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ener
lu-Öünç3 Çetinel4en6
Department of Pharmacology, School of Pharmacy, Marmara University, Istanbul, Turkey
1 Department of Radiation Oncology, School of Medicine, Marmara University, Istanbul, Turkey
2 Department of Medical Biology, School of Medicine, Marmara University, Istanbul, Turkey
3 Vocational School of Health Related Professions, Marmara University, Istanbul, Turkey
4 Department of Histology-Embryology, School of Medicine, Marmara University, Istanbul, Turkey
5 Division of Biochemistry, Kasimpasa Military Hospital, Istanbul, Turkey
6 Department of Physiology, School of Medicine, Marmara University, Haydarpa
a, Istanbul 34668, Turkey
(Requests for offprints should be addressed to B Ç Yeen; Email: byegen{at}marmara.edu.tr)
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Abstract |
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(TNF) were assayed in serum samples. Irradiation caused a significant decrease in GSH level, which was accompanied by significant increases in MDA levels, MPO activity, CL levels and collagen content of the tissues studied (P<0.05–0.001). Similarly, serum TNF and LDH were elevated in the irradiated rats as compared with the control group. On the other hand, PTU treatment reversed all these biochemical indices, as well as histopathological alterations induced by irradiation. Our results suggested that PTU-induced hypothyroidism reduces oxidative damage in the lung, hepatic, renal and ileal tissues probably due to hypometabolism, which is associated with decreased production of reactive oxygen metabolites and enhancement of antioxidant mechanisms. |
Introduction |
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Thyroid hormones are among the most important humoral factors involved in setting the basal metabolic rate (Guerrero et al. 1999). It is well known that thyroid hormones increase mitochondrial respiration through changes in the number and activity of mitochondrial respiratory chain components, providing a considerable impact on oxidative stress (Roodyn et al. 1965, Karbownik & Lewinski 2003). Accordingly, it was reported that the thyroid state might alter the changes in oxidant and antioxidant systems (Brzezinska-Slebodzinska 2001). High concentrations of thyroid hormones and thyrotoxicosis can facilitate the metabolism of oxygen in aerobic conditions, stimulate free radical generation and aggravate oxidant-mediated tissue injury (Asayama & Kato 1990, Venditti et al. 1997). On the other hand, metabolic depression brought about by hypothyroidism has been associated with decreased oxidant production and protection against tissue lipid peroxidation (Swaroop & Ramasarma 1985, Paller 1986).
Based on these reports, this study was designed to determine the possible protective effect of hypothyroidism against irradiation-induced oxidative damage of the gastrointestinal tissues in rats by evaluating the extent of tissue injury through biochemical and histological analyses.
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Materials and Methods |
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All experimental protocols were approved by the Marmara University School of Medicine Animal Care and Use Committee. Male Sprague–Dawley rats (200–250 g) were kept at a constant temperature (22 ± 1 °C) under 12 h light:12 h darkness cycles.
A total of 32 animals were exposed to irradiation performed with a LINAC (Saturne 42, General Electrics, France) producing 6 MV photons at a focus 100 cm distant from the skin. Under ketamine anaesthesia (100 mg/kg i.p.), each rat received a single whole-body X-ray irradiation of 800 cGy. Animals were returned to their home cages following irradiation. Control rats (n=6) received no irradiation or treatment.
Rats were pretreated daily with either 6-n-propyl-2-thiouracil (PTU; 10 mg/kg i.p.; Sigma Chemical Co., St Louis, MO, USA) or saline (1 ml/kg) for 15 days before irradiation. In the rats followed for 72 h after irradiation, saline or PTU injections were repeated once daily. Rats were decapitated at 6 h and 72 h after exposure to irradiation, trunk blood was collected and the serum was separated to measure the aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels and creatinine and blood urea nitrogen (BUN) as indicators of liver and kidney functions respectively. Lactate dehydrogenase (LDH) and tumour necrosis factor- (TNF) were also assayed in serum samples for the evaluation of generalized tissue damage. Tissue samples from the lung, liver, kidney and ileum were fixed in formaldehyde for histological analysis, while additional samples were stored at –80 °C for the determination of malondialdehyde (MDA) and glutathione (GSH) levels, myeloperoxidase (MPO) activity and collagen content. Formation of reactive oxygen species (ROS) in the tissue samples was monitored using the chemiluminescence (CL) technique with luminol and lucigenin probes. Additional ileal samples were obtained for the determination of DNA fragmentation as an index of apoptosis.
