Hepatocyte apoptosis in dairy cattle during the transition period

Abstract

The objective of this study was to investigate hepatocyte apoptosis in dairy cows during the transition period. Four clinically healthy, pregnant dairy cattle were used. The cows had no clinical diseases throughout this study. Blood samples were collected and livers were biopsied from the cows at 3 different times: 3 weeks before expected partition (wk −3); during parturition (wk 0), and 3 weeks (wk +3) after parturition. The damage to deoxyribonucleic acid (DNA) caused by hepatocytes was evaluated by comet assay. The apoptotic features of hepatocytes were examined by immunohistochemistry and electron microscopic analyses. The hepatic triglyceride content markedly increased at wk 0 and wk +3 compared with the values at wk −3. The results of the comet assay showed increases in the mean tail moment values of hepatic cells after parturition in all cows, which suggested increased DNA damage. Histopathologically, the hepatocytes began to contain lipid droplets at wk 0 and were severely opacified at wk +3. Caspase-3-positive and single-stranded DNA-(ssDNA)-positive cells were first detected in the liver after parturition. Condensation of nuclear chromatin, a typical sign of apoptosis, was confirmed by transmission electron microscopy after parturition. These results suggest that apoptosis is induced in hepatocytes of dairy cows around parturition and may result from lipotoxicity in hepatocytes.

Introduction

The transition period, which is typically defined as from 3 weeks before to 3 weeks after parturition (1,2), is regarded as a critical time in the lactation cycle of a dairy cow. During this period, the cow experiences a series of nutritional, physiological, and social changes and is more vulnerable to infectious and metabolic diseases (3). One of the major challenges faced by the cow at this time is obtaining sufficient energy to support the onset of lactation, especially as feed intake tends to be suppressed around the time of calving (2). A general decline in dry matter intake (DMI) beginning 3 weeks before calving has been reported (2), followed by a gradual increase in DMI in the weeks after calving (4). During the transition phase, the cow must adapt to a dramatic and several-fold increase in nutrient uptake by the mammary gland, which is associated with lactogenesis, compared with the much smaller nutrient requirement of the growing conceptus in late gestation. The periparturient period is thus associated with an increased incidence of metabolic and production-related diseases, including fatty liver and ketosis, arising because of inadequate homeorhetic adaptation of the metabolism (5–7). These conditions, together with changes in hormonal equilibrium, lead to an increase in fat mobilization, which increases levels of plasma non-esterified fatty acids (NEFA) and fatty acid uptake by the liver (1).

In our previous experiment on fasting-induced hepatic lipidosis, excessive mobilization of NEFA to the liver impaired the secretion of very low-density lipoproteins from the liver. The lipotoxicity of low-density lipoproteins to hepatocytes was considered a negative effect of NEFA (8). In humans, nonalcoholic fatty liver diseases (NAFLD) are characterized by insulin resistance and an increased concentration of serum NEFA (9). In addition, human NAFLD is accompanied by NEFA-mediated hepatocyte apoptosis and its induction is considered to contribute to the pathogenesis of nonalcoholic steatohepatitis.

During the transition period, increased NEFA has been known to facilitate hepatic lipidosis in dairy cattle. Hepatic apoptosis may therefore be induced during this period. The objective of this study was to test whether or not hepatocyte apoptosis could be detected in dairy cows during the transition period by indicators of damage to deoxyribonucleic acid (DNA), histopathology, immunohistochemistry, and electron microscopic analyses, and to assess associations with metabolites, including triglyceride (TG) content.

