Acrylamide-induced effects on general and neurospecific cellular functions during exposure and recovery.

Basal cytotoxicity, morphological changes and alterations in cell physiological and neurochemical functions were studied in differentiated human neuroblastoma (SH-SY5Y) cells during exposure to acrylamide and during a subsequent recovery period after cessation of exposure. Acrylamide induced a 20% reduction in the number of neurites per cell at 0.21 mmol/L and 20% decrease in the protein synthesis rate at 0.17 mmol/L after 72 h of exposure. Furthermore, the basal level of intracellular calcium concentration ([Ca2+]i) and receptor-activated (carbachol, 0.1 mmol/L) Ca2+ fluxes increased by 49% and 21%, respectively, at 0.25 mmol/L. These observations were made at noncytotoxic acrylamide concentrations, signifying specific neurotoxic alterations. Forty-eight hours after cessation of acrylamide exposure, the SH-SY5Y cells had recovered, i.e., the number of neurites per cell as well as the basal level of [Ca2+]i and rate of protein synthesis were comparable to those of control cells. The general calpain inhibitor calpeptin decreased the acrylamide-induced (0.5 mmol/L) neurite degeneration, determined as reduction in number of neurites per cell, from 52% to 17% as compared to control cells, which further supports the hypothesis that an increased [Ca2+]i plays a significant role for acrylamide-induced axonopathy.

Protection by glutathione of neutrophils against the toxic effects of peritoneal dialysis fluid.

The possibility of reducing the cytotoxic effect of heat-sterilized peritoneal dialysis (PD) fluid by addition of antioxidants/scavengers during incubation of titanium-adhering cells was investigated. Capillary blood from healthy donors was placed in drops on commercially available titanium pieces and incubated in a humidified chamber at 37 degrees C for 60min. After incubation the adherent polymorphonuclear leukocytes were immersed for 1-4h in PD-fluid, pH 7.4, containing 2.5% glucose with glutathione (GSH), superoxide dismutase, catalase or dithiothreitol (DTT). Luminol- or isoluminol-amplified chemiluminescence was used to measure the zymosan- and phorbol myristate acetate (PMA)-stimulated respiratory burst activity, as an indicator of the cytotoxicity of the PD-fluids. Heat sterilized PD-fluid had inhibitory effect on zymosan-induced respiratory burst and impaired both the extracellular and intracellular PMA-induced respiratory burst. Addition of GSH to the PD-fluid resulted in reduction of cytotoxical effects on the zymosan-induced and extracellular PMA-induced respiratory burst. The intracellular respiratory burst was not affected. The present results show that GSH and DTT have the ability to protect polymorphonuclear leukocytes against the cytotoxic effects of the PD-fluid by keeping the cell membrane in a reduced state.

Degradation in peritoneal dialysis fluids may be avoided by using low pH and high glucose concentration.

OBJECTIVE: When glucose is present in a medical fluid, the heat applied during sterilization leads to degradation. The glucose degradation products (GDPs) give rise to bioincompatible reactions in peritoneal dialysis patients. The extent of the degradation depends on a number of factors, such as heating time, temperature, pH, glucose concentration, and catalyzing substances. In the present work, we investigated the influence of pH and concentration in order to determine how to decrease the amounts of GDPs produced. DESIGN: Glucose solutions (1%-60% glucose; pH 1-8) were heat sterilized at 121 degrees C. Ultraviolet (UV) absorption, aldehydes, pH, and inhibition of cell growth (ICG) were used as measures of degradation. RESULTS: Glucose degradation was minimum at an initial pH (prior to sterilization) of around 3.5 and at a high concentration of glucose. There was considerable development of acid degradation products during the sterilization process when the initial pH was high. Two different patterns of development of UV-absorbing degradation products were seen: one below pH 3.5, dominated by the formation of 5-hydroxy-methyl-2-furaldehyde (5-HMF); and one above, dominated by degradation products absorbing at 228 nm. 3-Deoxyglucosone (3-DG) concentration and the portion of 228 nm UV absorbance not caused by 5-HMF were found to relate to the in vitro bioincompatibility measured as ICG; there was no relation between 5-HMF or absorbance at 284 nm and bioincompatibility. CONCLUSION: In order to minimize the development of bioincompatible GDPs in peritoneal dialysis fluids during heat sterilization, pH should be kept around 3.2 and the concentration of glucose should be high. 5-HMF and 284 nm UV absorbance are not reliable as quality measures. 3-DG and the portion of UV absorbance at 228 nm caused by degradation products other than 5-HMF seem to be reliable indicators of bioincompatibility.

