Indian J Gastroenterol 2010(January–February):29(1):23–27
ORIGINAL ARTICLE
Ethanol-induced changes in lipid peroxidation of enterocytes across the crypt–villus axis in rats Amandeep Kaur Kalra · Shiffalli Gupta · Aasma Turan · Safrun Mahmood · Akhtar Mahmood
Abstract Background Reactive oxygen species (ROS) have been implicated in the turnover of epithelial cells in the rat intestine. The metabolism of ethanol generates ROS, which are implicated in cellular injury, but the levels of lipid peroxidation in intestine in chronic alcoholism are unknown. Aim To investigate the effects of ethanol ingestion on lipid peroxidation, and anti- and pro-oxidant enzyme systems in enterocytes across the crypt–villus axis in intestine. Methods Wistar rats (90–100 g) were administered 1 mL of 30% ethanol daily for 39 days. Intestinal epithelial cells were isolated in fractions. Malondialdehyde levels, and activities of glutathione-S-transferase (GST), glutathione reductase (GR), superoxide dismutase (SOD) and catalase were determined in various cell fractions. Incorporation of H3thymidine into DNA of enterocytes was also determined. Results Lipid peroxidation was elevated by two- to threefolds in both villus and crypt cells in ethanol-fed animals compared to controls. The activities of GST and GR were four- to six-folds higher in villus tip cells compared to crypt base cells. A. K. Kalra1 · S. Gupta1 · A. Turan1 · S. Mahmood2 · A. Mahmood1 1
Department of Biochemistry, Punjab University, Chandigarh 160 014, India
2
Department of Experimental Medicine and Biotechnology, Postgraduate Institute of Medical Education and Research, Chandigarh 160 012, India A. Mahmood () e-mail:
[email protected] Received: 1 May 2009 / Revised: 7 July 2009 / Accepted: 17 October 2009 © Indian Society of Gastroenterology 2010
Indian J Gastroenterol 2010(January–February):29(1):23–27
The activities of SOD and catalase were five- to seven-fold higher in crypt base cells compared to villus tip cells. Ethanol feeding elevated the activities of SOD (76-190%) and catalase (20-150%) in enterocytes all along the crypt–villus axis compared to the controls. H3 thymidine incorporation into DNA of enterocytes was reduced by half in ethanol-fed rats compared to controls. Conclusions There is a gradient in the concentration of lipid peroxides in enterocytes across the crypt–villus axis, being high at the villus tip and low at the crypt base in the rat intestine. Ethanol feeding enhanced lipid peroxidation in both villus and crypt cells. Keywords testine
Chronic alcoholism · lipid peroxidation · rat in-
Introduction Chronic alcoholism affects intestinal morphology,1 enzymes2,3 and transport functions in the mammalian intestine.4 The intestinal lumen is lined with a single layer of epithelial cells, which divide in the crypt base, differentiate and migrate to the villus tip, whereupon apoptosis they are detached from the underlying muscular tissue.5,6 We recently demonstrated that reactive oxygen species (ROS) may be involved in the turnover of enterocytes in rat intestine, since malondialdehyde (MDA) formed as a consequence of lipid peroxidation in epithelial cells at the villus tip was 10- to 12-fold higher compared to that in the immature crypt cells.7,8 There is evidence that ethanol is oxidized by the mucosa or by bacterial enzymes to generate high levels of free radicals in the colon.9 Metabolic degradation of ethanol to acetaldehyde, with the generation of free radicals in the liver, has been implicated in ethanol-induced liver injury.10 The present study was undertaken to test the hypothesis that ethanol feeding affects lipid peroxidation of enterocytes, by examining the effects of ethanol ingestion on lipid peroxidation and on anti- and pro-oxidant enzyme
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systems in cells distributed across the crypt–villus axis of the rat intestine. Methods The experimental protocol was approved by the Ethical Committee of the Institute on the use of laboratory animals. Experiments on animals were performed in accordance with the guidelines for use of laboratory animals, approved by the Indian Council of Medical Research, New Delhi. Male albino rats (Wistar strain) weighing 90–100 g obtained from the central animal house of Punjab University, Chandigarh were kept on standard rat pellet diet (Hindustan Lever Ltd., Ghaziabad, India), and had free access to water. The rats were randomized into two groups with eight to 10 animals in each group. Animals in the test group were administered 1 mL of 30% ethanol intragastrically by Ryles tube (Thermo Fisher Scientific Inc. USA) without anesthesia daily between 9 am and 10 am for 39 days. Control animals were given 1 mL of isocaloric amounts of glucose. Body weights of animals were recorded on alternate days in each group. At day 40 of the commencement of alcohol treatment, overnight-fasted animals were sacrificed by cervical dislocation. Starting from the ligament of Treitz, the proximal 20 cm of the intestine was removed, washed with ice-cold saline, and used for the isolation of various cell fractions.
