Mutagenesis, Vol. 18, No. 3, 283-286,
May 2003
© 2003 UK Environmental Mutagen Society/Oxford University Press
Lack of clastogenic activity of aniline hydrochloride in the mouse bone marrow
Health Assessment, Syngenta CTL, Alderley Park, Macclesfield, Cheshire SK10 4TJ, UK
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Abstract |
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Aniline has been reported to be positive in the mouse bone marrow micronucleus test. This finding is inconsistent with its lack of carcinogenicity in this species. Micronuclei can arise by mechanisms that do not involve direct interaction with DNA, e.g. induction of aneuploidy or stimulation of erythropoiesis. However, clastogenic materials would be expected to demonstrate an increased level of chromosomal damage in dividing precursor erythroblasts. In the present study we have investigated the ability of aniline HCl to induce chromosome aberrations in bone marrow metaphase cells. No evidence of clastogenicity was observed in this study. This suggests that the activity seen in earlier micronucleus assays may have arisen by a mechanism not involving direct DNA interaction. Aniline is known to be toxic towards the erythropoietic system and the possibility exists that micronuclei may be produced as a result of this toxicity.
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Introduction |
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Aniline is produced globally on a large scale (
2 800 000 metric tonnes in 2000). Most aniline produced (80%) is used in the manufacture of methylene di-p-phenylene isocyanate, a component of rigid urethane foam. Other uses of aniline include the manufacture of rubber processing chemicals and the production of agrochemicals and dyestuffs. Overall the potential for human exposure is high. Whilst IARC classifies the available evidence for the human carcinogenicity of aniline as inadequate (IARC, 1987
), the results from rat studies indicate there is still cause for concern.
A major target for aniline toxicity in the rodent is the erythrocyte. Aniline induces methaemoglobinaemia, with consequent increase in the level of free iron, which in turn leads to damage to the spleen. In the NTP bioassays aniline HCl is reported as non-carcinogenic in the mouse but in the rat it produced fibrosarcomas, sarcomas and haemangiosarcomas of the spleen and peritoneal cavity (NTP, 1978). The overall IARC classification of aniline is that there is limited evidence for carcinogenicity to animals (IARC, 1987).
Aniline has been tested in a range of in vitro genotoxicity assays. It has been shown to be consistently negative in the standard Salmonella typhimurium gene mutation assay (see for example Haworth et al., 1983; Dunkel et al., 1985). Aniline has been tested in a wide range of cultured mammalian cell assays using a variety of end-points. Positive responses have been reported in a number of these assays, including the L5178Y Tk+/- assay (Amacher et al., 1980), in vitro cytogenetics assay in CHL cells (Ishidate, 1983) and in vitro sister chromatid exchange assay in Chinese hamster DON cells (Abe and Sasaki, 1977). Reported positive responses have mainly been observed at higher (>1000 µg/ml) concentrations of aniline.
In vivo assays have concentrated on clastogenic end-points, in particular the induction of micronuclei in rodent bone marrow. Witt et al. (2000) reported a positive response in the mouse in a peripheral blood micronucleus test following 90 days dietary administration of aniline HCl. However, the increases observed were very small and do not support a clear conclusion of positive in the assay. George et al. (1990) report positive findings in a rat bone marrow micronucleus test following a single oral dose of aniline HCl. These authors used a staining method that is not DNA-specific, therefore, the possibility exists that observed micronuclei may be non-DNA-containing bodies. A positive response in the mouse bone marrow micronucleus test was first reported by Westmoreland and Gatehouse (1991) following a single administration of aniline HCl using both oral and i.p. routes. Ashby et al. (1991) investigated the i.p. route further and found a clear response in the mouse following two administrations of aniline 24 h apart. The lack of species specificity for the reported clastogenic activity is in contrast to the clear species specificity for carcinogenicity demonstrated in the NTP studies.
A positive response in the rodent bone marrow micronucleus test is usually taken as prima facie evidence of genotoxic activity in vivo. However, the possibility of micronuclei arising by a non-genotoxic mechanism should not be discounted. One such mechanism, which has been reported in the literature to increase the incidence of micronuclei, is stimulation of erythropoiesis: either by administration of erythropoietin (Suzuki et al., 1989), splenectomy, induction of haemolysis (Steinheider et al., 1985) or removal of blood (Steinheider et al., 1985; Hirai et al., 1991). An established target for the toxicity of aniline is the erythropoietic system. Therefore, the possibility exists that micronuclei observed in the mouse may not have arisen through a genotoxic mechanism but be a result of toxicity towards the erythropoietic system. A clastogenic material would be expected to produce chromosome aberrations in the dividing precursor cells of the erythrocytes. The lack of such damage would be strong evidence that any micronuclei observed had arisen as the result of a non-genotoxic mechanism.