Glucose degradation products in peritoneal dialysis fluids: how they can be avoided.

OBJECTIVES: A patient on peritoneal dialysis (PD) uses 3-7 tons of PD fluid every year. The result is considerable stress on the peritoneal tissue. Aspects of PD fluids that have been considered responsible for bioincompatibility are low pH, high osmolality, high glucose and lactate concentrations, and the presence of glucose degradation products (GDPs). However, the relative importance of each factor in PD fluid has so far not been investigated. Discovering their relative importance was the aim of the present study. METHODS: Two main methods for investigating biocompatibility were used in this study: cytotoxicity measured as in vitro inhibition of cell growth, and in vitro AGE formation measured as albumin-linked fluorescence. RESULTS: The two most important factors for determining in vitro bioincompatibility of PD fluids were the presence of GDPs, which caused both severe cytotoxicity and strong AGE promotion, and low pH, which induced severe cytotoxicity. CONCLUSIONS: The biocompatibility of PD fluids can be monitored through fairly simple in vitro methods such as cell proliferation and AGE formation. Bioincompatibility of PD fluids is caused mainly by the presence of GDPs and low pH. These findings correlate well with known clinical bioincompatibility.

Glucose degradation products in peritoneal dialysis fluids may have both local and systemic effects: a study of residual fluid and mesothelial cells.

OBJECTIVE: When peritoneal dialysis (PD) fluids are heat sterilized, glucose is degraded to carbonyl compounds. These compounds are known to interfere with many cellular functions and to promote the formation of advanced glycation end-products. However, little is known about what actually happens with glucose degradation products (GDPs) after infusion into the peritoneal cavity. The aim of the present study was to investigate possible targets for GDPs in the peritoneal cavity. DESIGN: In vitro reactions between residual fluid and GDPs were studied by incubating unused PD fluid with overnight dialysate. Confluent monolayer cultures of human mesothelial cells were used as a model to study the reactions of GDPs with the cells lining the peritoneal cavity. METHODS: Samples were analyzed, using high pressure liquid chromatography, for the presence of formaldehyde, acetaldehyde, 5-hydroxymethyl-2-furaldehyde (5-HMF), methylglyoxal, and 3-deoxyglucosone (3-DG). Cytotoxicity was determined as inhibition of proliferation of cultured fibroblasts. RESULTS: None of the analyzed GDPs reacted with overnight dialysate. Formaldehyde and methylglyoxal, in contrast to 3-DG and 5-HMF, reacted with the cultured mesothelial cells. CONCLUSIONS: Low molecular weight carbonyls such as formaldehyde and methylglyoxal most probably react with the mesothelial cells lining the peritoneal cavity, and could be responsible for the disappearance of these cells during long-term treatment. 3-Deoxyglucosone showed remarkably low reactivity and was most probably transported within the patient.

Toxicity testing of polymer materials for dialysis equipment: reconsidering in vivo testing.

