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World Journal of Nephrology & Urology

Case Report

Volume 6, Number 1-2, June 2017, pages 14-17

Do Sterilization Processes Really Make Difference in Dialysis-Induced Genotoxicity?

Hemodialysis (HD) is the routine treatment for chronic renal failure [1, 2]. The major problem associated with this therapy is the bioincompatibility of dialyzer membranes [3]. Several polymeric membranes have been used as materials for HD and these require sterilization processes before utilization. The methods currently used for sterilization of dialyzers include treatment with ethylene oxide and gamma irradiation. Meanwhile, alternative sterilization modes such as steam sterilization have been suggested [4, 5]. In this study, we tried to check if the sterilization process really makes any difference in dialysis-induced genotoxicity.

This study involved one HD patient (man, aged 38 years) with end-stage renal disease, who had undergone chronic HD treatment for 4 h, three times a week for 6 years. The patient has given his informed consent. For this study, before sample collecting, the patient underwent dialysis with every membrane type for at least 3 months. Results were then compared with those from one age-matched healthy subject from the general population.

Blood venous samples (5 mL) were drawn from HD patient at the beginning (T0), the middle; after 2 h (T2) and the end; after 4 h (T4) of HD session and were placed in heparinized tubes. Serum was obtained by centrifugation at 1,500 × g for 10 min at 4 °C.

Dialyzer membranes include: 1) F6-HPS: polysulfone dialyzer, surface area 1.3 m² sterilized by steam (M1) and from Fresenius Medical Care AG, Bad Homburg, Germany. 2) APS 650: polysulfone dialyzer, surface area 1.3 m² sterilized by gamma rays (M2) and from Asahi Kasei Medical, Tokyo, Japan.

All dialyzers are high-flux dialyzers.

Lipid peroxidation was carried out by the measurement of malondialdehyde (MDA) according to the method of Ohkawa et al (1979) [6]. Serum (100 µL) was combined with 0.2 mL of 8.1% SDS, 1.5 mL of 20% acetic acid adjusted to 3.5 of pH and 1.5 mL of 0.8% thiobarbituric acid. The mixture was brought to a final volume of 4 mL with distilled water and heated at 95 °C for 2 h. After being cooled at room temperature, 5 mL of n-butanol and pyridine mixture (15:1, v/v) was added to each sample and the whole was shaken vigorously. After centrifugation at 15,000 rpm for 10 min, the supernatant fraction was isolated and the absorbance was measured at 532 nm. The concentration of MDA was determined according to a standard curve.

DNA was extracted from peripheral blood leucocytes using blood DNA purification kit (Promega) and was quantified by UV spectrophotometry at 260 nm.

Loading buffer (0.25% bromophenol blue and 40% saccharose) was added to 5 µg of DNA for each sample. Samples were then analyzed by electrophoresis on a 1% agarose gel (1 h at 80 V/30 mA) with a Tris-Borate-EDTA running buffer (44 mM Tris-HCl, 44 mM boric acid, 50 mM EDTA, pH 8.0), stained with ethidium bromide and photographed with a SynGene GeneStore system.

DNA fragmentation was measured according to the method described by Sandau et al (1997) [7]. Diphenylamine solution (320 µL) (in 10 mL glacial acetic acid: 150 mg diphenylamine, 150 mL H₂SO₄ and 50 mL acetaldehyde 16 mg/mL) was added to each sample tube containing 5 µg of DNA, followed by incubation overnight at room temperature. The optical density was determined at 600 nm. DNA fragmentation was calculated as follows: % fragmented DNA = (OD supernatant/OD supernatant + OD pellet) × 100.

In the uremic state, many factors could be involved in DNA damage, in particular an enhanced generation of oxidative stress [8, 9]. The increased DNA damage in cells of patients with chronic renal insufficiency and long-term HD therapy may be causally linked to the high cancer incidence, in particular, cancer of the kidney, prostate, liver, and uterus, compared with the general population [10, 11]. In the present study, we have demonstrated that the sterilization processes of dialyzer membranes can interfere with the oxidative and genotoxic status of HD patient. As compared to healthy subject, there was a rising generation of MDA in patient blood during HD session. Nevertheless, this MDA production differs with the sterilization process. Indeed, at the end of HD session (T4), the serum MDA amounts were about 5 and 15 folds respectively for steam sterilized M1 and gamma rays sterilized M2 as compared to the MDA amount in control (Fig. 1). Consequently, utilization of radiation as sterilization process induced more ROS than the steam procedure. Even so, the steam method for sterilization is still the most biocompatible one. In fact, we have previously shown that steam-sterilized membranes improve endothelial cell viability and limit lipid peroxidation enhancement when compared to ethylene oxide or gamma rays-sterilized ones [12].

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Figure 1. Lipid peroxidation levels in patient and healthy subject sera measured by production of malondialdehyde (MDA), at the beginning (T0), the middle (after 2 h, T2) and the end (after 4 h, T4) of HD session and using different dialyzer sterilization processes: M1 (sterilized by steam) and M2 (sterilized by gamma rays). MDA amounts (µg/mL) were determined according to a standard curve. Results were expressed as mean ± SD from three independent experiments.

Slight DNA fragmentation was observed with dialyzer sterilized by steam (M1). The DNA fragmentation profile was more pronounced with dialyzer sterilized by gamma rays (M2). DNA fragments (DNA ladders) showing varying sizes between 750 and 2,000 base pairs were clearly visible after agarose gel electrophoresis, but no specific DNA fragments were detected when controls were analyzed (Fig. 2). Using a quantitative analysis of DNA fragmentation, a significant effect was observed after peripheral blood leucocytes exposure to different sterilized dialyzer membranes. This DNA fragmented level reached at the end of HD session 19±0.624% and 28±0.975%, respectively for steam sterilized M1 and gamma rays-sterilized M2 (Fig. 3). However, these genotoxic effects should be consequences of oxidative injury. Our finding is in concordance with other data that report a significant DNA fragmentation level in HD patient lymphocytes [13, 14].

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Figure 2. DNA fragmentation induced by exposure to different dialyzer membranes sterilization mode: M1 (sterilized by steam) and M2 (sterilized by gamma rays), in patient leucocytes at the end (after 4 h, T4) of HD session and revealed by agarose gel electrophoresis.

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Figure 3. Percentage of DNA fragmentation caused by exposure to different dialyzer membranes sterilization mode: M1 (sterilized by steam) and M2 (sterilized by gamma rays), in patient leucocytes at the beginning (T0), the middle (after 2 h, T2) and the end (after 4 h, T4) of HD session and determined using the diphenylamine assay. Results were expressed as mean ± SD from three independent experiments.

Finally, steam sterilization of dialyzers offers considerable safety advantages to the patient and is preferred to other sterilization modes, such as gamma irradiation.

Acknowledgments

This research was supported by Le Ministere Tunisien de l’Enseignement Superieur, de la Recherche Scientifique et de la Technologie, through the Laboratoire de Recherche sur les Substances Biologiquement Compatible.

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World Journal of Nephrology and Urology is published by Elmer Press Inc.