Research in Veterinary Science 93 (2012) 1281–1286

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Research in Veterinary Science

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Hepatocyte apoptosis in dairy cows with fatty infiltration of the liver

Mohamed Tharwat a,⇑, Daiji Endoh b, Shin Oikawa c

a Department of Animal Medicine, Faculty of Veterinary Medicine, Zagazig University, Egyptb Departments of Veterinary Radiology, School of Veterinary Medicine, Rakuno Gakuen University, 582 Bunkyodai-Midorimachi, Ebetsu, Hokkaido 069-8501, Japanc Departments of Veterinary Herd Health, School of Veterinary Medicine, Rakuno Gakuen University, 582 Bunkyodai-Midorimachi, Ebetsu, Hokkaido 069-8501, Japan

a r t i c l e i n f o

Article history:Received 23 September 2011Accepted 25 March 2012

Keywords:ApoptosisComet assayCowsFatty liverImmunohistochemistry

0034-5288/$ - see front matter � 2012 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.rvsc.2012.03.011

⇑ Corresponding author. Address: Department of VeAgriculture and Veterinary Medicine, Qassim Univers

E-mail address: mohamedtharwat129@gmail.com

a b s t r a c t

The objective of the present study was to analyse the apoptotic process of liver cells in dairy cows withfatty infiltration of the liver using indicators of DNA damage and immunohistochemistry. For this pur-pose, sixteen dairy cows with fatty liver were examined. On clinical examination, the physical conditionof the animals was fair in nine and poor in seven cows. The most dominant clinical signs were reducedruminal motility, inappetance and/or anorexia and recumbency. Postmortem examination, in sevencases, revealed enlarged liver (18–33 kg), icteric carcasses and distended gallbladder. Laboratory resultsincluded neutrophilia, hypochloraemia, decreased concentrations of total bilirubin and increased concen-trations of b-hydroxy butyric acid, non-esterified fatty acids and insulin. The activities of aspartate ami-notransferase, c-glutamyl transpeptidase, creatine kinase and lactate dehydrogenase were high.Histopathological examination of hepatic specimen showed lipid drops in cytosol with indistinct cellularmembranes. In control hepatic cells, the DNA was tightly compressed and maintained the circular dispo-sition of the normal nucleus. However, in the diseased cows, the damaged DNA migrated from the coretoward the anode, forming a tail of a comet. Compared to controls, numerous ssDNA and caspase-3-posi-tive cells were detected in the liver. To the authors’ knowledge, this is the first study to document accel-erated apoptosis of hepatocytes in dairy cows with fatty infiltration of the liver.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Fatty liver is a major metabolic disorder of animals (Gruffatet al., 1996; Goff and Horst, 1997; Bobe et al., 2003a,b, 2004; Thar-wat, 2012). It develops when the hepatic uptake of lipids exceedsthe oxidation and secretion of lipids by the liver. Excess lipidsare stored as triglycerides (TG) in the liver and are associated withdecreased metabolic functions of the liver (Grummer, 1995; Drack-ley, 1999; Mulligan and Doherty 2008). Fatty liver occurs primarilyin the first 4 week after calving (Grummer, 1993); when up to 50%of cows have some accumulation of TG in the liver (Jorritsma et al.,2001). One reason is that dietary intake is insufficient to meet theincreased requirements of energy for maintenance and lactation(Goff and Horst, 1997; Herdt, 2000). Thus, non-esterified fattyacids (NEFA) are mobilized from the adipose tissue, often inamounts greater than are needed by cows, and the excess is trans-ported to the liver, especially in obese cows (McNamara, 2000).Hormonal changes and greater incidence of infections during par-turition are other reasons for increased mobilization of NEFA fromadipose tissue (Goff and Horst, 1997).

ll rights reserved.

terinary Medicine, College ofity, Saudi Arabia.(M. Tharwat).