The rationale for preclinical testing of plastic materials for medical devices is the protection of patients from leachable toxic substances. A controversial and costly part of this testing is the use of animal in vivo procedures. The objective of the present study was to analyse the importance of in vivo tests in relation to the decision to approve or not to approve materials for use. A total of 1044 plastic materials were analysed by employing chemical, in vitro and in vivo tests: 5708 in vivo tests were performed. In only one out of 2650 systemic injection tests on mice did a material fail. As that material also failed in chemical tests, the systemic injection test had no influence on the decision not to approve the material. Intradermal irritation (2644 tests), implantation (398 tests) and sensitivity (11 tests) procedures on rabbits and guinea-pigs were the other in vivo tests. However, in all except three cases, the same decision on whether or not to use a material would have been reached without any of these in vivo tests. Thus, little security appears to be gained from the in vivo tests, and abandoning them would save resources, probably without any additional risk.

Neurite degeneration in differentiated human neuroblastoma cells.

We have studied neurite degeneration in differentiated human neuroblastoma (SH-SY5Y) cells. The axonopathy-inducing potency in vitro of caffeine, diazepam, methylmercury chloride (MeHg), triethyltin chloride (TET) and acrylamide (ACR) was elucidated. After 72 hours of exposure the neurite degeneration was determined (by morphological quantification) as well as the total protein content (general cytotoxicity). The concentrations that caused 20% reduction of number of neurites (ND(20)) for ACR (250+/-36 mum) and TET (0.097+/-0.03 mum) was significantly lower, 63% and 35%, respectively (P

Toxicity of 20 Chemicals from the MEIC Programme Determined by Growth Inhibition of L-929 Fibroblast-like Cells.

The Multicentre Evaluation of In vitro Cytotoxicity (MEIC) programme is an international project aimed at evaluating the relevance of in vitro tests in predicting human toxicity. We have screened 20 chemicals (MEIC codes 31-50) from the programme, by using a cytotoxicity test based on growth inhibition of the mouse fibroblast-like L-929 cell line. Inhibition of cell growth was determined by the neutral red uptake method, which is well established and is used for screening the cytotoxicity of chemicals and plastics for pharmaceuticals and medical devices. The concentrations causing 50% inhibition of cell growth after a 72-hour exposure period varied from 3.1µM for hexachlorophene, to 1.4mM for caffeine. This is within the same range as results recently obtained with five other cell models. However, with some chemicals (chloroform, carbon tetrachloride and dichloromethane), no reliable results were obtained. These substances could not be dissolved in a reproducible way in any of the solvents used and, furthermore, they were highly volatile, which led to difficulties in maintaining the concentrations.

Increased amounts of C3a and the terminal complement complex at high dialysis blood-flow: the relation with dialysis efficiency.

To estimate the influence of blood-flow on complement generation and the relation with dialysis efficacy (KT/V) 10 patients underwent cuprophan hemodialyses for 6 h using low (200 ml/min) or high (400 ml/min) blood-flow (n = 40). Dialysis with high blood-flow compared to low induced a more rapid drop in leukocyte count and a more pronounced leukocyte rebound. Net generation of C3a (microgram/min) was also larger at all 15 measuring points during high blood-flow dialysis and there was significantly larger total generation of C3a (after 3 h p < 0.05, after 6 h p < 0.05) as compared to low blood-flow. Reaching a KT/V of 1.2-1.4 with high blood-flow induced a 40% larger total net generation of C3a and 21% more of TCC than with low blood-flow. Net generation of TCC (AU/min) was higher during the first and last parts of high blood-flow dialysis compared to low. In conclusion, increased blood-flow in order to increase dialysis efficiency (KT/V 1.2-1.4) results in larger net infusion of complement products with possible impact on immune response and complement-associated pathophysiological mechanisms.

Are aldehydes in heat-sterilized peritoneal dialysis fluids toxic in vitro?