Blood assays
BUN (Talke & Schubert 1965), serum AST, ALT (Moss et al. 1987) and creatinine (Slot 1965) concentrations and LDH levels (Martinek 1972) were determined spectrophotometrically using an automated analyzer. TNF was evaluated by an RIA-IRMA method. All samples were assayed in duplicate using a commercial kit (Biosource Europe S.A., Nivelles, Belgium). The activity of radioactive assays was measured by a 
counter (LKB WALLAC 1270 RACK Gamma Counter, Turku, Finland). TNF in the serum samples is expressed as pg/ml.
MDA and GSH assays
Tissue samples were homogenized with ice-cold 150 mM KCl for the determination of MDA and GSH levels. The MDA levels were assayed for products of lipid peroxidation by monitoring thiobarbituric acid reactive substance formation as described previously (Beuge & Aust 1978). Lipid peroxidation is expressed in terms of MDA equivalents using an extinction coefficient of 1.56 x 105 M/cm and results are expressed as nmol MDA/g tissue. GSH measurements were performed using a modification of the Ellman procedure (Beutler 1975). Briefly, after centrifugation at 3000 g for 10 min, 0.5 ml supernatant was added to 2 ml 0.3 mol/l Na2HPO4. 2H2O solution. A 0.2 ml solution of dithiobisnitrobenzoate (0.4 mg/ml 1% sodium citrate) was added and the absorbance at 412 nm was measured immediately after mixing. GSH levels were calculated using an extinction coefficient of 1.36 x 105 M/cm. Results are expressed in µmol GSH/g tissue.
Measurement of MPO activity
MPO is an enzyme that is found predominantly in the azurophilic granules of polymorphonuclear leukocytes (PMN). Tissue MPO activity is frequently utilized to estimate tissue PMN accumulation in inflamed tissues and correlates significantly with the number of PMN determined histochemically in tissues (Bradley et al. 1982). MPO activity was measured in tissues in a procedure similar to that documented by Hillegas et al.(1990). Tissue samples were homogenized in 50 mM potassium phosphate buffer (PB; pH 6.0), and centrifuged at 41 400 g (10 min); pellets were suspended in 50 mM PB containing 0.5% hexadecyltrimethylammonium bromide. After three freeze and thaw cycles, with sonication between cycles, the samples were centrifuged at 41 400 g for 10 min. Aliquots (0.3 ml) were added to 2.3 ml reaction mixture containing 50 mM PB, o-dianisidine and 20 mM H2O2 solution. One unit of enzyme activity was defined as the amount of MPO present that caused a change in absorbance measured at 460 nm for 3 min. MPO activity is expressed as U/g tissue.
Tissue collagen measurement
Tissue collagen was measured as a free radical-induced fibrosis marker. Tissue samples were cut with a razor blade, immediately fixed in 10% formalin in 0.1 M phosphate buffer (pH 7.2) in paraffin, and approximately 15 µm thick sections were obtained. Evaluation of collagen content was made according to the method published by Lopez de Leon & Rojkind (1985), based on selective binding of the dyes Sirius Red and Fast Green to collagen and non-collagenous components respectively. Both dyes were eluted readily and simultaneously by using 0.1 M NaOH–methanol (1:1, v/v). Finally, the absorbances at 540 and 605 nm were used to determine the amount of collagen and protein respectively.