The study reported here was designed to investigate whether aniline HCl is clastogenic in the mouse bone marrow by examining metaphase cells for aberrations. The design of the experiment was based on that used by Ashby et al. (1991), in this laboratory, for the micronucleus assay, as they reported the largest observed increase in the frequency of micronucleated erythrocytes. The same strain and sex of mice was used and the animals received two i.p. injections of aniline separated by 24 h. Both reports of positive responses in the mouse micronucleus test had a clear response at 24 h and no response at 48 h. Maturing mouse erythroblasts eject their nuclei ~6 h after the last cell division (Jenssen and Ramel, 1978). Therefore, in the present studies we used sampling times covering the period 16–24 h after the second dose, as this is when any underlying chromosomal damage would be expected to be observed in the erythroblasts. This time period also covers 1.5 cell cycles.
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Materials and methods |
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Chemicals
Aniline HCl was supplied by BASF AG as a white crystalline solid with a stated purity of 100%. Cyclophosphamide (ASTA Medica) was used as a positive control. Both materials were dissolved in sterile double deionized water.
Animals and husbandry
Male CBA mice in the age range 13–15 weeks were used for the study. The animals were supplied by Harlan (Bicester, Oxon, UK). On arrival the mice were housed in groups of 5 animals/cage and given food (Rat and Mouse No. 1 Maintenance Diet; Special Diets Services) and water ad libitum. Animals were maintained in accordance with the Code of Practice for the Housing and Care of Animals Used in Scientific Procedures issued by the UK Home Office.
Animal treatment
Groups of five, randomly allocated, non-fasted, male mice were dosed with the vehicle control, positive control or the test substance. Animals in the vehicle control and test substance groups received two i.p. administrations separated by 24 h. Animals were killed and the bone marrow sampled 16, 20 and 24 h after the second dose. The positive control group received a single oral administration (65 mg/kg) and the animals were killed and the bone marrow sampled 24 h later. The dose levels of aniline HCl used were 380, 300 and 220 mg/kg, expressed in terms of aniline base.
Approximately 2 h prior to scheduled termination, the mice were dosed via the i.p. route with colchicine at 3 mg/kg. The animals were killed by overexposure to halothane Ph. Eur. (Fluothane; AstraZeneca). Animals were examined internally for abnormalities of organs/tissues.
Slide preparation
For each animal both femurs were removed, stripped clean of muscle and both ends were removed. The bone marrow was aspirated from the femurs using Hank’s balanced salt solution (containing colchicine at 100 µg/ml). The cells were centrifuged and the supernatant discarded. The cells were resuspended in 0.075 M potassium chloride solution and left at room temperature for ~20 min. The samples were then centrifuged and the supernatant discarded. The samples were fixed in freshly prepared 3:1 methanol/glacial acetic acid fixative at room temperature. After three subsequent changes of fixative, metaphases were prepared by dropping the cell suspension onto labelled clean, moist microscope slides. Flame drying of the slides was used to aid spreading of the metaphases.
The slides were stained in a pre-filtered 10% solution of Giemsa stain in buffered deionized water (pH 6.8) for 5 min. The slides were rinsed in water, air dried and mounted with glass coverslips in DPX.
Slide analysis
Slides were examined to determine that they were of suitable quality and were coded prior to analysis. One hundred cells in metaphase were analysed from each animal, according to the principles of the criteria recommended by Scott et al. (1990). All cells analysed had 2n ± 2 centromeres. The mitotic index was determined by examining 1000 cells/culture and calculating the percentage of cells in metaphase.
Data evaluation
The percentages of aberrant metaphases were calculated for each treatment scored, excluding cells with only gap-type aberrations. The proportion of aberrations, excluding gaps, was considered by analysis of variance following the double arcsine transformation of Freeman and Tukey (1950). Each treatment group mean was compared with the control group mean at the corresponding sampling time using a one-sided Student’s t-test, based on the error mean square in the analysis.
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Results |
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Reactions to treatment were observed for animals dosed with aniline HCl, including: decreased activity; tremors; eyes closed; discharge from eyes; reduced righting reflex; increased response to sound. The reactions demonstrated a dose-related increase in severity. One animal died (380 mg/kg aniline HCl, 16 h) following administration of colchicine. These clinical signs indicate that the dose level of 380 mg/kg can be considered to be the maximum tolerated dose (MTD) of aniline HCl for the dosing route and regime used.
The group mean aberrant cells per animal are shown in Table I together with the mitotic index data. A small decrease in mitotic index was noted at the two highest dose levels at the 16 h sampling time. This can be taken as evidence of exposure of the target cell population.
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The positive control material, cyclophosphamide, induced a statistically significant increase in the percentage of aberrant cells, confirming the sensitivity of the model for the detection of clastogenic materials.