OBJECTIVE: Chemical analysis of several brands of peritoneal dialysis fluids (PD fluids) has revealed the presence of 2-furaldehyde, 5-HMF (5-hydroxymethylfuraldehyde), acetaldehyde, formaldehyde, glyoxal, and methylglyoxal. The aim of this study was to investigate if the in vitro side effects caused by glucose degradation products, mainly formed during heat sterilization, are due to any of these recently identified aldehydes. DESIGN: Cell growth media or sterile filtered PD fluids were spiked with different concentrations of thealdehydes. MEASUREMENTS: In vitro side effects were determined as the inhibition of cell growth of cultured mouse fibroblasts or stimulated superoxide radical release from human peritoneal cells. RESULTS: Our results demonstrate that the occurrences of 2-furaldehyde, 5-HMF, acetaldehyde, formaldehyde, glyoxal, or methylglyoxal in heat-sterilized PD fluids are probably not the direct cause of in vitro side effects. In order to induce the same magnitude of cell growth inhibition as the heat-sterilized PD fluids, the concentrations of 2-furaldehyde, glyoxal, and 5-HMF had to be 50 to 350 times higher than those quantified in the PD fluids. The concentrations of acetaldehyde, formaldehyde, and methylglyoxal observed in the heat-sterilized PD fluids were closer to the cytotoxic concentrations although still 3 to 7 times lower. CONCLUSION: Since none of these aldehydes caused in vitro toxicity at the tested concentrations, the toxicity found in PD fluids is likely to be due to another glucose degradation product, not yet identified. However, it is possible that these aldehydes may still have adverse effects for patients on peritoneal dialysis.

On complement net generation in fast hemodialysis: are high blood flow rates bioincompatible?

Arterial and venous concentrations of complement (C3a) and leukocyte count were determined in 17 patients during 201 hemodialysis sessions by 12 different treatment modes executed in random order using cuprophan, hemophan, or polyamide membranes with small or large membrane areas and high blood flow (Qb) (400 mL/min) for 2 hours or low Qb (200 mL/min) for 4 hours. With all membrane types, the number of leukocytes was significantly higher after 120 minutes of dialysis and by the end of treatment at high Qb compared with low Qb. C3a concentrations (microgram/mL) in the arterial and venous blood lines were significantly higher during cuprophan dialysis compared with hemophan and polyamide dialyses (P < 0.001). In addition, the net generation of C3a (microgram/min) was significantly higher during hemodialysis with cuprophan compared with hemophan and polyamide (P < 0.001). After 2 hours at high Qb for each of the three membranes, the net generation of C3a was significantly higher compared with low Qb (P < 0.05 for all comparisons). Possible reasons for the increase in the net generation of C3a (microgram/min) at high Qb are less protein deposition on the membrane at high Qb or the fact that the protein coat is stripped off in the dialyzer, thereby recreating a less biocompatible surface. Hemodialysis at high Qb may thus be less biocompatible than dialysis at low Qb.

Development of toxic degradation products during heat sterilization of glucose-containing fluids for peritoneal dialysis: influence of time and temperature.

OBJECTIVE: Fluids for peritoneal dialysis (PD) cause cytotoxic reactions in many different in vitro systems. The low pH, the high osmolality of the fluids, and the glucose degradation products formed during heat sterilization have been considered responsible. In the present study, we investigate the influence of temperature and time during heat sterilization of PD fluids and glucose solutions on glucose degradation and cytotoxicity of the solutions. DESIGN: Ampoules containing PD-fluid or glucose solution were heated in an oil bath to predetermined F0 values (combinations of time and temperature giving equal energy/bacterial lethality). Cytotoxicity of the solutions was measured as growth inhibition of cultured L-929 fibroblasts. Glucose degradation was measured as UV absorbance at 228 and 284 nm. RESULTS: The same general pattern was seen in both PD fluid and glucose solution. Cytotoxicity decreased from 90% to 15% when the sterilization temperature was increased from 115 degrees to 140 degrees C and concomitantly the length of time shortened in order to maintain equal bacterial lethality. Under the same conditions, degradation products, measured as UV absorbance at 284 nm, decreased from 0.2 to 0.02. CONCLUSION: To minimize the development of cytotoxic breakdown products, high temperatures over short periods of time should be used to heat-sterilize PD fluids. Even as small an increase as 5 degrees C at around 120 degrees C will improve the quality of the solutions.

Individual differences in biocompatibility responses to hemodialysis.