Fatty liver is associated with increased veterinary costs, longercalving intervals, decreased milk production, and decreased aver-age lifetime of cows. The exact costs for fatty liver are difficult toestimate, because fatty liver currently can be diagnosed only by li-ver biopsy. The average costs and incidence rate of primary ketosis,a metabolic disorder that is closely associated with fatty liver(Veenhuizen et al., 1991), however, are estimated to be $145/case(Guard, 1994). The disease is evaluated either by chemical or histo-logical analysis of liver samples for liver TG or total lipid (Woltowet al., 1991). Generally, fatty liver can be defined on the basis ofpercentage of liver TG or lipid that is associated with decreasedhealth status, well-being, productivity, or reproductive perfor-mance of cows (Wensing et al., 1997).

Single-cell gel (SCG) electrophoresis or ‘‘Comet’’ has been usedto detect DNA damage in a variety of cell types (Tice et al., 2000).The comet assay has proven to be very sensitive, simple, and inex-pensive, and quantification of DNA damage is possible using com-puter assisted image analysis (Endoh et al., 2002; Tharwat et al., inpress). The cells are sandwiched in agarose on a slide and subjectedto lysis followed by electrophoresis under specified conditions. Un-der lytic conditions the cells lose their proteins and are believed tobe incapable of repairing damaged DNA. During electrophoresis,the damaged and fragmented DNA migrates away from the nucleusand into the gel toward the anode. The amount of migrated DNA is

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a measure of the extent of damage. Cells containing damaged DNAhave the appearance of a comet with a bright head and tail. In con-trast, undamaged DNA remains tightly coiled and appears after theelectrophoresis as an intact nucleus with no tail, thus allowing thecells’ differentiation from those that have incurred DNA damage(Tice et al., 2000).

In human beings nonalcoholic fatty liver diseases (NAFLD) arecharacterised by insulin resistance and an increased serum NEFAconcentration (Malhi and Gores, 2008). In addition, human NAFLDis accompanied by NEFA-mediated hepatocyte apoptosis and itsinduction is considered to contribute to the pathogenesis of nonal-coholic steatohepatitis. In dairy cattle with fatty liver, increasedNEFA has been known to facilitate hepatic lipidosis; therefore, he-patic apoptosis may be induced. The present study was thereforedesigned to evaluate if hepatocytes apoptosis in dairy cows withfatty liver could be detected by comet assay and immunohisto-chemistry and, to assess associations with metabolites, includingTG content.

2. Materials and methods

2.1. Animals, clinical examination and treatment

Sixteen lactating Holstein cows, with fatty liver, that weighing650 ± 120 kg and aged 3.5–6 years were examined. Their body con-dition score ranged from 3.75 to 4.50 based on a 5-pint scale(Edmonson et al., 1989). Cows had referred to Rakuno Gakuen Uni-versity, Veterinary Teaching Hospital because of anorexia, de-creased milk production and recumbency. All animals underwenta thorough physical examination (Radostits et al., 2000), which in-cluded general behavior and condition, auscultation of the heart,lungs, rumen and intestine, measurement of heart rate, respiratoryrate and rectal temperature, swinging auscultation, percussionauscultation of both sides of the abdomen and rectal examination.All cattle were maintained under the Laboratory Animal ControlGuidelines of Rakuno Gakuen University, which basically conformto the Guide for the Care and Use of Laboratory Animals of the Na-tional Institutes of Health in the USA (NIH publication No. 86–23,revised 1996). Treatment had consisted of daily 10 L parental glu-cose 10% together with 1 L of propylene glycol for five successivedays. Ten clinically healthy Holstein lactating cows weighing590 ± 70 kg were used as controls.

2.2. Liver biopsy and preparation of liver specimens

Under ultrasound guidance, liver biopsy was carried out as pre-viously described (Mohamed et al., 2002). Briefly the procedure isas follows: the areas over the intercostal spaces 7–12 on the rightside were surgically prepared. All animals were evaluated ultraso-nographically for haemorrhage immediately prior to the procedure(Mohamed et al., 2003, 2004a,b; Mohamed and Oikawa, 2008). Anormal platelet count, prothrombin time, and activated partialthromboplastin time were prerequisites for enrolment into liverbiopsy. Biopsy site was first infiltrated with 10 mL of 2% procainehydrochloride. Prior to biopsy, a small incision was made immedi-ately adjacent to the transducer through the skin and abdominalwall with the point of a scalpel blade. With a free-hand technique,a 14G � 150 mm spinal biopsy needle (Kurita Co., Ltd., Tokyo, Ja-pan) was advanced through the hepatic parenchyma under directultrasound control. When the needle is considered to be in the cor-rect position, the plain stylet was withdrawn and a notched partinserted and advanced. Often the needle can be identified on ultra-sound within the renal parenchyma while the specimen is beingobtained, thus confirming the location of biopsy. Liver TG content