CL assay
To assess the role of ROS in radiation-induced tissue damage, luminol and lucigenin CLs were measured as indicators of radical formation. Measurements were made at room temperature using a Junior LB 9509 luminometer (EG&G, Berthold, Germany). Specimens were put into vials containing PBS–HEPES buffer (0.5 M PBS containing 20 mM HEPES, pH 7.2). ROS were quantitated after the addition of enhancers such as lucigenin or luminol at a final concentration of 0.2 mM. Luminol detects a group of reactive species, i.e. OH, H2O2, HOCl radicals, and lucigenin is selective for O2– (Ohara et al. 1993, Davies et al. 1994, Haklar et al. 2002). Counts were obtained at 1-min intervals and the results are given as the area under curve for a counting period of 5 min. The counts were corrected for wet tissue weights and expressed as relative light units (Haklar et al. 2002).
DNA fragmentation assay
Mucosal samples from ileal tissues were homogenized in 10 volumes of a lysis buffer (5 mM Tris–HCl, 20 mM ethylene diamine tatra-acetic acid (EDTA) and 0.5% (v/v) t-octylphenoxypolyethoxyethanol (Triton-X 100); pH 8.0). Two separate samples of 1 ml each were taken from the sample and centrifuged at 25 000 g for 30 min to separate the intact chromatin in the pellet from the fragmented DNA in the supernatant (Wyllie 1980). The supernatant was taken out to be saved and the pellet was re-suspended in 1 ml Tris-Ethylendiamintetraacetat buffer (pH 8.0) (10 mM:1 mM). Both the supernatant and the re-suspended pellet were then assayed for DNA content determination by the diphenylamine reaction described by Burton (1956).
Histopathological analysis
Samples of the tissues were fixed in 10% (v/v) buffered p-formaldehyde and prepared for routine paraffin embedding. Tissue sections (6 µm) were stained with haematoxylin and eosin and examined under a light microscope (Olympus-BH-2, Tokyo, Japan) by an experienced histologist who was unaware of the treatment conditions.
Statistics
Statistical analysis was carried out using GraphPad Prism 4.0 (GraphPad Software, San Diego, CA, USA). All data are expressed as means±S.E.M. Groups of data were compared with an ANOVA followed by Tukey’s multiple comparison tests. Values of P<0.05 were regarded as significant.
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Results |
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BUN and creatinine concentrations were studied to assess the renal functions, while serum AST and ALT levels were determined to evaluate the hepatic functions. As shown in Table 1
, ALT levels were significantly higher in both irradiated groups decapitated at 6 or 72 after irradiation, when compared with those of the control group (P<0.05–0.01), while PTU treatment decreased these elevations. AST levels were increased (P<0.05) at only 72 h following irradiation and PTU treatment reversed this alteration back to control levels. However, blood creatinine levels were not changed significantly at either 6 or 72 following irradiation (Table 1). Similarly, BUN levels were not different from the control group at 6 h after irradiation, but they were found to be elevated at 72 h of irradition (P<0.001). On the other hand, this elevation in BUN level was abolished in the PTU-treated irradiated group (P<0.001).
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levels were significantly increased (P<0.001) when compared with the control group, while this irradiation-induced rise in serum TNF levels was abolished (P<0.05–0.01) with PTU treatment (Fig. 1b). Serum LDH activity, as a marker of generalized tissue damage, showed a significant increase in irradiated animals at both time-points (P<0.05–0.01), while PTU administration reversed this effect (P<0.05–0.01; Fig. 1a).
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), while treatment with PTU led to replenishment of the tissue GSH contents (P<0.05–0.001). Except for the lipid peroxidation of the lung tissue, which showed a tendency to increase at 6 h of irradiation without reaching statistical significance, MDA levels were significantly higher in both irradiation groups (Fig. 3) as compared with the control group (P<0.05–0.001). However, treatment with PTU significantly (P<0.05–0.001) reversed irradiation-induced elevations in MDA levels back to the control levels in all the tissues.
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). Except for the ileum, this early neutrophil accumulation was not any more evident at 72 h of irradiation. On the other hand, PTU treatment abolished irradiation-induced increases in MPO activity at the early period of irradiation in all the tissues (P<0.01), while sustained elevation in MPO activity of the ileum was also suppressed by PTU treatment.