Materials and methods

Animals

Five pregnant, clinically healthy Holstein dairy cows (2.8 ± 1.0 y old; parity, 1.6 ± 0.9) on Rakuno Gakuen University Farm in Hokkaido, Japan (milk yield of the herd, approximately 9500 kg/y/cow) were selected based on physical examination and a complete blood (cell) count (CBC). All cows had a body condition score (BCS) of 3.25 to 3.75 based on a 5-point scale (10). Because the oldest cow (4.5 y old) died from gangrenous mastitis and hypocalcemia just after parturiton, only the remaining 4 dairy cows were used in this study. These cattle had no clinical diseases throughout the study. All cattle were maintained in tie-stall barns under the Laboratory Animal Control Guidelines of Rakuno Gakuen University, which basically conform with those of the National Institutes of Health in the United States (11).

Blood sampling and preparation of liver specimens

Sampling for blood and liver specimens was carried out 3 wk before expected parturition (wk −3, −16.5 ± 3.1 d from actual calving), at parturition (wk 0, 1.5 ± 1.3 d), and 3 weeks after parturition (wk +3, 21 d). All blood samples were collected from the jugular vein prior to liver biopsy. Separated serum specimens were stored at −40°C until assay.

Liver biopsy was carried out 3 times as previously described (12). Briefly, the procedure was as follows: the areas over intercostal spaces 7 to 12 on the right side were surgically prepared. With a free-hand technique, a 14-ga × 150-mm spinal biopsy needle (Kurita, Tokyo, Japan) was advanced through the hepatic parenchyma under direct ultrasound control. Single cells from part of the fresh liver sample were prepared as previously described (13). One liver biopsy was mixed with 1 mL of 0.9% sodium chloride (NaCl). The hepatic sample was then homogenized in 10 volumes of the homogenization buffer [75 mM NaCl, 24 mM ethylenediamine tetra-acetic acid (EDTA), pH 7.0] with a Potter homogenizer at 700 rpm and centrifuged at 2700 rpm for 5 min. After centrifugation, the supernatant was discarded and the sediment was suspended in 100 mL of the homogenization buffer. The remaining intact sample was used to determine liver triglyceride content.

Comet assay of hepatocytes

Comet assay was performed according to the method of Endoh et al (14). Briefly, liver cells were embedded in 1% low-melting-point agarose (Life Technologies, Japan) and deposited on top of a 1% agarose base layer (Nakarai, Osaka, Japan) on fully frosted slides (Matsunami Glass Industry, Tokyo, Japan). After the top layer of agarose had solidified, the slides were placed in lysis buffer (2.5 M NaCl, 100 mM EDTA, 10 mM Tris-HCl, 1% Na-sarcosinate, 10% dimethyl sulphoxide, and 1% Triton X-100, pH 10.0) for 1 h at 4°C in a dark room.

The cell membrane and cytosol were then lysed and isolated nuclei remained in the agarose. The slides were incubated in an electrophoretic buffer [0.3 M sodium hydroxide (NaOH), 1 mM EDTA] for 30 min. Electrophoresis was carried out at 25 V and approximately 400 mA for 25 min at room temperature. The slides were neutralized in 0.4 M Tris-HCl solution (pH 7.5) for 20 min, stained with propidium iodide (PI), and then photographed under a fluorescent microscope (Olympus Optical, Tokyo, Japan). Images were captured with a Sony CCD camera and saved using Image-Pro Plus software. ImageJ (open source, available free of charge for multiple operating systems at http://rsb.info.nih.gov/ij/) was used to quantify the different parameters of the images. Generally, 100 images were analyzed per slide. The migration lengths of nuclei and total lengths, including the nucleus and tail, were determined and the tail length was determined for each cell. Breaks in DNA strands measured by this assay are expressed as the “tail moment,” which is the product of the fraction of DNA that has exited the nucleus, multiplied by the distance migrated and expressed as the extent of DNA damage.

Histopathology and immunohistochemical detection of caspase-3 and ssDNA

Biopsied liver tissues were fixed in 4% paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.4, for 12 h at 4°C and then embedded in paraffin wax according to standard procedures. Sections (8 μM) were carefully dewaxed and dehydrated with xylene and ethanol. These sections were used for hematoxylin & eosin (H & E) staining and immunohistochemical staining to observe the morphological changes and distribution of apoptotic cells.

