Mutagenesis Advance Access originally published online on November 13, 2007
Mutagenesis 2008 23(1):27-33; doi:10.1093/mutage/gem034
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Assessment of the genotoxicity of trichloroethylene and its metabolite, S-(1,2-dichlorovinyl)-L-cysteine (DCVC), in the comet assay in rat kidney
Syngenta Central Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire, SK10 4TJ, UK
Trichloroethylene (TCE) has been reported to give a small, but significant, increase in renal tumours in the rat. These tumours were always associated with nephrotoxicity which is most likely caused by the metabolism of TCE to S-(1,2-dichlorovinyl)-L-cysteine (DCVC) which accumulates in the proximal tubules. The genotoxicity of TCE and DCVC have been evaluated in vivo using the comet assay to assess DNA breakage in the proximal tubules of rat kidneys. Rats were exposed to TCE by inhalation or to DCVC by oral gavage at dose levels in excess of those which produced effects in long-term bioassays. Cell suspensions were produced from proximal tubules isolated from the kidneys of treated rats and the level of DNA damage assessed in these cells using the pH >13 comet assay. In vitro and in vivo positive controls were included and demonstrated the sensitivity of the assay. TCE gave a clearly negative response in the assay at all dose levels as did DCVC at the 16-h sampling time and at the 2-h sampling time with the lower dose level. At the 2-h sampling time following administration of DCVC at the higher dose level (10 mg/kg), there was limited evidence of DNA damage in a small number of animals, but this was considered insufficient to indicate a positive response in this assay. These data support an overall conclusion, based on these and other published data, that the renal tumours seen in bioassays are non-genotoxic in origin.
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Introduction |
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Trichloroethylene (TCE) is widely used in cleaning and degreasing and is also a chemical intermediate in the production of fluorochemicals. Low incidences of renal tumours have been reported in several lifetime studies in which rats were exposed to TCE either by gavage or by inhalation (1
). A minor metabolic pathway of TCE (<0.05% of the dose) has been reported which leads to the formation of S-(1,2-dichlorovinyl)-L-cysteine (DCVC), a nephrotoxin in the rat and a bacterial mutagen in Salmonella typhimurium (2). The balance of evidence suggests that rat kidney tumours are induced by TCE as a result of a prolonged damage and repair cycle (3). However, direct interaction between reactive products of TCE or DCVC metabolism and DNA might indicate that risks of kidney cancer exist below dose levels of TCE that induce frank renal toxicity. A number of epidemiological studies of TCE-exposed workers failed to demonstrate a link between exposure and renal tumours, although there is evidence that renal cell cancer may be associated with unusually high occupational exposures (4), a finding compatible with the hypothesis that prolonged kidney damage is causative. This study was designed to establish whether genotoxicity is demonstrable in rat kidney following administration of TCE in vivo at high dose levels or following oral doses of DCVC leading to much greater levels in the kidney than could be generated from TCE itself.
In cancer bioassays, TCE is considered to have caused a very low incidence of renal tubular cell tumours in rats, predominantly males, following inhalation or oral exposures (3). Severe nephrotoxicity has been observed in all rat bioassays (3,5). Not all rat strains tested showed development of renal tumour and kidney tumours were not seen in any of a number of mouse studies (3,5). In a study by Maltoni et al. (6) in which Sprague–Dawley rats were exposed daily to TCE atmospheres of 0, 100, 300 and 600 ppm for 104 weeks and then followed until death, there were no renal tumours in the control or 300-ppm groups. A single (0.3%) renal tubular cell adenoma was found in the 100-ppm group and a total of four (3.1%) adenocarcinomas were present in the 600-ppm group. Among other rat strains, the male Fischer 344 rat was found to be susceptible to the development of kidney tubular tumours following oral administration of TCE in a 2-year bioassay (7,8): Two (4%) tubular adenomas were seen in the 500 mg/kg-day group and three (6%) tubular adenocarcinomas in the 1000 mg/kg day and no kidney tumours were seen in the control group. Adjusted for survival, the incidence in the higher dose group was 18.8% and reduced survival was considered to be due to kidney toxicity.