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); but no changes in collagen contents were observed at the earlier post-irradiation period. On the other hand, PTU-induced hypothyroidism prevented the increases in collagen levels, returning these levels back to control levels (P<0.05–0.01).
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DNA fragmentation was elevated in the ileal mucosae of saline-treated groups at both early and later post-irradiation periods (P<0.001), while PTU treatment significantly prevented the DNA damage (P<0.05) (Fig. 6).
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). PTU treatment led to the reversal of degenerations in both irradiated groups where the structure of sinusoids was re-established and congestion of the sinusoids was reduced (Fig. 7B and D).
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). In lung tissues, severe interstitial oedema with bronchiolar haemorrhage observed in the irradiated group at 72 h was ameliorated by PTU treatment, revealing the reversal of degeneration and interstitial oedema compared with the saline-treated group (Fig. 8C and D).
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). PTU-treated irradiated groups showed better organized tubular and glomerular structures with well-established epithelia which resembled that of the control group (Fig. 9B and D).
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). These findings were in accordance with those found in the intestines, where severe epithelial loss of the villi and inflammatory cell invasion in the lamina propria of the group treated for 72 h were replaced by regular tissue integrity and many goblet cells in the epithelium of the PTU-treated group (Fig. 10C and D).
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Discussion |
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In addition to its inhibitory effects on thyroid hormone synthesis, PTU has potential immunomodulatory (Wilson et al. 1988) and antioxidant effects (Hicks et al. 1992). Accordingly, PTU has been shown to act as a highly efficient scavenger of OH radicals, to be a potent inhibitor of lipid peroxidation in model membranes (Hicks et al. 1992) and to inhibit H2O2 production in neutrophils (Imamura et al. 1986) or Graves’ retroocular fibroblasts (Heufelder et al. 1992). It has also been observed that hyperthyroid patients treated with PTU have decreased serum MDA levels, suggesting that PTU may scavenge ROMs or may directly protect lipids from peroxidative attack (Videla et al. 1988). In accordance with these results, in the present study tissue MDA was increased in the lung, liver, kidney and ileum of whole-body irradiated rats, indicating the presence of radiation-induced oxidative damage. Peroxidation of membrane lipids can disrupt membrane fluidity and cell compartmentation, which may contribute to impaired cellular function and necrosis (Reiter et al. 2001). The present findings have shown that irradiation impaired the renal and hepatic functions, while PTU treatment prevented elevations in tissue MDA and attenuated the impairments in the liver and kidney functions. Furthermore, luminol- and lucigenin-enhanced CL levels were reduced in PTU-treated rats, implicating that amelioration of radiation-induced oxidative injury by PTU involves reduction in oxygen metabolism and scavenging of reactive oxygen radicals. Although it is difficult to quantitate oxygen radicals because of their reactive nature and short lives, luminol- and lucigenin-enhanced CL is a simple and reproducible tool indicating the involvement of free radicals. Despite the selectivity of the probes used in the study (Ohara et al. 1993, Davies et al. 1994, Haklar et al. 2002), our CL data support the notion that tissue injury induced by irradiation involves toxic oxygen metabolites without any selectivity.
GSH, a well-known antioxidant, provides major protection in oxidative injury by participating in the cellular system of defence against oxidative damage (Ross 1988). It was reported that tissue GSH levels and the activities of GSH reductase and GSH peroxidase, which are critical constituents of the GSH–redox cycle, were significantly reduced due to oxidative stress, permitting enhanced free radical-induced tissue damage (Reiter et al. 2001). In the present study, the decrease in tissue GSH levels may be due to its consumption during the oxidative stress induced by irradiation. On the other hand, treatment with PTU prevented GSH depletion by decreasing free radical generation. There is a good deal of evidence to indicate that metabolic depression brought about by hypothyroidism is associated with a decrease in free radical production and subsequent protection against oxidative damage (Asayama et al. 1987). In contrary, high concentrations of thyroid hormones were shown to increase the metabolism of oxygen in aerobic conditions and stimulate free radical generation (Fernandez & Videla 1989). Thus, reduced demand for oxygen and concomitant suppression of free radical formation in hypothyroidism appears to serve as a protective factor in radiation-induced oxidative injury.
