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Inhalation of formaldehyde does not induce systemic genotoxic effects in rats
Mutation Research 677 (2009) 76–85
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Mutation Research/Genetic Toxicology and Environmental Mutagenesis journal homepage: www.elsevier.com/locate/gentox Community address: www.elsevier.com/locate/mutres
Inhalation of formaldehyde does not induce systemic genotoxic effects in rats Günter Speit a,∗ , Jasmin Zeller a , Oliver Schmid a , Azeddine Elhajouji b , Lan Ma-Hock c , Simone Neuss a a
Universität Ulm, Institut für Humangenetik, D-89069 Ulm, Germany Novartis Pharma AG, Preclinical Safety, CH-4002 Basel, Switzerland c BASF SE, Experimental Toxicology and Ecology, D-67065 Ludwigshafen, Germany b
a r t i c l e
i n f o
Article history: Received 27 March 2009 Received in revised form 14 May 2009 Accepted 18 May 2009 Available online 17 June 2009 Keywords: Comet assay DNA–protein cross-links Sister chromatid exchange test Micronucleus test Peripheral blood
a b s t r a c t Male Fischer-344 rats were exposed to formaldehyde (FA) by inhalation for 4 weeks (6 h/day, 5 days/week). Groups of six rats each were exposed to the target concentrations of 0, 0.5, 1, 2, 6, 10 and 15 ppm. Potential systemic genotoxic effects were investigated as part of a comprehensive study on local and systemic toxic and genotoxic effects. For this purpose, peripheral blood samples were obtained by puncturing the retroorbital venous plexus at the end of the exposure period. Blood sampling was carried out in a randomized sequence and samples were coded by sequence number to ensure blind evaluation. Blood samples were used for the comet assay, the sister chromatid exchange test (SCE test) and the micronucleus test (MNT). DNA migration in the comet assay was measured both directly and after irradiation of the blood samples with 2 Gy gamma-radiation. The latter modification of the comet assay was included to increase its sensitivity for the detection of DNA–protein cross-links (DPX). The following positive control groups were included: one group (six animals) was treated with 50 mg/kg methyl methanesulfonate (MMS) once by gavage 4 h before blood sampling. Another group (six animals) was treated twice orally with 10 mg/kg cyclophosphamide (CP) with an interval of 24 h. The last application of CP was 24 h before blood sampling. For the comet assay, four slides were analysed from each blood sample, two without and two with irradiation. From each slide, 50 randomly selected cells were measured by image analysis, and tail intensity (% tail DNA) and tail moment were evaluated. For the SCE test, blood was cultured for 56 h in the presence of BrdU (10 g/ml for the last 35 h) and SCE were counted in 30 second-division metaphases per sample. The MNT with peripheral blood was performed according to the instructions for the micronucleus analysis kit MICROFLOW (Litron Laboratories). Approximately 20,000 cells per sample were analysed by flow cytometry and the percentage of reticulocytes with micronuclei (MN) was determined. The positive control substances induced a significant effect in the genotoxicity tests and thus demonstrated the sensitivity of the test systems. FA did not induce any significant effect in any of the genotoxicity tests performed. It can be concluded that inhalation of FA in a 28-day study with FA concentrations up to 15 ppm does not lead to systemic genotoxic effects in the blood of rats. © 2009 Elsevier B.V. All rights reserved.
