animals, which grow continuously in rodents.

In order to make the residue of chow CR-1 useful, the waste powder was dissolved in water and agar generating a vehicle in which ethanol could be added to establish a semi-solid diet. The idea that the powder chow could be used as a vehicle for ethanol diet was based on studies that ethanol could be successfully administered to animals through a liquid diet called the Lieber-DeCarli with agar. [11],[12] So, C57BL/6 and A/J mice were fed for 4 weeks with a diet based on the powder chow (Nuvilab Powder Chow [NPChow]) containing 5% of ethanol or glucose as equicaloric control. Unexpectedly, we did not find serum or hematological disorders, neither liver damage induced by the chronic treatment with ethanol. We also observed that NPChow containing ethanol was palatable to both mice strains. However, our data shows that it is possible to use the powder chow, which is usually discarded. It could be used as a new approach to ethanol administration to mice in order to not induce liver damage. Therefore, NPChow can be used for the administration of solutes soluble in ethanol (for example, antibiotics, medicines, vitamins) in oral form, providing an improvement in animal welfare.


   Materials and METHODS Top


Animals

C57BL/6 (n = 5 or 6 per group) and A/J (n = 3 per group) mice were purchased from the Animal Care Unit of the Institute of Biomedical Sciences, University of Sγo Paulo, Brazil. Male mice (10-12-week-old) were used in all experiments. Animals were provided with food (NPChow) and water ad libitum throughout the experiments. All procedures were previously approved by the Committee for Ethics in Animal Experimentation from the Institute of Biomedical Sciences (CEUA #057), University of Sγo Paulo, Brazil.

Nuvilab powder chow with agar

The Nuvilab CR-1 chow autoclavable is widely used as a food source for mice in animal houses. It is basically composed of 55% carbohydrate, 22% protein, 4.5% fat, 18.5% others (fibrous, vitamins, and minerals) raw materials. The powder of Nuvilab CR-1 was autoclaved for 30 min and then diluted in warm water with 1% (w/v) agar (Select Agar Gibco/BRL). Various concentrations of agar were tested: 0.5%, 1%, 1.5%, and 2%. The concentration that reached a nice consistency was 1%.

The diet was prepared as follow: 1 g of agar was melted in 50 mL of water and cooled to be added to 34 g of sieved powdered chow and ethanol (95% Merck, 7 kcal/g) or glucose (4 kcal/g) were added to complete 100 mL. The 34 g were used to control chow calories, 34 g = 1 kcal. The extra calories had as source the ethanol (5%) and glucose as equicaloric control (70.4 g/L). These diets were distributed in 50 ml Falcon tubes with an opening of approximately 2.0 × 2.0 cm and kept in the refrigerator for hardening of the agar.

The ethanol was gradually added to the diet. As an adaptation time, diet was presented to mice without alcohol or glucose for the 1 st and 2 nd days. The alcohol concentration was increased from 2% to 5% every 2 days and kept for a month (4 weeks). Fresh diet was made every 2 days. The tube with the diet was fixed to the cage [Figure 1]. One animal per cage was kept for greater control of diet consumption.
Figure 1: Falcon tube presentation with Nuvilab Powder Chow. The tube was fixed to the pellet grid inside the cage as indicated. Fresh diet was provided daily

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Blood leukocyte counting and liver enzyme assays

Four weeks after treatment, mice were anaesthetized with ketamin and xilazin (100 mg/kg and 10 mg/kg, respectively, i.p.). Peripheral blood samples were harvested from the orbital plexus using a glass capillary tube. [13] Total and differential leukocytes were counted using Turk solution and blood smear, respectively. Serum levels of alanine aminotransferase (ALT) (cat. K035) and aspartate aminotransferase (AST) (cat. K034) were determined using commercial kits (Bioclin Quibasa, Quνmica Bαsica Ltda. Belo Horizonte, M.G., Brazil).

Liver histopathology analysis

Liver fragments from each mouse treated with NPChow containing ethanol or glucose were collected, kept in buffered formalin, embedded in paraffin, sliced in 6 μm sections and stained with hematoxylin/eosin and analyzed under optical microscopy, ×20 magnification using a Nikon Eclipse E200 microscope (Nikon Instruments Inc.). All images were captured using a Nikon DXM1200C digital camera. We chose a representative histopathology from each treatment to present.

Statistical analysis

Treatments were compared by Mann-Whitney test. The weight gain was compared by ANOVA two-way and Bonferroni was used as a post test. Differences were considered significant when P ≤ 0.05.