Lipid peroxidation Lipid peroxidation was measured as MDA formation using thiobarbituric acid reaction, following the method of Buege and Aust.19 In vitro lipid peroxidation under induced (Fe2+/ascorbate) or uninduced conditions was measured as described by Dudeja and Brasitus.20 Protein content was determined by the method of Lowry et al21 using bovine serum albumin as the reference standard. Enzyme activities and MDA were expressed per milligram protein. Radioisotope incorporation studies The incorporation of 3H thymidine (Bhabha Atomic Research Center, Mumbai, India) into DNA was studied by injecting I.P., 100 μCi tritium-labeled thymidine into each animal. Intestinal cell fractions containing 300 μg of protein were digested with 10% KOH for 30 minutes in boiling water bath. After adding 8 mL of scintillation fluid, the radioactivity was measured using Packard Tri-Carb liquid scintillation analyzer.22 The radioactivity incorporated was expressed as cpm/mg DNA. Statistical analysis Statistcal analysis of the data was carried out using Student’s t-test.
Preparation of cell fractions
Results
Epithelial cells from intestine were isolated following the method of Weisser.11 Starting from the villus tip, fractions 1–3 (villus cells), 4–6 (mid-villus cells), and 7–9 (crypt cells) were isolated. The cell fractions were suspended in 50 mM sodium maleate buffer (pH 6.8) and homogenized in the same buffer to prepare 10% homogenate (w/v). Cell fractions were characterized by the assay of marker enzymes, sucrase, and alkaline phosphatase and were used for various biochemical analyses. DNA content was estimated by diphenylamine method as described by Burton.12
The isolated cell fractions were validated by assaying the marker enzymes sucrase and alkaline phosphatase, which were four- to six-fold higher in villus tip cells compared to crypt base cells (results not shown). The level of MDA was four- to five-fold higher in villus tip cells compared to crypt base cells both under uninduced and induced conditions (Table 1). In ethanol-fed animals, there was two- to three-fold increase in MDA concentration in villus tip cells compared to corresponding cells in the control animals. In all epithelial cell fractions (villus and crypt) lipid peroxidation was 130–180% more in ethanol-fed rats compared to controls. Glutathione-S-transferase activity did not differ between control and ethanol-fed animals. The gradient of activity being four-fold higher in villus tip cells compared to crypt base cells. Glutathione reductase activity was six-fold higher in villus tip cells compared to crypt base cells (Table 1). Ethanol administration elevated the enzyme activity in all cell fractions compared to the controls. The free radical scavenging enzyme, SOD, was five- to seven-fold higher in activity in crypt base cells compared to villus tip cells both in control and ethanol fed animals (Table 2). The enzyme activity was nearly 99%, 142% and 34% higher in the villus tip, mid-villus and crypt base regions respectively in ethanol fed animals,
Assay of enzymes Previously described methods were used for measurement of activity of alkaline phosphatase [EC. 3.1.3.1],13 sucrase [EC.3.2.1.48],14 glutathione reductase [EC.1.6.4.2],15 glutathione-S-transferase [EC.2.5.1.18],16 superoxide dismutase (SOD) [EC.1.15.1.1];17 catalase [EC.1.11.1.6] activity was assayed by monitoring the disappearance of H2O2 at 240 nm.18 Three milliliters of reaction mixture contained potassium phosphate buffer (pH 7.0), H2O2 and an appropriate amount of the enzyme. A blank lacking H2O2was run simultaneously. A decrease in absorbance at 240 nm was measured.