No statistically significant increases in the percentages of aberrant cells, above the vehicle control values, were recorded in animals treated with aniline HCl at any dose level or sampling time investigated.
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Discussion |
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We have demonstrated that aniline HCl does not induce chromosomal aberrations in the mouse bone marrow. The dosing regime and mouse strain used were the same as used in the mouse bone marrow micronucleus assay by Ashby et al. (1991)
, as they had demonstrated that the use of two i.p. injections produced a larger response than the single administration used by Westmoreland and Gatehouse (1991). Both authors obtained a response at a 24 h sampling time but not at 48 h. Our sampling regime was designed to maximize the possibility of observing any underlying chromosomal damage in the precursor cells of erythrocytes that would have been analysed at 24 h in the micronucleus test. Our findings show that aniline HCl does not exhibit clastogenic activity in vivo in the mouse bone marrow. This suggests that the micronuclei observed by others have arisen by a non-genotoxic mechanism. Aniline is reported to induce methaemoglobinaemia in rodents and this can lead to the formation of Heinz bodies (iron-containing inclusions), which can be mistaken for micronuclei. A non-DNA-specific stain, such as May– Gruenwald/Giemsa, as used initially by Westmoreland and Gatehouse, can lead to the erroneous scoring of such artefacts as micronuclei. However, they also stained slides with a DNA-specific stain (Feulgen–Light Green) and confirmed the DNA content of the micronuclei seen. In this laboratory Ashby et al. used a DNA-specific stain (acridine orange). Therefore, the micronuclei reported by both groups can be taken to be true (i.e. DNA-containing) micronuclei.
Micronuclei can arise either from fragments of chromosomes following breakage events or from whole chromosomes which have lagged behind during mitosis and been excluded from the main nucleus. Micronuclei that contain whole chromosomes tend to be large and may exhibit abnormal morphology (micronuclei generally being spherical). Tinwell and Ashby (1991) did not observe an increase in the proportion of large or morphologically abnormal micronuclei following treatment with aniline. Conversely, Westmoreland and Gatehouse (1991) did report the occurrence of large and abnormally shaped micronuclei in their study. No large micronuclei were noted by George et al. (1991) in the rat micronucleus study and there appears to be no other evidence to suggest that aniline is an aneugen.
Both Ashby et al. and Westmoreland and Gatehouse report positive responses at dose levels that appear to be the MTD for their test systems (as demonstrated by the reporting of clear clinical signs). At lower dose levels no increases in the incidence of micronucleated polychromatic erythrocytes were observed. The literature also contains a report of a negative micronucleus test (Harper et al., 1984) that used lower dose levels (up to 250 mg/kg). It appears that the induction of micronuclei in the erythrocytes of the bone marrow of the mouse is highly correlated with high, i.e. toxic, dose levels.
A major consequence of aniline intoxication is the induction of methaemoglobinaemia and haemolysis. The resulting tissue hypoxia could result in the production of erythropoietin, which would stimulate erythropoiesis. Stimulation of erythropoiesis by the induction of haemolysis has been shown to increase the incidence of micronucleated erythrocytes (Steinheider et al., 1985). Therefore the possibility exists that the micronuclei seen in mouse bone marrow could have arisen by a mechanism related to the imbalance in the erythropoietic system caused by aniline.
Suzuki et al. (1989) speculated that erythropoietin-induced acceleration of erythroblast maturation could reduce the efficiency of DNA repair and lead to an increase in micronucleated erythrocytes. The work of Hirai et al. (1991), which involved bleeding to stimulate erythropoiesis, gives further evidence to support this hypothesis. However, both authors recognize that this mechanism should also lead to the presence of chromosomal aberrations in the dividing precursor cells, which is not the case with aniline. Nor does the induction of aneuploidy seem a likely explanation for the discrepancy between micronucleus and chromosomal aberration assays. It is possible that aniline may interfere with another aspect of erythropoiesis, e.g. enucleation.
The data from the present study suggest that aniline, although an in vitro clastogen and inducer of micronuclei in vivo, is non-genotoxic in the mouse. The positive response in the micronucleus test is most likely caused by disruption of erythropoiesis. The lack of in vivo clastogenicity is consistent with the lack of carcinogenicity in this species in the NTP study.
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Acknowledgments |
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The authors are grateful to Dr Barry Elliott and Prof. John Ashby for helpful discussions. This work was sponsored jointly by BASF AG (Germany), Bayer AG (Germany) and Huntsman Polyurethanes (Belgium).
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Notes |
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1 To whom correspondence should be addressed. Tel: +44 1625 517633; Fax: +44 1625 590249; Email: eryl.jones{at}syngenta.com
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References |
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Received on September 30, 2002; revised on January 13, 2003; accepted on January 16, 2003.
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