was determined in hepatic tissue as described previously (Moham-ed et al., 2004b).

Single cells from parts of the fresh liver sample were preparedas described previously (Tice et al., 2000). Briefly, about one liverbiopsy was mixed with 1 mL of 0.9% NaCl. The hepatic samplewas then homogenised in 10 vol of the homogenisation buffer(75 mM NaCl, 24 mM EDTA, pH 7.0) with a Potter homogeniserat 700 rpm and centrifuged at 2700 rpm for 5 min. After centrifu-gation, the supernatant is discarded and the sediment is suspendedin 100 lL of the homogenisation buffer. The remaining intact sam-ple was used for the determination of liver TG content.

2.3. Blood sampling and biochemical analysis

Two blood samples were collected by puncture of the jugularvein, one on EDTA and the other without anticoagulant. A completeblood count (haematocrit and total and differential leucocytecount) was carried out on the first blood sample. After centrifuga-tion of the second blood sample, serum samples were collected andthen frozen for later analysis of clinical chemistries. In the serum,commercial kits were used to determine the concentrations of totalprotein, calcium, phosphorus, glucose, total cholesterol, total bili-rubin, sodium, potassium, chloride, blood urea nitrogen, insulin,b-hydroxy butyric acid (BHBA) and NEFA. The activities of aspar-tate aminotransferase (AST), c-glutamyl transpeptidase (GGT), cre-atine kinase (CK) and lactate dehydrogenase (LDH) were alsomeasured in serum samples.

2.4. Comet assay of hepatocytes

Comet assay was performed as described previously (Endohet al. 2002; Tharwat et al., in press). Briefly, liver cells were embed-ded in 1% low-melting-point agarose (Life Technologies Co., Ltd.,Japan) and deposited on top of a 1% agarose base layer (NakaraiTechs Co., Ltd., Osaka, Japan) on fully frosted slides (MatsunamiGlass Indust. Ltd., Tokyo, Japan). After solidification of the top layerof agarose, the slides were placed in lysis buffer (2.5 M NaCl,100 mM EDTA, 10 mM Tris–HCl, 1% Na-sarcosinate, 10% dimethylsulphoxide and 1% Triton X-100, pH 10.0) for one hour at 4 �C ina dark room. After this, the cell membrane and cytosol were lysedand isolated nuclei remained in the agarose. The slides were incu-bated in an electrophoretic buffer (0.3 M NaOH, 1 mM EDTA) for30 min. Electrophoresis was carried out at 25 V and approximately400 mA for 25 min at room temperature. The slides were neutra-lised in 0.4 M Tris–HCl solution (pH 7.5) for 20 min, stained withpropidium iodide (PI), and then photographed under a fluorescentmicroscope (Olympus Optical Co., Ltd., Tokyo, Japan). Images werecaptured with a Sony CCD camera and saved using Image Pro Plussoftware. Image J, open source and available free of charge for mul-tiple operating systems at http://rsb.info.nih.gov/ij/, was used toquantify the different parameters of the images. Generally, 100images were analysed per slide. The migration lengths of nucleiand total lengths including the nucleus and tail were determined,and then tail length was determined for each cell. DNA strandbreaks measured by this assay are expressed as the ‘‘tail moment’’which is the product of the fraction of DNA that has exited the nu-cleus multiplied by the distance migrated and expressed as the ex-tent of DNA damage.