1. Introduction The possibility that inhaled formaldehyde (FA) may induce systemic (distant-site) toxicity and lympho-hematopoietic malignancies has been raised repeatedly over the years and is still a matter of debate [1–3]. With regard to risk assessment, it is of particular interest whether FA is able to induce systemic genotoxic and mutagenic effects. There is no doubt that FA induces a variety of genotoxic and mutagenic effects in directly exposed proliferating mammalian cells in vitro [4–8] and also induces DNA alterations (DNA–protein cross-links, DPX) in the nasal mucosa of rats and the upper respiratory tract of monkeys exposed to FA by inhala-
∗ Corresponding author. Tel.: +49 731 500 65440; fax: +49 731 500 65402. E-mail address: [email protected] (G. Speit). 1383-5718/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2009.05.020
tion [9,10]. However, a large body of evidence suggests that FA is not delivered to distant sites and, therefore, should not be able to induce systemic (geno-)toxic effects [2,3,11]. Systemic genotoxic effects of FA have been studied in the context of human bio-monitoring and in animal experiments. Contradictory results – positive and negative – were reported for humans exposed to FA by inhalation in human bio-monitoring studies. Some studies reported genotoxic effects in peripheral blood, including increased frequencies of DPX, sister chromatid exchange (SCE) and micronuclei (MN). However, in most of the studies, the descriptions of exposure of the human subjects to FA are quite vague and information on potential co-exposure and other confounding factors is incomplete. A critical evaluation of these studies is hampered by these shortcomings. Based on toxicokinetic data, results from ex vivo studies and theoretical considerations, the reliability of these results has been questioned [2,7].
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Table 1 DNA migration (tail moment) in peripheral blood of rats exposed to formaldehyde (FA) by inhalation measured by the standard alkaline comet assay. Dose [ppm]
Seq. no.a
Without irradiation
MMS
3 5 16 20 37 39
15.38 19.65 25.06 20.16
19.24 16.31 23.44 19.31
17.31 17.98 24.25 19.74
0
23 24 29 36 52 54
0.17 0.16 0.17 0.27 0.08 0.37
0.17 0.15 0.22 0.26 0.08 0.19
0.17 0.16 0.20 0.27 0.08 0.28
0.5
2 4 19 31 40 48
0.25 0.20 0.15 0.39
0.20 0.16 0.17 0.43
0.23 0.18 0.16 0.41
1
1 18 44 45 47 53
0.37 0.14 0.38 0.14 0.21
0.15 0.11 0.40 0.13 0.20
0.26 0.13 0.39 0.14 0.21
2
8 11 22 26 28 43
0.21 0.17 0.15 0.17 0.14 0.17
0.17 0.12 0.13 0.23 0.11 0.16
6
6 9 13 14 27 34
0.18 0.12 0.20 0.12 0.12 0.09
10
7 15 21 30 32 38
15
10 12 17 25 41 50
a b
Mean
Group mean (±S.D.)b
P-value (t-test)
19.82 (3.13)
0.0011
0.19 (0.07)
0.24 (0.11)
0.28
0.22 (0.11)
0.48
0.19 0.15 0.14 0.20 0.13 0.17
0.16 (0.03)
0.49
0.12 0.11 0.18 0.12 0.10 0.10
0.15 0.12 0.19 0.12 0.11 0.10
0.13 (0.03)
0.17
0.10 0.12 0.08 0.48 0.11
0.12 0.27 0.11 0.22 0.10
0.11 0.20 0.10 0.35 0.11
0.17 (0.11)
0.67
0.12 0.15 0.15 0.24 0.17 0.20
0.13 0.19 0.25 0.16 0.18 0.13
0.13 0.17 0.20 0.20 0.18 0.17
0.17 (0.03)
0.67
Sequence number of the blood sample. Standard deviation.
There are only a relatively small number of studies available that investigated systemic genotoxicity of FA in experimental animals. Three inhalation studies with FA doses up to 10 or 15 ppm did not find an induction of DPX and cytogenetic effects (chromosomal aberrations, SCE) in peripheral blood or bone-marrow cells [12–14]. One study reported increased frequencies of chromosomal aberrations in bone-marrow cells after exposure of rats to FA (0.42–1.25 ppm) for 4 months [15]. However, this study suffers from severe methodological shortcomings and is not sufficiently reliable. Therefore, to clarify the uncertainties with regard to systemic genotoxic effects, we now performed an inhalation study with rats under GLP conditions and measured genotoxic effects in the comet assay, the SCE test and the MNT with peripheral blood. These investigations are part of a comprehensive study on local and systemic toxic and genotoxic effects after inhalation of FA.