The presence and distribution of activated caspase-3 and single-stranded DNA (ssDNA) were examined immunohistochemically. Monoclonal antibodies specific exclusively to the activated forms of caspase-3 (dilution 1:100; Cell Signaling Technology, Beverly, Massachusetts, USA) were used, as well as a polyclonal antibody against ssDNA (Dako Cytomation, Kyoto, Japan). Sections embedded in paraffin wax were dewaxed and pretreated in a microwave oven with sodium acetate buffer (pH 6.0). Sections were pretreated with 0.3% Triton X-100 in phosphate-buffered saline (PBS; pH 7.4) for 30 min and with 0.03% hydrogen peroxide (H₂O₂) in methanol to inhibit endogenous peroxidase activity. Immunoreactivity was detected by the avidin-biotin complex (ABC) method.

After treatment with normal 10% rabbit or goat serum for 30 min, the sections were incubated overnight with 2 antibodies (caspase-3, diluted 1:100 in PBS and 1:50 ssDNA) at room temperature in a humidified chamber. The antigen-antibody reactions were detected by incubation with biotin-labeled rabbit anti-mouse IgG, IgM, and IgA for the caspase-3 antibody or goat anti-rabbit IgG for the ssDNA antibody (Histofine DAB-3S Kit; Nichirei Biosciences, Tokyo, Japan). The enzyme reaction was developed with a mixture of 0.04% diaminobenzidine (DAB) and 0.02% H₂O₂ in 0.05 M Tris-HCl buffer (pH 7.6). The sections were counterstained with hematoxylin and mounted with Canada balsam (Kanto Chemical, Tokyo, Japan).

Transmission electron microscopy (TEM)

Biopsied liver tissues were cut into pieces (1 × 1 × 5 mm) and fixed with glutaraldehyde in 0.1 M phosphate buffer (PB) (pH 7.2) at 4°C for 2 h. After washing in PB, samples were post-fixed in 1% osmium tetroxide for 1 h at room temperature. The samples were then dehydrated with alcohol, embedded in Quetol 812, sectioned with a diamond knife, stained with 1% uranyl acetate and 1% lead citrate, and examined with a transmission electron microscope (JME-1220; JEOL, Tokyo, Japan) at an acceleration voltage of 80 kV.

Blood analyses

The serum concentrations of non-esterified fatty acids (NEFA) and β-hydroxybutyric acid (BHBA) and aspartate transaminase activity were determined using a clinical biochemistry autoanalyzer (BioMajesty JCA-BM 2250; JEOL) in the Kishimoto Clinical Laboratory, Sapporo, Japan. The tryglyceride (TG) content of the liver was measured on a wet basis by a colorimetric assay (12).

Statistical analysis

All statistical analyses were performed using computer software (SPSS version 17.0; SPSS, Chicago, Illinois, USA). The data were evaluated by repeated measures analysis of variance (ANOVA). The significance of differences between the means at wk −3 and wk 0 or wk +3 were analyzed by Dunnett’s test. Values were expressed as mean ± standard error.

Results

As shown in Table I, body weight decreased dramatically at wk 0 (P < 0.05) and at wk +3 (P < 0.01), compared with the value at wk −3. The body condition score at wk +3 was significantly lower (P < 0.01) than at wk −3. The NEFA concentration was higher in cows at wk 0 and wk +3 than at wk −3, although the differences did not reach statistical significance because of an outlier (0.58 mEq/L; the remaining 3 cattle, < 0.4 mEq/L). After parturition, the BHBA concentration gradually increased. The activity of aspartate transaminase (AST) was higher at wk 0 than the value at week −3.

Table I.