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Table 1 Effect of ethanol administration on lipid peroxidation and glutathione-s-transferase in enterocytes across crypt–villus axis in rat intestine Lipid peroxidation Cell fraction
Uninduced
Control 1. Villus tip 2. 3. 4. Mid-villus 5. 6. 7. Crypt base 8. 9.
0.32 0.24 0.22 0.20 0.15 0.12 0.09 0.07 0.06
Induced
Ethanol-fed
Control
(0.09) 0.83 (0.03)* (0.10) 0.76 (0.02)* (0.09) 0.58 (0.03)* (0.08) 0.50 (0.01)* (0.03) 0.12 (0.03)* (0.03) 0.17 (0.03)** (0.02) 0.10 (0.02) (0.03) 0.089 (0.06) (0.03) 0.077 (0.04)
0.37 0.26 0.26 0.22 0.18 0.10 0.09 0.07 0.06
(0.14) (0.08) (0.08) (0.07) (0.06) (0.01) (0.01) (0.01) (0.003)
Glutathione-S-transferase
Ethanol-fed 1.86 1.25 1.17 0.97 0.69 0.57 0.43 0.31 0.21
(0.04)* (0.03)* (0.05)* (0.03)* (0.02)* (0.04)* (0.05) (0.018)* (0.04)*
Control 7.19 6.72 6.40 5.23 4.25 3.40 2.06 1.79 1.44
Glutathione reductase
Ethanol-fed
(0.19) (0.21) (0.20) (0.10) (0.11) (0.12) (0.09) (0.08) (0.11)
7.44 6.55 6.49 5.20 4.41 4.79 3.03 2.13 1.33
Control
(0.99) (0.42) (0.41) (0.36) (0.72) (0.16)* (0.79)* (0.37) (0.40)
4.62 3.78 3.38 2.28 1.80 1.22 0.90 0.79 0.73
(0.12) (0.11) (0.10) (0.14) (0.12) (0.10) (0.08) (0.05) (0.06)
Ethanol-fed 7.24 6.25 5.91 4.77 3.85 3.37 2.16 1.83 1.27
(0.36)* (0.75)* (1.21)* (0.78)* (0.42)* (0.28)* (0.19)* (0.22)* (0.39)**
Values are mean (SD) of five observations, expressed as micromoles of MDA formed per milligram of protein. Fractions 1-3 represent villus tip cells; fractions 4-5: mid villus and fractions 7-9: crypt base *p < 0.001, **p < 0.05 as compared to controls Table 2 Effect of ethanol administration on superoxide dismutase and catalase activities of cells across crypt–villus axis in rat intestine Cell fraction