We used Fischer-344 rats because FA induced squamous cell carcinoma in Fischer-344 rats after exposure to 6 and 15 ppm for 24 months, whereas 2 ppm did not induce tumours. We tested a broad range of concentrations (i.e., from 0.5 to 15 ppm) to include the carcinogenic concentrations but also lower, non-carcinogenic concentrations because induction of genotoxic effects might already occur under non-carcinogenic conditions. The genetic endpoints DPX/comet assay, SCE and MN were chosen because FA very efficiently induced these endpoints in in vitro studies with mammalian cells [4–7] and positive effects were reported for these genetic endpoints in several human bio-monitoring studies [16–24]. Our results clearly show that inhalation of FA for 4 weeks at concentrations between 0.5 and 15 ppm does not induce genotoxic effects in peripheral blood of rats. This comprehensive study supports previous investigations suggesting that (geno-)toxic effects of FA at distant sites are highly unlikely and confirms the view that
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Table 2 DNA-migration (tail moment) in peripheral blood of rats exposed to formaldehyde (FA) by inhalation measured by the alkaline comet assay with irradiation. Dose [ppm]
Seq. no.a
With irradiation
Mean
Group mean (±S.D.)b
P-value (t-test)
MMS
3 5 16 20 37 39
28.92 27.48 28.07 30.90 24.28 26.90
28.69 23.28 28.60 30.50 29.64 24.84
28.81 25.38 28.34 30.70 26.96 25.87
27.68 (2.00)
0.0001
0
23 24 29 36 52 54
2.55 2.58 1.82 3.22 2.42 3.13
2.23 2.51 2.25 3.04 2.30 2.28
2.39 2.55 2.04 3.13 2.36 2.71
2.53 (0.34)
0.5
2 4 19 31 40 48
3.07 3.57 2.10 2.32 3.23 3.39
2.97 3.26 3.06 2.76 3.42 3.26
3.02 3.42 2.58 2.54 3.33 3.33
3.03 (0.39)
0.19
1
1 18 44 45 47 53
3.93 2.06 1.21 3.15 1.51 2.30
3.68 2.45 1.19 3.71 1.35 2.78
3.81 2.26 1.20 3.43 1.43 2.54
2.44 (1.04)
0.83
2
8 11 22 26 28 43
2.87 0.91 2.41 2.24 1.85 3.75
2.41 1.14 2.85 2.71 1.94 3.14
2.64 1.03 2.63 2.48 1.90 3.45
2.35 (0.82)
0.65
6
6 9 13 14 27 34
2.36 1.00 2.61 2.44 2.39 2.45
1.37 1.02 2.33 2.48 2.64 2.14
1.87 1.01 2.47 2.46 2.52 2.30
2.10 (0.59)
0.26
10
7 15 21 30 32 38
2.86 2.07 2.75 1.96 2.86 2.34
2.37 2.07 2.64 2.17 2.55 2.45
2.62 2.07 2.70 2.07 2.71 2.40
2.42 (0.30)
0.79
15
10 12 17 25 41 50
0.84 1.24 2.18 2.14 2.71 1.90
1.02 1.39 2.74 2.82 2.83 1.91
0.93 1.32 2.46 2.48 2.77 1.91
1.98 (0.73)
0.15
a b
Sequence number of the blood sample. Standard deviation.
positive results obtained in human bio-monitoring studies are not related to exposure to FA.
The animals were approximately 9 weeks old at the beginning of the experiment. The study was approved by the local authorizing agency for animal experiments (Landesuntersuchungsanstalt Koblenz, Germany).
2. Materials and methods 2.1. Animals
2.2. Inhalation procedure and analysis
Male Fischer-344 (F-344/DuCrl) rats, specific pathogen-free, approximately 7 weeks old, were purchased from Charles River Laboratories (Research Models and Services GmbH; Sulzfeld, Germany). Animals were maintained in an AAALAC-accredited facility. All procedures for animal care and exposure were conducted under the rule of the German Animal Welfare Act (1998). The animals were housed socially (3–8 animals per cage) in H-Temp (PSU) cages (floor area 610 mm × 435 mm × 215 mm; TECNIPLAST, Germany) in accommodation maintained at 20–24 ◦ C, with a relative humidity of 30–70% and a light/dark cycle of 12 h (beginning 6:00 AM). The rats were allowed to acclimatize to these conditions for approximately 2 weeks before the start of the study. In home cages the animals were allowed free access to rat laboratory diet (Provimi Kliba SA, Basel, Switzerland) and water, but not during the exposure.