   Results Top


Before starting the tests with the NPChow, two ethanol administration approaches were tested. In the first approach, mice were treated with ethanol or glucose by gavage daily for 2 weeks. All mice developed aversion to procedure, and therefore this approach was aborted. With the gavage method it is possible to control more precisely the volume of ethanol administered, but it is extremely uncomfortable to mice as a daily treatment. For the second approach, ethanol was diluted in water (5%) and provided to C57BL/6 and A/J mice in bottles, since the voluntary ethanol consumption is well reported in the literature for C57BL/6 mice. [7],[8] The consumption was relatively low in the bottles containing ethanol. However, the bottles containing glucose were emptied rapidly (within hours). The administration of ethanol in bottles is apparently an easy practice. On the other hand, the control of ethanol volume consumed is problematic. The simple fact of removing the mice cage from the shelf favors the leakage of the water (or ethanol) from the bottle. Therefore, regarding the difficulty in finding appropriate forms of alcohol administration to animals our aim was to take advantage of the NPChow to make a new diet for the administration of ethanol to mice [Figure 1].{Figure 1}

C57BL/6 and A/J mice used are inbred and certifiably specific pathogens free (Unit care of isogenic mice from Department of Immunology, Institute of Biomedical Sciences, USP). These animals were bred and maintained throughout the experiment in micro isolators and in ventilated shelf. The exchanges of diet and mice weight were performed in laminar flow. The treatment groups were divided into NPChow-Etanol (C57BL/6, n = 6; A/J, n = 3) and NPChow-Glucose (C57BL/6, n = 5; A/J, n = 3) as an equicalorical control (pair-fed). All mice were fed for 4 weeks with each diet. In both diet groups, the dietary intake and animal weight were followed weekly. After 4 weeks both mice strain were investigated regarding: Organs weight, number of circulating leukocytes in peripheral blood, liver function (by serum ALT and AST enzymes) and liver integrity (by histopathology).

All mice had the diet intake monitored daily (once a day using a balance portable with precision scales of proximate 0.01 g - BEL). The weekly consumption of NPChow-ethanol was 7.8 g ± 0.58 and 8.40 g ± 1.24 and of NPChow-Glucose was 6.75 g ± 0.62 and 8 g ± 0.8 to C57BL/6 and A/J mice, respectively [Figure 2]a]. Significant difference was identified between the diet consumption of NPChow-Ethanol and NPChow-Glucose after 4 weeks of treatment only for C57BL/6 mice [Figure 2]a]. The consumption of NPChow was satisfactory, since C57BL/6 and A/J mice usual consumption of CR-1 pelleted chow is an average 5 g/day and NPChow diet is less concentrated than that. The weight gain over the weeks of treatment was accompanied. For C57BL/6 mice strain it was observed that the NPChow-Glucose fed group had a significant increase in weight when compared to the NPChow-Ethanol fed [Figure 2]b]. The A/J mice maintained the same weight observed at the beginning of treatment.
Figure 2: Nuvilab Powder Chow (NPChow) consumed by C57BL/6 and A/J mice and weight gain during 4 weeks of treatment. (a) C57BL/6 mice were fed with NPChow-glucose (n = 5) or NPChow-ethanol (n = 6) and consumed similar amounts of diet. Likewise, A/J mice fed with NPChow-glucose (n = 3) or NPChow-ethanol (n = 3) showed the same results. (b) Mice body weights were measured weekly. Only C57BL/6 mice fed with NPChow-glucose significantly gained weight during the treatment when compared with C57BL/6 mice fed with NPChow-ethanol. Statistical differences between the diet consume were evaluated by Mann-Whitney test and the weight gain by ANOVA two-way test followed by Bonferroni post-test, *P < 0.05 and **P < 0.001. Data represents values of mean and standard deviation

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Variations in organ weight of animals are generally associated with pathological changes and effect of the treatment in toxicological studies. [14] Therefore, as a new diet was tested it is important to evaluate weight of different organs: Liver, spleen, kidneys, and heart [Table 1]. No significant change was observed in organ weights. Furthermore, three parameters associated with abnormal liver functions, which are evaluated in models of alcoholic liver disease: Hepatomegaly, serum ALT and AST increase were evaluated. In alcoholic chronic models, the hepatomegaly is accompanied by increased serum ALT and AST, [15] and sometimes the ration ALT/AST is an indicative of alcoholic liver disease. [16] Therefore, we evaluated liver weight to body weight and serum concentrations of ALT and AST [Figure 3]a]. No significant difference was found for any of the parameters evaluated. However, it was quite evident the difference between C57BL/6 and A/J mice when considering the ratio between liver to body weight [Figure 3]a], reinforcing an intrinsic difference of strains.
Figure 3: Evaluation of liver weight to body ratio, hepatic function and inflammatory parameters in C57BL/6 and A/J mice after Nuvilab Powder Chow (NPChow) treatment for 4 weeks. (a) Liver to body weight, serum levels of liver enzymes alanine aminotransferase and aspartate aminotransferase were analyzed for C57Bl/6 and A/J mice fed with NPChow-glucose or NPChow-ethanol. (b) Number of peripheral white blood cells and blood percentage of mononuclear and polymorphonuclear cells after treatments. Results are presented as mean and standard deviation (*P < 0.05)

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Table 1: Mean and standard deviation of C57BL/6 and A/J mice organs weight after 4 weeks of treatment with NPChow