There are very few reports in the literature on individual differences in the response to dialysis treatment. We studied the influence of the individual patient, dialysis membrane quality, blood-flow (Qb) and surface area on leukocyte activation and complement generation (C3a) during 234 hemodialysis treatments using Cuprophan (CU), hemophane (HE) and polyamide (PA) dialyzers. The most common reaction was a decrease in leukocyte count and an increase in C3a after 15-minutes of treatment. Leukocyte overshoot by the end of dialysis was observed at high Qb for all three membranes but at low Qb only during CU treatments. The reaction patterns were influenced by the quality of the membrane, area and Qb. Analysis of each individual patient showed for a large number of treatments reaction patterns corresponding to those described in the literature. However, some patients reacted differently. In four patients (20%), the nadir in leukocyte count and maximum in C3a concentration was reached considerably later during CU-dialysis. Three patients were devoid of pronounced early leukocyte response but presented with the late overshoot during CU-dialysis. Three other patients reacted with an early drop in leukocyte count and a rapid increase in C3a generation during PA treatments but not during HE treatments. Three other patients reacted vice versa. A particular mode of dialysis treatment may thus be biocompatible for some patients but not necessarily for all. In the case biocompatibility is desired the individual response to the particular dialysis mode needs to be identified. The underlying mechanisms warrant further studies.

Patient related factors leading to slow urea transfer in the body during dialysis.

We studied the trans compartmental speed of urea transfer by comparing concentration changes of blood urea nitrogen to mass changes of urea during 80 dialyses in six patients. The speed of urea transfer was studied as a dependent factor of 15 patient characteristics: age; gender; fluid overload; and pre and post values of and change in pulse and temperature, calcitonin gene related peptide, and mean arterial blood pressure. Concentration changes in blood urea nitrogen were measured as pre and post dialysis urea concentration, the total urea in the body was measured by pre dialysis urea and tritium total body water determinations, and the actual mass of urea removed by collecting all dialysate. As a mean, concentration of blood urea nitrogen fell 54% but the mass urea removed was only 40% for a mean ratio of 1.41. Nine factors were associated with the speed of urea transfer. Patients with fast transfer had more normal fluid balance, a normal pulse rate, body temperature, calcitonin gene related peptide values, and blood pressure both before and after dialysis. The patients with a slower transfer of urea had a lower blood pressure before and after dialysis and a more labile pulse rate and body temperature. Patients with unpredictable urea transfer were the most edematous and had the most labile blood pressure. It is important to know which patients have slow urea transfer. Such patients should not be treated by fast dialysis, and those with the slowest rates may do particularly well on continuous ambulatory peritoneal dialysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Toxicity testing of polymer materials for dialysis equipment: is there any need for in vivo testing?

In an earlier work, slightly more than 650 plastic materials, intended for use in medical devices, were tested with a battery of chemical, as well as in vitro and in vivo biological tests. An analysis showed that only a limited number of the tests used were actually necessary to obtain the same pass or fail decision as that obtained using the full test battery. This prompted us to prescreen all new materials with a small test battery consisting of the two most discriminating chemical tests and an in vitro cell growth inhibition test. The present work is a report of our findings after testing another 155 materials using this prescreen system. For each single one of the 155 tested materials the same decision on whether or not to use the material in the intended medical device would have been reached without any in vivo testing. In no single case in a total of 851 in vivo tests did an eluate that had passed the in vitro cell test give rise to a reaction in vivo. Thus, among the tests on living systems, the cell test alone seems to be sensitive enough to provide sufficient information. Nothing appears to be gained from the in vivo animal tests. However, some of the materials that passed the prescreening tests later failed in one or several of the chemical tests. Both nonspecific chemical tests and tests for specific molecules seem to detect undesirable levels of leachable substances not detected by the prescreening system. Therefore these tests should not be abandoned. Abandoning unnecessary in vivo testing, on the other hand, would save considerable costs.

Heat sterilized PD-fluids impair growth and inflammatory responses of cultured cell lines and human leukocytes.