Animals were randomly divided in seven groups of six rats/group. The rats were exposed (whole-body) to 0, 0.5, 1, 2, 6, 10 or 15 ppm formaldehyde for 4 weeks (5 days/week, 6 h/day). The exposures were conducted in 1.4-m3 stainless-steel and glass inhalation chambers, where the animals were maintained individually in wire cages (DK III, BECKER & Co., Castrop-Rauxel, Germany). To generate the test atmosphere, constant amounts of formalin were supplied to tempered vaporizers by means of metering pumps. The vapors were mixed with streams of conditioned air and introduced into the chamber air supply and were removed later by an exhaust air system. The air change rate was 20 times per hour. Formaldehyde concentrations were measured using a calibrated Fourier-transform infrared spectrophotometer Gasmet DX4000 (Ansyco, Karlsruhe, Germany). Formaldehyde concentrations in the analytical chambers were measured during the 4 weeks of exposure and averaged
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Table 3 DNA migration (tail intensity) in peripheral blood of rats exposed to formaldehyde (FA) by inhalation measured by the standard alkaline comet assay. Dose [ppm]
Seq. no.a
Without irradiation
MMS
3 5 16 20 37 39
44.89 51.64 59.12 55.94
51.43 47.24 58.45 53.54
48.16 49.44 58.79 54.74
0
23 24 29 36 52 54
1.65 1.80 2.24 2.40 1.76 2.67
2.15 1.99 2.50 2.28 1.80 1.94
1.90 1.90 2.37 2.34 1.78 2.31
0.5
2 4 19 31 40 48
2.41 2.24 1.91 3.17
2.15 1.75 1.92 2.98
2.28 2.00 1.92 3.08
1
1 18 44 45 47 53
2.28 1.90 2.95 1.86 2.37
1.83 1.66 2.65 1.76 2.03
2.06 1.78 2.80 1.81 2.20
2
8 11 22 26 28 43
2.39 2.47 1.79 1.72 2.16 1.81
1.90 2.09 1.69 2.58 1.63 1.68
6
6 9 13 14 27 34
1.96 2.14 2.33 1.57 1.72 1.33
10
7 15 21 30 32 38
15
10 12 17 25 41 50
a b
Mean
Group mean (±S.D.)b
P-value (t-test)
52.78 (4.91)
0.0002
2.10 (0.27)
2.32 (0.53)
0.39
2.13 (0.41)
0.90
2.15 2.28 1.74 2.15 1.90 1.75
1.99 (0.23)
0.64
1.48 2.10 1.95 1.67 1.52 1.56
1.72 2.12 2.14 1.62 1.62 1.45
1.78 (0.29)
0.16
1.48 1.61 1.31 3.69 1.65
1.48 2.29 1.65 2.12 1.54
1.48 1.95 1.48 2.91 1.60
1.88 (0.60)
0.36
2.09 2.51 1.84 2.29 1.87 2.21
2.28 3.24 2.71 1.66 1.93 1.70
2.19 2.88 2.28 1.98 1.90 1.96
2.19 (0.36)
0.67
Sequence number of the blood sample. Standard deviation.