2.5. Histopathology and immunohistochemical detection of caspase-3and ssDNA

Biopsied liver tissues were fixed in 4% paraformaldehyde in0.1 M sodium phosphate buffer, pH 7.4, for 12 h at 4 �C and thenembedded in paraffin wax according to standard procedures. Sec-tions (8 lM) were carefully dewaxed and dehydrated with xylene

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and ethanol. These sections were used for haematoxylin–eosin(HE) staining, and immunohistochemical staining to observe themorphological changes and distribution of apoptotic cells.

The presence and distribution of activated caspase-3 andssDNA were examined immunohistochemically (Tharwat et al.,in press). Monoclonal antibodies exclusively specific to the acti-vated forms of caspase-3 (dilution 1:100; Cell Signaling, Beverly,MA, USA) and a polyclonal antibody against ssDNA (Dako Cyto-mation, Kyoto, Japan) were used. Paraffin wax-embedded sec-tions were dewaxed and pretreated in a microwave oven withsodium acetate buffer (pH 6.0). Sections were pretreated with0.3% Triton X-100 in phosphate-buffered saline (PBS; pH 7.4)for 30 min and with 0.03% H2O2 in methanol to inhibit endoge-nous peroxidase activity. Immunoreactivity was detected by theavidin–biotin complex (ABC) method. After treatment with nor-mal 10% rabbit or goat serum for 30 min, the sections were incu-bated overnight with two antibodies (caspase-3, diluted 1:100 inPBS, and 1:50 ssDNA) at room temperature in a humified cham-ber. The antigen–antibody reactions were detected by incubationwith biotin-labeled rabbit anti-mouse IgG, IgM and IgA for thecaspase-3 antibody or goat anti-rabbit IgG for the ssDNA anti-body (Histofine Kit; Nichirei, Tokyo, Japan). The enzyme reactionwas developed with a mixture of 0.04% diaminobenzidine (DAB)and 0.02% H2O2 in 0.05 M Tris–HCl buffer (pH 7.6). The sectionswere counterstained with haematoxylin and mounted with Can-ada balsam (Kanto Chemical, Tokyo, Japan).

Fig. 1. Liver triglyceride content (TG) (A) and tail momentum (B) in dairy cows withfatty liver. ⁄⁄P < 0.01 compared to controls.

2.6. Statistical analysis

Data are presented as mean ± SD. All statistical analyses wereperformed using computer software (SPSS version 17.0, Chicago,Illinois, USA). The significance of differences between the meanswere compared between diseased and control cows using Stu-dent’s t-test. The level of significance was set at P < 0.05.

3. Results

The physical condition of the diseased cows was fair in nine andpoor in seven. They had a rectal temperature ranged from 38.3 to39.7 �C, respiratory rate of 18–44/min and pulse rate of 76–108/min. The mucous membranes were icteric in fourteen and pale intwo, and the episcleral vessels were swollen in seven. The mostdominant clinical signs were reduced ruminal motility, inappe-tance and/or anorexia and recumbency. Rectal examination re-vealed hydronephrosis in one cow. Other clinical findingsincluded muzzle necrosis (photosensitisation) in one cow, chronicmastitis in one cow, left abomasal displacement in one, arthritis inone and black tarry faeces in one animal. Abomasal displacementwas fixed surgically and abomasal ulceration was treated medi-cally. Nine cows recovered after treatment, however the conditionof seven animals deteriorated quickly and the owners preferredslaughter. At postmortem examination, the liver was enlargedand ranged from 18 to 33 kg. Other necropsy findings included ic-teric carcasses, distended gallbladder, hydronephrosis in one cowand abomasal ulceration in one cow.

Laboratory results included neutrophilia, hypochloraemia, de-creased concentrations of total bilirubin and increased concentra-tions of BHBA, NEFA and insulin. The activities of AST, GGT, CKand LDH were also high. Compared to controls, other haematolog-ical and serum biochemical parameters did not differ significantly.Compared to control values, the hepatic TG concentration was high(262 ± 77 vs. 51 ± 12) (P < 0.01) (Fig. 1). Histopathological exami-nation of hepatic specimen stained with H and E showed thathepatocytes contained lipid drops in cytosol with indistinct cellu-lar membranes. Fig. 1 shows tail moment values of hepatocytes

in cows with fatty liver compared to controls measured by the co-met assay. Hepatocyte tail moment values increased in cows withfatty liver compared to controls (P < 0.01).