Body weight, body condition score, and serum metabolite concentrations in dairy cattle at 3 weeks before parturition (wk −3), during parturition (wk 0), and 3 weeks after parturition (wk +3)

	Weeks relative to parturition
Variables	wk −3	wk 0	wk +3
BW (kg)	632.0 ± 34.8	598.0 ± 35.8*	556.0 ± 29.5**
BCS	3.44 ± 0.12	3.19 ± 0.12	2.88 ± 0.07**
NEFA (mEq/L)	0.41 ± 0.06	0.69 ± 0.11	0.59 ± 0.04
BHBA (mM)	0.444 ± 0.038	0.571 ± 0.135	0.624 ± 0.108
AST (IU/L)	85 ± 10	114 ± 12	98 ± 23

BW — body weight; BCS — body condition score; NEFA — non-esterified fatty acids; BHBA — β-hydroxybutyrate; AST — aspartate transaminase.

Data are expressed as mean ± standard error.

^*P < 0.05;

^**P < 0.01, compared with respective values at wk −3.

As shown in Figure 1, the triglyceride (TG) contents of the liver increased significantly (P < 0.05) at wk 0 and wk +3 compared with those at wk −3. The tail moment values of hepatocytes during the transition period, measured by the comet assay are shown in Figure 1B. Tail moment values increased at wk 0 and wk +3 compared to wk −3.

Figure 1.

A — Liver triglyceride (TG) contents in dairy cattle 3 weeks before parturition (wk −3), during parturition (wk 0), and 3 weeks after parturition (wk +3). B — Tail momentum in experimental dairy cattle at 3 weeks before parturition (wk −3), during parturition (wk 0), and 3 weeks after parturition (wk +3).

*P < 0.05, compared with value at wk −3.

Figure 2 shows DNA integrity in liver cells measured by the comet assay during the transition period. While the DNA within the core remained undamaged (A), broken DNA migrated from the core towards the anode, forming the tail of a comet (B). When cells processed for the comet assay were examined by fluorescence microscopy, fluorescent structures corresponded to the PI-stained nuclear DNA of the hepatic cells. In undamaged cells, the DNA was tightly compressed and maintained the circular disposition of the normal nucleus.

Figure 2.

Comet images of hepatocytes. A — In undamaged cells, the DNA is tightly compressed and maintains the circular disposition of the normal nucleus (wk −3). B — The damaged DNA migrates from the core toward the anode, forming the tail of a comet (wk +3).

Representative microscopic images of liver specimens during the transition period are shown in Figure 3. No cellular abnormalities were found in any of the hepatocytes at wk −3 (A). At wk 0, however, the hepatocytes contained some lipid drops in their cytosol (B) and at wk +3 (C), the cells were opacified severely, with indistinct cellular membranes [hematoxylin & eosin (H & E) staining]. Figure 3 also shows the results of immunohistochemical examination for caspase-3 (D–F) and ssDNA (G–I) by the ABC method for detecting apoptotic cells. Although no caspase-3- or ssDNA-positive cells were observed in the liver specimens at wk −3 (D, G), positive cells appeared first at wk 0 (E, H) and increased at wk +3 (F, I).

Figure 3.

Images in histopathological and immunohistochemical analyses of liver specimens.

A–C — hematoxylin and eosin (H & E) staining; D–F — staining for detecting activated caspase-3; G–I — staining for detecting single-stranded DNA (ssDNA). Arrows indicate positive cells.

Figure 4 shows representative TEM images of hepatocytes at wk −3 (A) and at wk +3 (B). No significant alterations were observed in hepatocytes at wk −3. Condensation of nuclear chromatin, however, which is a typical sign of apoptosis, was observed at wk +3.

Figure 4.

Transmission electron microscope images of hepatocytes in liver specimens.

A — wk −3; B — wk +3; Nu — nucleus; Ab — apoptotic body.