14.96 ± 0.05, 10.00 ± 0.39, 6.12 ± 0.34, 2.02 ± 0.15, 1.00 ± 0.11, 0.51 ± 0.05 and 0 ppm on a daily basis. 2.3. Study design This study was performed to investigate potential local (site of contact) and systemic toxic and genotoxic effects of FA exposure. Only results from genotoxicity tests with peripheral blood are reported here. In addition to the formaldehyde-exposed animals, two groups of positive control animals were included. One group of animals (six males) was treated twice orally, with an interval of 24 h, with 10 mg/kg bw/day cyclophosphamide (CP). This is the standard positive control for the in vivo MNT with peripheral blood. For the other endpoints (SCE test, comet assay), a second group of positive control animals was treated once by gavage with MMS (50 mg/kg bw) 4 h before blood samples were taken. The inhalation study was performed at BASF SE (Department of Experimental Toxicology and Ecology) in accordance with the OECD Principles of Good Laboratory Practice (GLP). During the exposure-free days, a check for moribund or dead animals was carried out twice per day on working days and once per day on weekends. In addition, a clinical inspection of the animals exposed to formaldehyde was performed at least
three times on exposure days, once a day during the acclimatization period, and on the day of necropsy. The positive control animals were examined once a day (except weekends) for clinical abnormalities. Signs and findings were recorded for each animal. Body weight was determined weekly for all animals. At the end of the exposure period, blood sampling was carried out in a randomized sequence and the samples were coded by sequence number. Blood samples were taken by puncturing the retro-orbital venous plexus and collected in tubes containing an anticoagulant (heparin) for the comet assay and the SCE test. These blood samples were stored on ice and immediately carried to the laboratory at Ulm University, where the comet assay and the SCE test were started immediately (i.e. about 4 h after blood sampling). For the MNT (Litron MicroFlow Rat Micronucleus Analysis Kit), approximately 120 l of blood were collected from each animal into a tube containing 300 l anticoagulant solution. Blood samples were fixed in ultra-cold methanol within 2 h according to the manufacturer’s recommendations and stored at −75 ◦ C until they were sent on dry ice to the laboratory at Novartis Pharma, where the test was performed. After blind evaluation of all tests, the complete raw data sets were sent to BASF, where they were decoded. After decoding, final assessment and statistical analysis of the test results was performed at the Ulm University (comet assay and SCE test) and at Novartis Pharma AG (MNT).
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Table 4 DNA migration (tail intensity) in peripheral blood of rats exposed to formaldehyde (FA) by inhalation measured by the alkaline comet assay with irradiation. Dose [ppm]
Seq. no.a
With irradiation
Mean
Group mean (±S.D.)b
P-value (t-test)
MMS
3 5 16 20 37 39
65.84 63.42 66.22 68.91 59.14 70.08
66.65 58.80 68.36 71.57 67.01 65.04
66.25 61.11 67.29 70.24 63.08 67.56
65.92 (3.30)
0.001
0
23 24 29 36 52 54
11.23 11.04 9.77 13.01 10.96 12.96
10.14 10.79 11.74 11.94 9.73 9.99
10.69 10.92 10.76 12.48 10.35 11.48
11.11 (0.77)
0.5
2 4 19 31 40 48
12.60 15.20 10.61 9.65 14.43 13.43
12.20 13.72 11.88 11.54 13.04 13.35
12.40 14.46 11.25 10.60 13.74 13.39
12.64 (1.50)
0.17
1
1 18 44 45 47 53
15.51 9.89 7.54 13.94 8.75 10.36
14.80 10.42 7.20 14.67 8.05 12.24
15.16 10.16 7.37 14.31 8.40 11.30
11.11 (3.13)
0.99
2
8 11 22 26 28 43
13.38 6.84 10.48 10.50 9.48 13.77
11.72 8.46 11.45 12.79 10.21 12.34
12.55 7.65 10.97 11.65 9.85 13.06
10.95 (1.98)
0.89
6
6 9 13 14 27 34
10.10 7.03 11.97 11.59 11.92 11.92
8.96 7.47 11.79 11.72 11.16 10.76
9.53 7.25 11.88 11.66 11.54 11.34
10.53 (1.82)
0.60
10
7 15 21 30 32 38
13.33 10.06 11.78 9.78 12.10 12.31
11.41 8.87 10.55 10.85 12.02 12.66
12.37 9.47 11.17 10.32 12.06 12.49
11.31 (1.22)
0.85
15
10 12 17 25 41 50
6.42 8.44 10.33 10.55 11.34 10.22
7.66 9.64 12.38 12.53 11.68 10.53
7.04 9.04 11.36 11.54 11.51 10.38
10.14 (1.80)
0.38
a b
Sequence number of the blood sample. Standard deviation.