DCVC has not been tested in a lifetime carcinogenicity bioassay. However, renal tumours were not induced in rats receiving an oral dose of 10 mg/kg-day DCVC for 46 weeks followed by observation to 87 weeks. Kidney toxicity was seen in this study (9) and the dose was estimated to be three orders of magnitude greater than the DCVC generated from TCE dose levels associated with renal tumourigenicity (5). Glutathione conjugation of TCE followed by metabolism by the enzymes of mercapturic acid pathway produces DCVC, which is accumulated in the proximal tubules. Cleavage of DCVC by β-lyase, which is present at high concentrations in this part of the nephron, produces a highly reactive thioketene (10–12). In addition, DCVC exerts marked specific nephrotoxicity in the kidney cortex in vivo, which is in line with the massive nephrotoxicity observed with TCE in the long-term National Toxicology Program study (13–16). Overall, the evidence suggests that TCE induction of kidney tumours in the rat is the result of a prolonged damage and repair cycle rather than direct interaction of DCVC with DNA (3). However, it is important to establish whether genotoxicity results from TCE administration since this could indicate that a finite risk of kidney tumour induction could exist below dose levels causing nephrotoxicity. In this study, the genotoxicity of both TCE and DCVC have been evaluated in the comet assay in proximal tubular cells following exposure of rats in vivo. TCE was examined at dose levels in excess of those shown to produce kidney tumours in a 2-year study and DCVC was examined at dose levels higher than those produced by exposure to TCE. The sampling times used were designed to maximize the detection of any genotoxic effects.
The in vivo comet assay (17–22) can be used to investigate the genotoxicity of chemicals. The principle of the comet assay is the migration of DNA through an agarose matrix under electrophoretic conditions. When viewed under a microscope, a cell has the appearance of a comet, with a head (the nuclear region) and a tail containing DNA fragments or strands. Among the various versions of the assay, the alkaline (pH13) method enables detection of the broadest spectrum of DNA damage and was used for this series of experiments. It can detect double- and single-strand breaks, alkali-labile sites that are expressed as single-strand breaks and single-strand breaks associated with incomplete excision repair.
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Materials and methods |
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Chemicals
TCE (99.5% pure) was obtained from BDH, Lutterworth, UK. DCVC was synthesized in house with a purity of >95%. N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and N-nitrosodimethylamine (N-DMA) were both obtained from Sigma, Gillingham, UK.
Animals and husbandry
Male CD (Sprague–Dawley) rats (273–335 g for Experiment 1 and 230–250 g for Experiment 2) were used for the study as male animals are known to be sensitive to the toxicity induced by these materials (19,20). The animals were supplied by Charles River, Margate, UK. On arrival, the rats were group housed and given food (Rat and Mouse No. 1 Maintenance diet, Special Diets Services) and water ad libitum. Animals were housed in animal rooms which were maintained within a temperature range of 19–25°C and within a relative humidity range of 30–70%. Lighting was controlled to provide 12 h artificial light followed by 12 h darkness. The animal room was under positive pressure with respect to the access corridor and had at least 15 air changes per hour. Animals received no food or water during the exposure period. All experiments were conducted under the provisions of the UK Animals (Scientific Procedures) Act, 1986.
Experimental design
During the production, storage and use of TCE, it can evaporate into the atmosphere and has been identified as a common contaminant of air. Exposure of the test animals to TCE by inhalation was therefore considered relevant to assess the genotoxicity of TCE. Dosing of DCVC was by oral gavage in order to achieve an appropriate dose level.
In the first experiment, groups of animals (5 animals per group) were exposed by inhalation to a range of concentrations of TCE or air control (6 h per day, 5 days), or received a single oral dose of DCVC or vehicle control. In a second experiment, groups of animals (5 animals per group) received a single oral dose of DCVC, vehicle control, or N-DMA. Animals exposed to TCE were killed immediately after the fifth exposure. Those dosed with DCVC or N-DMA were killed either 2 or 16 h after dosing. Kidney proximal tubular cells were prepared immediately after sacrifice and used to prepare slides for the evaluation of comet formation. Subsamples of cells isolated from vehicle control groups were treated in vitro with MNNG as an in vitro positive control.
The maximum exposure concentration used for TCE (2000 ppm) was 4-fold higher than that known to cause kidney tumours in 2-year studies (1,3,5). Lower concentrations of 1000 and 500 ppm were included in the study design. The highest dose of DCVC (10 mg/kg) was equivalent to approximately three orders of magnitude higher than the amounts of DCVC formed following exposure of rats to TCE. A lower dose level of DCVC (1 mg/kg) was also included. Groups of animals exposed to the two higher concentrations of TCE showed transient clinical signs only during exposure periods, but were fully recovered by the next day in each case. Rats dosed with DCVC or N-DMA were clinically normal throughout the experiment.
Dosing regime for TCE
Animals were exposed in short-term whole-body chambers which consisted of a circular PERSPEX section subdivided into 10 equal segments by wire mesh partitions. The chamber was covered by an aluminium lid and stood on an aluminium base plate. The animals were exposed for 6 h on each day of exposure. Temperature and relative humidity in the chamber were measured approximately every hour using a hand-held meter. Airflow through the system was monitored frequently and recorded approximately every 30 min. Atmosphere samples were taken hourly during each exposure period to assess the achieved concentrations of TCE. Control atmospheres and room air were also sampled and analysed. The mean analysed atmospheric concentrations of TCE were 484, 1035 and 1749 ppm for the groups exposed to nominal concentrations of 500, 1000 and 2000 ppm, respectively. No TCE was detected in control or room samples.