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Finally, we investigated whether the diet could influence the number of circulating leukocytes in the peripheral blood since we have previously shown that acute treatment with ethanol by gavage was able to reduce significantly the number of circulating leukocytes in C57BL/6 mice and A/J when compared to controls. [17] After 4 weeks of treatment, it was observed that there was a significant increase in total blood leukocytes only to A/J mice NPChow-Ethanol fed group [Figure 3]b]. Regarding the number of mononuclear (MN) and polymorphonuclear (PMN) leukocytes, no significant differences were found for any of mice strains. For A/J mice, the MN cells average was 92% and the PMN cells was 8% for NPChow-Ethanol fed group, and 90% of MN cells and 10% of PMN for NPChow-Glucose fed group. For C57BL/6 mice the MN cells average was 86% and the PMN cells was 14% for NPChow-Ethanol fed group and 89% of MN cells and 11% of PMN for NPChow-Glucose fed group. Confirming the previous results associated with liver function (ALT and AST) it was evident by histopathology that neither liver tissue nor damage was induced by ethanol after 4 weeks NPChow-Ethanol or -Glucose treatment [Figure 4].
Figure 4: C57BL/6 and A/J mice liver histopathological analysis after treatment with Nuvilab Powder Chow (NPChow)-glucose or NPChow-ethanol. Animals received the diet daily for 4 weeks. The liver was fixed in formalin and sections were prepared and stained with H and E. Both mice strain show intact liver architecture (×20)

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   Discussion Top


Several murine models of alcoholism feed mice with high-fat diet containing ethanol diet from 25 days to 4 or 6 weeks of treatment, for the induction of hepatic steatosis and inflammation. [18],[19] Possibly the time of 4 weeks was not sufficient to induce liver damage in our model [Figure 4]. When the treatment was extended for 6 weeks no induction of liver disease in C57BL/6 and A/J mice was observed (data not shown).

It is possible that the ethanol concentration was low or the period of feeding was short. Or, NPChow diet composition was inappropriate because the amount of lipid is 4.5% and it is rich in carbohydrate, unlike the studies that used a high-fat diet with 32-44% of their total lipid composition. [19],[20],[21] The lipid enrichment into liquid diet accelerates the development of steatosis. [11] Although NPChow diet did not induce the expected chronic ethanol pathologies it is possible to find many applications for this new diet developed. Many animal facilities need to perform procedures for the removal of endo- and ecto-parasites in mice and rats as part of health programs. In this case, NPChow diet could be used as a vehicle for the administration of anthelmintic or antiectoparasites medicines. Pregnant and lactating females mice or rats would not need to be handled and inoculated with substances (such as vitamins or antibiotics), they could receive medication diluted in NPChow, which was demonstrated by this work do not induce liver injury. There are specific diets rich in vitamins and minerals, however with the use of NPChow diet to the administration of extra nutrients could be customized to each situation.

The administration of drugs by oral voluntary consumption has been proposed by Zhang, [22] in which Rimonabant drug was chronically given to animals inserted into a gelatinous diet. Studying the effect of the plant Artemisia annua as antimalarial therapy Elfawal et al. [23] mixed dry plant extract with mouse powder chow. The extract consumed orally killed effectively more parasites than the pure plant extract supplied by oral gavage. Mice fed with a diet containing the powdered drug Roxithromycin in chow were protected when infected with a lethal dose of Toxoplasma gondii. [24] In type 1 diabetes model, mice treated with p38α selective mitogen-activated protein kinase inhibitor administered in the powder diet exhibited obesity prevention and became therapeutically a potent approach during the prediabetic period. [25] That is, the applications are numerous. In these reports, several substances were administered mixed with feed powder. Our diet offers the possibility of diluting substances in water or ethanol and control the consume intake.

In summary, both diets, with and without ethanol did not cause physiological changes in C57BL/6 and A/J mice. Our results contribute to the development of a new chow approach as vehicle to substances soluble in ethanol or water, which can be administrated orally to mice. Therefore, under conditions where substances can be added to the chow the observed effects will be referent just to the substance added and not to ethanol, as shown in our results. Besides the various applications mentioned here, it is clear the importance of the use of the feed powder that is currently being discarded.

Our work proposes a new approach for ethanol administration to animals, which does not induce liver damage neither hematological changes. We found a useful destiny to the powder waste from regular mice and rat chow, which can be a vehicle to administration of substances soluble in ethanol or water.


   Acknowledgment Top


This work was supported by the Fundaηγo de Amparo ΰ Pesquisa do Estado de Sγo Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Cientνfico e Tecnolσgico (CNPq). L. Bavia was recipient of graduate fellowships from FAPESP during her PhD. I would like to thank Dr. Lourdes Isaac (USP), Dr. Giovanny A. C. A. Mazzarotto (FIOCRUZ/ICC-PR) and Dr. Andrea Cristine Koishi (FIOCRUZ/ICC-PR) for the careful reading of the manuscript.

 
   References Top

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21.Pritchard MT, McMullen MR, Stavitsky AB, Cohen JI, Lin F, Medof ME, et al. Differential contributions of C3, C5, and decay-accelerating factor to ethanol-induced fatty liver in mice. Gastroenterology 2007;132:1117-26.  Back to cited text no. 21
    
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