Tissue macrophages and monocytes respond to any noxious event by secreting cytokines such as interleukin-1 and TNF (Schwacha 2003, Enomoto et al. 2004). As evidenced in the present study, irradiation resulted in increased serum TNF, indicating the role of this cytokine in irradiation-induced toxicity, while depression of the TNF response by PTU implicates the inhibitory effect of PTU on the release of this pro-inflammatory cytokine. In our observations, elevated MPO levels in the tissues indicate that neutrophil accumulation contributes to the irradiation-induced oxidative injury and PTU appears to have a preventive effect through the inhibition of neutrophil infiltration and subsequent release of pro-inflammatory mediators. Observations suggest that ROMs play a role in the recruitment of neutrophils into injured tissues, but activated neutrophils are also a potential source of ROMs (Kettle & Winterbourn 1997). Thus, it seems likely that the alleviation of irradiation-induced oxidative tissue damage by PTU involves the suppression of a variety of pro-inflammatory mediators produced by leukocytes and macrophages. It is known that PTU inhibits not only the catalytic activity of thyroid peroxidase, but also the other members of the mammalian peroxidases, such as MPO, lactoperoxidase, eosinophil peroxidase and salivary peroxidase (Taurog 1999). Although it was shown that PTU is a direct inhibitor of MPO function (Taurog & Dorris 1992), our data related to the effect of PTU on non-irradiated rats demonstrated that MPO activity was not different from the non-treated control rats. It appears that PTU does not have a significant impact on basal MPO activity, but it may have a direct inhibitory effect on inflammation-induced enhanced MPO activity. Thus, the protective effects of PTU on radiation-induced injury is not solely related to its anti-thyroidal action which reduces the oxidative metabolism but it may also involve the direct inhibitory effect of PTU on MPO activity.
In vital organs (such as the heart, kidney, lung or liver) tissue injury caused by a range of insults, including oxidative stress, can result in the development of progressive fibrosis leading to ultimate organ failure (Franklin 1997, Martin et al. 2000). The risk of irradiation-induced fibrosis, occurring in normal tissues several months to years after radiotherapy, remains a major factor in limiting the radiation dose that can be applied safely to cancer patients. In the current study, collagen contents of the studied tissues were significantly increased at 72 h following irradiation, indicating the presence of tissue fibrotic activity, while PTU treatment attenuated the fibrotic activity by its antioxidant properties. It has been shown that irradiation dramatically increases the low level of spontaneous apoptosis in intestinal crypts (Potten et al. 1994, Potten 1997), which normally occurs to protect against the survival and expansion of genetically damaged cells. Our results indicated that whole-body irradiation increased the apoptotic ratio of ileal epithelium, which seems to be responsible for severe epithelial loss of the villi. On the other hand, PTU treatment suppressed the high percentage of DNA fragmentation, while epithelial integrity was maintained. Although it was shown that PTU inhibits cell proliferation and migration by a phosphatidylinositol 3-kinase-mediated pathway (Chen et al. 2004), there is no direct evidence to support the idea that PTU has an impact on malignant cell proliferation. Since radiotherapy cannot be precisely targeted to facilitate the apoptosis of tumour cells without affecting the healthy cells, and the apoptosis of the normal tissue cells is inevitable, further studies are required to evaluate the anti-proliferative and protective effects of PTU on malignant and non-malignant cells respectively.
The current data have suggested that PTU reverses oxidant responses to irradiation and thereby reduces inflammation and subsequent organ failure by depressing neutrophil infiltration and the associated release of pro-inflammatory cytokines. In conclusion, PTU merits consideration as an antioxidant and anti-inflammatory agent in ameliorating the severity of irradiation-induced multiple organ injury.
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Acknowledgements |
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engöz and Z Özen for their support in conducting the irradiation procedure. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work. |
References |
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Received in final form 6 December 2005
Accepted 18 January 2006
Made available online as an Accepted Preprint 1 February 2006
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