2.4. Comet assay
2.5. Sister chromatid exchange test (SCE test)
The comet assay was performed according to our standard protocol for human blood [7] with 3 l whole rat blood per slide, a 25-min alkali denaturation period, and electrophoresis (0.86 V/cm) during 25 min at pH > 13. Determination of DPX by the reduction of induced DNA migration was accomplished by irradiating blood samples with 2 Gy ␥-rays (Cs-137; Gammacell 2000, Nuclear Data, Frankfurt, Germany) immediately before conducting the comet assay. Images of 50 randomly selected cells stained with ethidium bromide were analysed from each of two parallel slides. Measurements were made by image analysis (Comet Assay II, Perceptive Instruments). For all experiments, we evaluated two image-analysis parameters: tail intensity and tail moment. Tail moment (TM) is calculated according to the formula: TM = (tail intensity/total comet intensity) × (tail centre of gravity − peak position). Statistical analysis was performed for the results of the comet assay using the twotailed Student’s t-test and ANOVA (analysis of variance) followed by a Dunnett test. In addition, the non-parametric Wilcoxon rank test was performed which never led to a divergent result. A statistically significant difference between mean values of the negative control group and the groups exposed to FA or between the negative control group and the positive control group was set at P < 0.05.
SCE tests were performed as previously described for human blood [7] with some modifications. Rat blood samples were carefully washed with 1× PBS. 300 l of the washed blood were then added to 5 ml RPMI 1640 + GlutaMax medium into a T25 cell culture flask. Concanavalin A (20 g/ml) was added and cultures were incubated at 37 ◦ C and 5% CO2 . After 21 h, BrdU (10 g/ml final concentration) was added and cultures were incubated for another 35 h (including exposure to 2 × 10−7 M colcemid for the final 2 h). Chromosome preparation was done following standard procedures. Cells were centrifuged, resuspended in 0.56% KCl for 10 min and fixed three times in methanol:glacial acetic acid (3 + 1). For sister chromatid differentiation, air-dried slides were covered with Sörensen buffer (pH 6.8) and irradiated with an 8-W UV lamp (254 nm) at a distance of 10 cm for 30 min. Subsequently, slides were incubated in 2× SSC for 20 min at 58 ◦ C and then stained with 7% Giemsa in Sörensen buffer. SCE were scored in 30 well-spread and well-stained metaphases per sample. Statistical analysis was performed for the results of the SCE test using the one-tailed Student’s t-test and ANOVA (analysis of variance) followed by a Dunnett test. In addition, the non-parametric Wilcoxon rank test was performed which never led to a divergent result. A statistically significant difference between mean values of the negative
G. Speit et al. / Mutation Research 677 (2009) 76–85 control group and the groups exposed to FA or between the negative control group and the positive control group was set at P < 0.05. 2.6. Peripheral blood micronucleus test (MNT) The MNT with peripheral blood was performed by flow cytometric analysis using the MicroFlow Rat Micronucleus Analysis Kit [25]. On the day of analysis, blood samples were simultaneously treated with 1 mg/ml RNase and anti-CD71-FITC. Fluorescein isothiocyanate-conjugated antibody against the rat transferrin receptor anti-CD71-FITC was used to differentially label and score cells with high transferrin receptor content i.e., the youngest fraction of reticulocytes. The cells were incubated at 4 ◦ C for 30 min and then at 2 ◦ C for 90 min to ensure complete degradation of cellular RNA. The cells were then kept at 4 ◦ C until analysis. Immediately prior to analysis, 1 ml ice-cold propidium iodide staining solution (PI; 1.25 g/ml bicarbonate-buffered saline) was added to a malaria-infected blood sample (biological standard purchased from Litron, NY, USA) for flow cytometer set-up and calibration [26,27]. After the flow cytometer was calibrated, 1 ml cold PI was added to one sample at a time and each was analysed by high-speed flow cytometry using the settings guided by malaria-infected blood standards. The percentage of reticulocytes (RET) among all erythrocytes (RET plus normochromatic erythrocytes, NCE) and the percentage of reticulocytes with MN (MN-RET) were determined. Statistical evaluation was done with SAS V8.2 TS 2M0. ANOVA and linear trend test were performed. Since only males were used, “treatment group” was used as a fixed factor then 1-way ANOVA was performed. For the one-tailed linear trend test, a linear regression with dose as an independent variable was done. The significance level used was ≤0.05.