Dosing regime for DCVC and N-DMA
Rats were given a single oral dose at a dosing volume of 10 ml/kg. The dose level of N-DMA used was 20 mg/kg.
Isolation of cells
Proximal tubules were isolated from rat kidneys by the method of Vinay et al. (23) except that the percoll gradient was 35% and not 50% as described. Briefly, the kidney medulla was dissected out, the cortex incubated in buffer with collagenase and the cell suspension fractionated on a percoll gradient. The fraction containing proximal tubular cells was identified by microscopy and the viability of a sample of each of the cell preparations was estimated by trypan blue exclusion. Those cells excluding trypan blue were considered to be viable.
In vitro positive controls
A sample was taken from the cells isolated from the vehicle control animals and treated in triplicate with a final concentration of 5 µg/ml MNNG for 30 min after which they were centrifuged, the supernatant removed and the cells re-suspended in fresh medium. These cell suspensions were then treated in the same way as all other cell suspensions.
Slide preparation
Three slides were prepared from each cell suspension (a cell suspension was prepared from each animal and each positive control treatment) as follows: Each slide was pre-coated by adding 1 ml of 1% normal melting agarose to a fully frosted slide, scraping off and allowing to air dry. Eighty-five µl of 0.5% normal melting agarose was then added, coverslipped, gelled on ice and the coverslip removed. A further layer of 65 µl of 0.7% low melting agarose containing 2 x 104 cells was added, coverslipped, gelled on ice and the coverslip removed. Finally, a top layer of 75 µl 0.7% low melting agarose was added, coverslipped, gelled on ice and the coverslip removed.
The slides were then placed in lysis buffer for 1 h before being removed to an electrophoresis tank containing electrophoresis buffer at 4°C for 20 min. Electrophoresis in the same buffer at 4°C for 40 min at 25 V (1 V/cm) and 380 mA was followed by incubation in neutralization buffer for 5 min. The slides were stored in the dark at 4°C in humidified boxes until scored as below.
Slide analysis
The slides were stained with 20 µg/ml ethidium bromide solution in water, wet mounted and evaluated under 20x magnification using a fluorescence microscope. Data were collected from 150 cells per animal (normally from 50 cells per slide and three slides per animal). The first 50 clearly defined, non-overlapping cells per slide, starting at the top left of the slide, were evaluated using a Perceptive Instruments "Comet Assay II (V. 1.03)" image analysis system. Data collected from each cell included tail moment, tail length, head length, percent head DNA and percent tail DNA.
Data evaluation
A mean value for each parameter per animal was calculated; individual animals or cultures were the experimental units. From the individual animal data, mean and standard variation values were calculated for each treatment group. The primary measure of DNA damage was percent tail DNA (22). Group mean values for each parameter were compared to appropriate vehicle control values by single-sided Student's t-test. For a chemical to be considered to have given a positive response in this assay, it should show a dose-related change in the defined measurement between the control and test groups at least at a single sampling time or a change in the defined measurement in a single dose group at least at a single sampling time (24). Consistency of effect within a group and across experiments is also an important factor when assessing the data.
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Results |
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Cells isolated from most animals were of high viability (>70%) after the isolation procedures except for the cells from a single animal in the second experiment which showed a viability of 52%. In the first experiment, no cells from one of the 1000-ppm TCE animals and one of the DCVC 1 mg/kg, 16-h sampling time animals were present following the isolation procedures for technical reasons.
In the first experiment (Tables I–III), no significant increases in percent tail DNA were seen for any of the groups treated with TCE or 16 h after treatment with DCVC. In the groups of animals sampled 2 h after treatment with DCVC, isolated statistically significant increases in percent tail DNA were seen but there was no evidence of a dose response.
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In the second experiment (Tables IV and V), statistically significant increases in percent tail DNA were seen in groups of animals sampled both 2 and 16 h after treatment with 10 mg/kg DCVC. In the group of animals sampled 16 h after treatment with DCVC, these increases were small and considered not to be of biological significance. No increase in tail length was observed to support the other apparent changes in the comet tail and head parameters. DCVC is therefore considered not to have induced any consistent or significant changes to the comet parameters at the 16-h sampling time. This conclusion is supported by the results of the first experiment in which no changes to parameters measured were observed at the 16-h sampling time at either 1 or 10 mg/kg dose level.