Superoxide dismutase Control
1. Villus tip 2. 3. 4. Mid-villus 5. 6. 7. Crypt base 8. 9.
0.58 0.79 1.38 2.22 2.45 3.68 4.76 5.98 8.09
(0.10) (0.08) (0.08) (0.07) (0.12) (0.50) (0.10) (0.15) (0.22)
Catalase
Ethanol-fed 1.68 1.81 3.66 4.23 6.98 7.81 8.37 10.66 17.38
(0.23)* (0.14)* (0.15)* (0.13)* (0.21)* (0.15)* (0.23)* (0.34)* (0.28)*
Control 8.01 11.51 12.59 16.01 19.36 22.50 57.60 77.43 109.08
(0.52) (0.68) (0.33) (0.99) (0.72) (0.79) (1.28) (1.22) (2.34)
Ethanol-fed 12.01 23.02 28.95 40.01 48.40 51.75 86.40 108.4 130.8
(0.78)* (1.02)* (0.75)* (0.24)* (1.80)* (1.81)* (1.92)* (1.7)* (2.8)**
Values are mean (SD) of five observations, expressed as micromoles per hour per milligram of protein. Fractions 1-3 represent villus tip cells; fractions 4-5: mid villus and fractions 7-9: crypt base *p < 0.001, **p < 0.05 as compared to controls
compared to corresponding cell fractions in the controls. A similar pattern of the distribution of catalase activity was observed in control and ethanol-fed rats (Table 3). Catalase activity was 10- to 13-fold higher in crypt base compared to villus tip cells. Ethanol feeding enhanced catalase activity in villus tip (157%), mid-villus (144%) and crypt base (33%) cells compared to the control group. The incorporation of H3-thymidine into DNA revealed an average 42% (crypt base 50%, villus tip cells 20%) decrease in isotope incorporation in ethanol-fed animals compared to the controls (Fig. 1). Discussion In the present study, rats were administered 1 mL of 30% ethanol daily, which is equivalent to two ounces of whisky per day for a 70-kg man. This amount yields 6.5–9.4% and Indian J Gastroenterol 2010(January–February):29(1):23–27
5.7–6.4% concentrations of ethanol in the duodenum and jejunum, respectively.2 The present findings indicate that ethanol feeding enhanced the levels of MDA by two-fold in cells at the villus tip, which suggests high level of lipid peroxidation as a consequence of generation of free radicals in these cells. Jackson et al suggested23 that damage to cellular DNA by free radicals plays an important role in cellular injury and leads to apoptosis. The lipid peroxidation levels were relatively low in the crypt base, compared to those in the villus tip cells in ethanol-fed animals. This pattern of lipid peroxidation is similar to that reported earlier in fat-fed animals.7 The observed changes in lipid peroxidation were associated with high activities of glutathioneS-transferase and glutathione reductase activities in villus tip cells compared to those in the crypt base. However, the reverse pattern of distribution of free radical scavenger enzymes SOD and catalase activities was observed, where
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Acknowledgement Shiffalli Gupta was a recipient of senior research fellowship from University Grants Commission, New Delhi. References 1.
2. 3.
Fig. 1 The incorporation of tritium-labeled thymidine into DNA of enterocytes along the villus–crypt axis in control (_) and ethanol-fed (…..) rats. Values are mean from two animals each determined in triplicate
the enzyme levels were high in the crypt base and low in villus tip cells in ethanol-fed animals. The observed increase in SOD and catalase activities in ethanol-fed rats is similar to that observed in fat-fed animals.8 Kaur et al24 reported an increase in activity of GST in ethanol-fed rat intestine which is in agreement with the present finding. The balance between ROS generation and antioxidant capacity within the cells determines the oxidative status. When ROS exceeds the antioxidant capacity of cells, oxidative stress ensues. Oxidant capacity can initiate or mediate a number of signaling cascades leading to apoptosis. Ethanol is rapidly metabolized in the body and is known to generate free radicals, which have been implicated in tissue injury.25,26 Thus the generation of high levels of free radicals, as suggested by enhanced MDA contents, may induce apoptosis in cells, resulting in their detachment from the underlying muscular tissue.7 This may be responsible for observed changes in intestinal morphology and function in alcoholism.6 The present data also indicate that the incorporation of H3 thymidine into DNA was low in different cell fractions across the villus height in ethanolingested rats. This may lead to reduced number of epithelial cells across the villi, and explain decreased intestinal cell mass in chronic alcoholism as well as in rat pups born to alcohol-fed mothers.2,27 Peres et al28 have also described that ethanol administration affects the proliferation and differentiation of epithelial cells in intestine. The present data indicate a gradient in the distribution of lipid peroxidation and the activities of pro-oxidant enzymes, being high at the villus tip and low in the crypt base, while there was a gradient of anti-oxidant enzymes in the reverse pattern. Thus an increase in lipid peroxides at villus tip cells after ethanol ingestion may lead to detachment of the cells as a result of the generation of free radicals.
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