Fig. 2 shows DNA integrity in the liver cells of cows with fattyinfiltration of the liver compared to a control animal. In the controlcows, undamaged DNA remained within the core, however thedamaged DNA in the diseased cows migrated from the core towardthe anode, forming a tail of a comet. When cells processed for thecomet assay were examined by fluorescence microscopy, fluores-cent structures corresponding to the PI-stained nuclear DNA ofthe hepatic cells. In control cells, the DNA was tightly compressedand maintained the circular disposition of the normal nucleus.Fig. 3 shows results of immunohistochemical examination for cas-pase-3 and ssDNA. Numerous caspase-3- and ssDNA-positive cellswere detected in the liver.

4. Discussion

Fatty liver is a multifactorial, multifaceted disease that developswhen the hepatic uptake of lipids exceeds the oxidation and secre-tion of lipids by the liver and thereby causes accumulation of TG inthe liver, which is associated with decreased metabolic function ofthe liver. Most of the knowledge about the pathology of fatty liverhas been generated in the last 20 year by quantifying metabolicreactions in hepatocytes and adipocytes (Mulligan and Doherty,2008). Preventatives as well as treatments for fatty liver focus onprevention of excessive lipolysis of adipose tissue, increasing hepa-tic gluconeogenesis or glucose supply, and increasing glucose up-take of extrahepatic tissues (Nafikov et al., 2006). Fatty liver isassociated with altered biochemical parameters. In the presentstudy, changes of NEFA, BHBA, AST, GGT and total bilirubin werereported previously (Mulligan and Doherty, 2008; Tharwat,2012). The elevated CK and LDH activities may be explained be-cause of the long-term recumbency in some cows before admissionfor examination. Elevated hepatic TG accumulations were also

Fig. 2. Representative comet image of hepatocytes in a dairy cow with fatty liver. Incontrol cow (A), the DNA is tightly compressed and maintained the circulardisposition of the normal nucleus. The damaged DNA in the diseased cow (B)migrates from the core toward the anode, forming a tail of a comet (arrow).

Fig. 3. Immunohistochemical examination by liver staining of ssDNA (A) andcaspase-3 (B) in cows with fatty liver. Numerous ssDNA and caspase-3-positivecells are detected.

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measured. These findings were confirmed by histological examina-tion of the liver specimens.

Apoptosis is a programmed physiological process of cell deathcharacterised by a distinct set of morphological and biochemicalchanges, including cytoplasmic membrane blebbing, apoptoticbody formation, nuclear condensation and chromosomal DNA frag-mentation. Apoptosis can be triggered in a wide variety of cell linesby diverse stimuli, ranging from extracellular signals to intracellu-lar events (Chimienti et al., 2001). In the present study, we used co-met assay to evaluate the degree of apoptosis in cows with fattyinfiltration of the liver and the effect on DNA integrity. The cometassay is a straightforward visual method for the detection of DNAdamage in interphase cells. This technique is especially sensitivein detecting DNA single-strand breaks (SSBs), alkaline-labile dam-age and excision repair sites in individual cells. Compared withother classical methods of detection of DNA damage, comet assayhas the advantage of showing DNA damage in individual cells. Itis a direct, sensitive, simple, rapid and powerful technique usedin toxicological studies. This method can be used for small biopsysamples and even for small numbers of cells with relatively highsensitivity and provides quantitative index for DNA damage(McKelvey-Martin et al., 1993; Mitchelmore and Chipman 1998;Cotelle and Férard, 1999; Tharwat et al., in press). The clear advan-tage of comet assay over other techniques that measure DNAstrand breaks is its ability to measure heterogeneity within com-plex populations. In the comet assay a damaged cell takes on theappearance of a comet, with head and tail regions.