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The group of animals sampled 2 h after treatment with 10 mg/kg DCVC showed statistically significant increases in the group mean values for percent tail DNA. Examination of the individual animal data (Table IV and Figure 1), however, shows that the changes in percent tail DNA are primarily due to increases in two out of the five animals, one of these having a viability of the cell preparation of only 52%. If these two animals are excluded from the analysis, then no statistically significant increase in percent tail DNA is observed for this group. No such increases in these parameters were observed in the first experiment with DCVC at the same dose level or sampling time. It should also be noted that the group mean values observed for percent tail DNA and tail moment with DCVC treatment at the 2-h sampling time in this study are within the control values for the parallel experiment examining the 16-h time point. It is therefore considered that the changes observed with DCVC at the 2-h sampling time indicate limited evidence of DNA damage in individual animals. Taken with the results of the first experiment, these data are considered insufficient to indicate a positive response for DCVC in this assay. The 10 mg/kg DCVC dose level used in these studies was approximately one half of that (25 mg/kg) known to cause cytotoxicity to the rat kidney (25
) and is therefore considered to be a maximal dose for this assay. It is noteworthy that a dose of 10 mg/kg DCVC is approximately 1000-fold higher than the amount which is formed by the metabolism of TCE at cancer bioassay dose levels, typically 500–600 ppm (1,3,5).
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Statistically significant increases in percent tail DNA were seen in all the in vitro and in vivo treated positive control groups.
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Discussion |
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Considering the data together from both experiments, it can be concluded that the assay is capable of detecting DNA damage (as measured by comet formation) in rat kidney proximal tubules following treatment both in vitro (with MNNG) and in vivo (with N-DMA). Under the conditions of this test, TCE did not induce DNA damage in rat kidney proximal tubules in vivo. DCVC (1 and 10 mg/kg) induced no significant DNA damage in rat kidney proximal tubules at the 16-h sampling time or after 1 mg/kg DCVC at the 2-h sampling time. At the 2-h sampling time following administration of DCVC at 10 mg/kg, there was limited evidence of DNA damage in a small number of animals (2 out of 10), but this was considered insufficient to indicate a positive response in this assay.
These results stand in contrast to the findings of Robbiano et al. (26) which showed DNA damage and increased micronuclei in the rat kidney 20 h following a single dose (half LD50, 3591 mg/kg body weight) of TCE. These investigators also reported positive responses for DNA damage and induction of micronuclei for human and rat kidney cells in vitro following incubations with TCE of 20 h (DNA damage) and 48 h (micronuclei). The concentrations of TCE required to yield positive results in the in vitro experiments were extremely high and outside any conceivable level achieved through in vivo exposure. The relationship between the results of Robbiano et al. (26) and effects of lower repeated doses is not clear, and it is possible that a mechanism operates at extremely high levels, possibly related to the direct solvency of TCE, that is not effective at lower dose levels. The results obtained here also contrast with those of Jaffe et al. (27). These research workers studied DNA single-strand breaks in rabbit kidney employing DCVC administered by intraperitoneal and intravenous injections, by perfusion of isolated kidneys and by exposure of isolated tubules in vitro. In all exposure modes, increases in single-strand breaks were observed at dose levels that caused tubular damage. The level of DCVC achieved in the rabbit kidney appears to have been very high and the single-strand breaks may have associated with the severe toxicity to renal tubular cells rather than a true genotoxic response.
A recent review of TCE and renal toxicity and renal cancer (3) concluded that tumours following repeated exposures to TCE were likely to be the result of direct cytotoxicity and sustained tubule cell regeneration, a non-genotoxic mechanism. Supporting evidence for a non-genotoxic mechanism for TCE induction of rat kidney tumours has been reported in a sophisticated recent study (28) which explored the ability of TCE and DCVC to induce pre-neoplastic kidney lesions in a susceptible rat strain, the Eker rat. Unlike positive controls, a 13-week oral administration of TCE at up to 1000 mg/kg body weight (5 days per week) did not induce tumours or pre-neoplastic lesions. High concentrations of DCVC in vitro which reduced cell survival by 50% were able to transform kidney epithelial cells but no carcinogen-specific mutations were found in the kidney tumour suppressor genes von Hippel-Lindau (VHL) or tuberous sclerosis 2 (Tsc-2). The authors concluded that kidney carcinogenicity mediated by TCE may be secondary to continuous toxic injury and sustained regenerative cell proliferation. The results of the study reported here also support the conclusion that the renal tumours seen in the bioassay are non-genotoxic in origin.
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Halogenated Solvents Industry Alliance, Inc.; European Chlorinated Solvents Association.
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* To whom correspondence should be addressed. Tel: +44 0 1625 515461; Email: phil.clay{at}mac.com
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Received on October 19, 2006; revised on July 12, 2007; accepted on August 4, 2007.
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