We have recently demonstrated that commercial PD-fluids inhibit the growth of a cultured mouse fibroblast cell line. Toxic substances produced during heat sterilization were believed to be the probable cause of the growth inhibition. The aim of the present study was to investigate if heat sterilized PD-fluids affect other cell types and other cellular functions than the growth of fibroblasts. The effect of three commercially and one laboratory made PD-fluid on cell growth of a mouse macrophage cell line (RAW) and a human neuroblastoma cell line (SH-SY5Y) was examined. The influence on stimulated release of tumour necrosis factor alpha (TNF alpha) from the macrophage cell line and stimulated superoxide generation from freshly prepared human leukocytes were also investigated. Compared to the filter sterilized PD-fluid, we found that heat treated PD-fluids significantly inhibited the growth of the two cell lines and impaired the stimulated release of TNF alpha and superoxide radicals. These results demonstrate that heat sterilization of PD-fluids produces substances that are cytotoxic regardless of the cell species, the cell type or the cell function tested.

Beta 2-microglobulin generation and removal in long slow and short fast hemodialysis.

We studied the influence of different modes of hemodialysis (HD) on plasma levels of beta 2-microglobulin (P-beta 2-m) and its correlation to changes in leukocyte count, complement activation (C3a), and elastase generation. The influence of dialyzer membrane, membrane surface area, duration of treatment, and blood flow was analyzed with respect to post-HD levels of P-beta 2-m. Twenty patients underwent 12 modes of bicarbonate hemodialysis in random order (n = 252) using three different membranes (Cuprophan [CU], hemophan [HE], or polyamide [PA], two dialyzer areas, and fast (400 mL/min) or slow (200 mL/min) blood flow (Qb) for 2 or 4 hours, respectively. All dialysate was collected and beta 2-m was analyzed (D-beta 2-m). After correction for hemoconcentration, P-beta 2-m concentrations were found to have decreased significantly during treatment with all three membranes (CU, 0.9 +/- 0.3 mg/L, P = 0.002; HE, 1.2 +/- 0.3 mg/L, P < 0.001; and PA, 8.3 +/- 0.3 mg/L, P < 0.001). Elimination of P-beta 2-m was influenced by type of membrane (P < 0.001) and ultrafiltration volume (P = 0.0019) but not by membrane area or Qb. The largest reduction in P-beta 2-m (-10.4 mg/L) was achieved by the following treatment combination: PA membrane, large dialyzer area, and low Qb for 4 hours. P-beta 2-m decreased more during PA dialysis at low Qb for 4 hours (-9.9 +/- 0.5 mg/L) than during high Qb for 2 hours (-6.8 +/- 0.5 mg/L, P < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Spatial memory and stereotypic behaviour of animals in radial arm mazes.

The symmetric radial arm maze, described by Olton in 1976, has developed into an important tool for the study of spatial memory. In a typical test an animal is placed in the centre of the maze, which contains some small piece of food at the end of each arm. The sampling behaviour of the animal is then recorded. In such studies the score (number of choices of arms which still contain food) of the animal is normally compared with the score of an imaginary animal which changes arms entirely at random. In a new method of analysis the non-random score of the animal is split into two parts, one depending on memory and one on stereotypic choice behaviour. Even mild departures from randomness are shown to alter considerably the expected 'random' score in an eight-armed maze. The part of the score claimed to depend on memory was shown to increase when the animals learn to search the maze, the stereotypic part did not. The general effect from stereotypic choice behaviour is shown to result, in most animals, in an increase in the total score. In an eight-armed maze this increase may amount to more than 20% of the total non-random score, even in a well-trained animal. The effect is less pronounced in mazes with 16 arms. It has been proposed that hippocampal lesions produce a stereotypic behaviour. We propose, based on our analysis, that the stereotypic behaviour is not produced but revealed by hippocampal lesions which destroy almost completely the memory-guided behaviour masking the stereotypic behaviour in the intact animal.