Discussion

The importance of the transition period has been highlighted in several review articles because dairy cattle suffer dramatically decreased nutritional energy due to marked changes in the endocrine status, the onset of lactation, and physiological reduction of feed intake at this time (1). Based on the finding that NEFA concentrations are higher after calving, it is concluded that the cows in this study fell into negative energy balance (Table I). This imbalance of energy status was also demonstrated by decreased BCS and body weight. In the cow excluded from this study due to death, the decrease in body weight was 17.5% compared with wk −3 (5.4% in the remaining 4 dairy cattle), which may indicate a dramatically increased NEFA concentration.

The TG content of the liver increased after parturition (Figure 1A). The sharp increase of liver TG agreed with a previous report (15). The pathogenetic degree is considered to be mild fatty liver, which was consistent with a report that most cows in commercial dairy herds develop mild fatty liver (16). It seems that the mildly elevated BHBA concentrations observed in the present study as well as in another study (17) were similar to those occurring in cows with mild fatty liver during the transition period.

In this study, the extent of single-strand breaks (SSBs) of DNA (18) was evaluated by the average tail lengths of comet images. Because the tail length reflects the number of single-strand breaks in the DNA, the percentage of DNA in the tail provides a quantitative measure of the damaged DNA. We detected increased tail moment after parturition in all dairy cattle compared with the tail moment at wk −3 (Figure 2). When assessed by immunohistochemical staining, it was concluded that the DNA damage was hepatocyte apoptosis. Briefly, activated caspase-3 and ssDNA were not observed at wk −3 (Figure 3). Positive cells (apoptotic cells) were first detected in the liver specimens at wk 0, however, and increased at wk +3. Analysis of the hepatocyte ultrastructure in all dairy cattle using TEM showed no significant alterations at wk −3, but condensed nuclei with a crescent or circular shape, which are typical signs of apoptosis, were detected after parturition (Figure 4). The ultrastructure of the hepatocytes was not consistent with necrosis, as none of the usual features such as dissolution of the cell membranes and lysis of organelles (19) was observed in this study. These results also support the conclusion that the change of hepatocytes was apoptosis.

It has been reported that non-esterified fatty acids (NEFAs) induce hepatocyte apoptosis in human nonalcoholic fatty liver diseases (NAFLD) (9). Briefly, NEFAs can modulate both the extrinsic and intrinsic pathways of apoptosis. Saturated fatty acids such as palmitic acid and stearic acid cause c-Jun N-terminal kinase-dependent activation of the proapoptotic protein Bax, which then leads to mito-chondrial permeabilization with release of cytochrome c, activation of effector caspases, and apoptosis. Palmic acid can also activate the lysosomal pathway of apoptosis via Bax activation and Bax-dependent lysosomsal permeabilization. The monounsaturated fatty acid, oleic acid, imparts sensitivity to the death receptor-mediated extrinsic pathway of apoptosis. Contreras et al (20) reported that significantly increased palmitic acid and higher oleic acid concentrations in plasma NEFA were observed in dairy cows during transition.

According to our data, the palmitic acid and oleic acid proportions of liver tissue were significantly higher in cows with fatty liver than in healthy cows (21). The hepatocyte apoptosis detected in this study might have been induced by increased NEFA with high proportions of palmitic acid and oleic acid. On the other hand, a subset of patients with NAFLD develop progressive nonalcoholic steatohepatitis (NASH) in which disease activity correlates with hepatocyte apoptosis (22). Tumor necrosis factor α(TNFα) promotes hepatocyte apoptosis in experimental fatty liver models of mice (23). It has been reported that serum TNFα activity increases in cows with naturally occurring fatty liver (24). Although it is controversial because we did not deterimine TNFα activity in these cows, TNFα may be implicated as another inducing factor in hepatocyte apoptosis.

In conclusion, this study detected the presence of hepatocyte apoptosis during parturition. This is the first report of such detection to our knowledge. The induction of apoptosis probably resulted from lipotoxicity toward cells (8,9). Additional research is required, however, in order to determine the mechanism of apoptosis induction.