It is reported that in human NAFLD NEFA induces hepatocyteapoptosis (Malhi and Gores, 2008). Briefly, NEFA can modulateboth the extrinsic and intrinsic pathways of apoptosis. Saturatedfatty acids such as palmitic acid and stearic acid cause c-jun N-ter-minal kinase-dependent activation of the proapoptotic protein Bax,which then leads to mitochondrial permeabilization with releaseof cytochrome c, activation of effector caspases, and apoptosis. Pal-mic acid can also activate the lysosomal pathway of apoptosis viaBax activation and Bax-dependent lysosomsal permeabilization.The monounsaturated fatty acid, oleic acid, imparts sensitivity tothe death receptor-mediated extrinsic pathway of apoptosis. Con-treras et al. (2010) reported that significantly increased palmiticacid and higher oleic acid concentrations in plasma NEFA were ob-served in dairy cows during transition.

According to our previously published data (Sato et al., 2004),the palmitic acid and oleic acid proportions of liver tissue were sig-nificantly higher in cows with fatty liver than in healthy cows. Thehepatocyte apoptosis detected in this study might have been in-duced by increased NEFA having high proportions of palmitic acidand oleic acid. On the other hand, a subset of patients with NAFLDdevelops progressive nonalcoholic steat-hepatitis in which diseaseactivity correlates with hepatocyte apoptosis (Feldstein et al.,2003a). Tumor necrosis factor-a (TNF-a) promotes hepatocyteapoptosis in experimental fatty liver models of mice (Feldsteinet al., 2003b). It has been reported that serum TNF-a activity in-creases in cows with naturally occurring fatty liver (Ohtsukaet al., 2001). Although it is controversial because we did not deter-mine TNF-a activity in the cows with fatty liver, TNF-a may beimplicated as another inducing factor in hepatocyte apoptosis.

The use of an anti-ssDNA antibody to detect apoptotic cells innormal as well as tumor tissues has already been reported. Frank-furt et al. (1996, 1997) developed a novel immunohistochemicalprocedure for the staining of apoptotic cells using a monoclonalantibody (MAb) to ssDNA. This MAb successfully applied for thedetection and precise quantitation of apoptotic cells in solid tu-mors, such as breast carcinoma (Frankfurt et al., 1996, 1997). Acommon event in the pathway leading to apoptosis is the activa-tion of certain cysteine proteases called caspases (Cohen 1997).Caspase-3 holds a key position in this activation process and is

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responsible for the cleavage of PARP (Prasad et al., 1998). Detectingthe characteristic fragments resulting from cleavage of PARP bycaspases-3 has been established as a method to detect apoptosis(Duriez and Shah 1997; Simbulan-Rosenthal et al., 1998).

In this study, activated caspase-3 and ssDNA were detected inlarge numbers in apoptotic hepatic cells. As proved with the cometassay results, these findings prove also that immunohistochemicaldetection of activated caspase-3 and ssDNA is a sensitive and spe-cific marker for detecting hepatocytes apoptosis and is suitablealso to identify the early stages of apoptosis. The high sensitivityfor early apoptosis can be explained by the ability of the antissDNA mAb to stain apoptotic cells that have only a low numberof high molecular weight DNA breaks characteristically for theearly stages of apoptosis. In the classic process of apoptosis, nucle-ar condensation precedes budding of the nucleus and of the cell it-self into several small apoptotic bodies (Kerr et al., 1995).

The comet assay performed under alkaline conditions primarilydetects also SSBs and alkali-labile sites in DNA (Endoh et al., 2002;Tharwat et al., in press). In this study, the hepatocytes DNA integ-rity was completely different from the control hepatocytes. Hepa-tocytes apoptosis may reflect excessive amount of TGaccumulations. From the results obtained in this study, it is sug-gested that increased levels of TG in the liver of diseased cowsmay play a role in the pathogenesis of hepatocyte apoptosis. Thistheory is supported here by the increased activities of AST, GGTand total bilirubin in diseased cases. It is likely that TG accumula-tion in liver may enhance oxidative stress-induced hepatocytesDNA damage. In a recent study for our group, an increased levelof hepatocyte apoptosis was detected in dairy cows during thetransition period (Tharwat et al., in press). In the later study, hepa-tocyte apoptosis was also associated with a high level of liver TGconcentrations.

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