3. Results 3.1. Clinical examination During the whole study period there were no substance-related clinical signs of toxicity. The body-weight development of the animals exposed to formaldehyde up to 6 ppm was comparable with that of the control animals, so was that of animals receiving CP and MMS. Only the body-weight gain of rats exposed to 10 ppm (day 7 only) and 15 ppm FA was transiently decreased (−16.7% to −32.7%) at the beginning of the exposure period. This effect was no longer statistically significant toward the end of the exposure, although the average body-weight gain of the 15-ppm group was still slightly lower than in the control group. 3.2. Comet assay Tables 1–4 summarize the results for the comet assay with blood from rats exposed to FA by inhalation. Slides from six animals (2 of the positive control group, 2 of the 0.5-ppm group, 1 of the 1-ppm group and 1 of the 10-ppm group) could not be evaluated because of a technical problem during electrophoresis. Tables 1 and 2 comprise the tail-moment results in the absence and presence of gamma irradiation and Tables 3 and 4 those for the tail intensity, respectively. In the absence of irradiation, the effects in the comet assay are either due to DNA strand breaks (increased migration in comparison with the negative control) or cross-links (DPX in the case of FA) indicated by a decreased DNA migration. Because the background levels of DNA migration under standard conditions are low, the detection of DPX is difficult. Therefore, parallel slides were irradiated (2 Gy gamma-radiation) to induce a defined amount of strand breaks and to increase DNA migration. In the presence of DPX, a reduction in gamma ray-induced DNA migration (relative to the negative control after gamma irradiation) is expected. Under standard conditions, the positive control substance MMS caused a clear and statistically significant increase in DNA migration and this effect was enhanced by gamma irradiation. No statistically significant effect in the comet assay was measured for any of the dose groups either in the absence or in the presence of gamma irradiation (ANOVA: tail moment without irradiation: P = 0.2823; tail moment with irradiation: P = 0.1749; tail intensity without irradiation: P = 0.3478; tail intensity with irradiation: P = 0.4137). These results indicate that under the conditions of this study, FA concen-
81
trations up to 15 ppm in a 28-day inhalation study did not induce genotoxic effects in the comet assay. The results excluded a DNA strand breaking effect, the induction of alkali-labile sites (ALS) as well as a cross-linking effect of FA in leukocytes of rats exposed to FA by inhalation. 3.3. SCE test The results for the SCE test with cultured rat lymphocytes are summarized in Table 5. Blood cultures from six animals (2 of the positive control group, 2 of the 6-ppm group and 2 of the 10-ppm group) could not be evaluated due to problems with blood coagulation. The positive control substance MMS clearly induced SCE. The mean SCE frequency (9.7/cell) was about twice the mean value of the negative control group (4.6/cell). There was no statistically significant increase in the SCE frequency in any of the groups exposed to FA (ANOVA: P = 0.8642). Consequently, FA did not induce SCE in cultured lymphocytes of rats exposed by inhalation. 3.4. MNT The results for the MNT with peripheral blood are shown in Table 6 and the statistical analysis of the MNT is summarized in Table 7. Samples from three animals, no. 2 (negative control group), no. 8 (0.5-ppm FA group) and no. 50 (cyclophosphamide group) were excluded from the MNT analysis due to poor quality. Biometric analysis revealed no statistically significant difference in the percentages of RET between treated groups and control animals and no significant difference in the trend test. The percentage of MN-RET in the positive control (CP, 2 × 10 mg/kg) group was 0.44 ± 0.11%. Biometric analysis also showed no statistically significant difference between the mean micronucleus frequencies in the treated groups and the negative control group (0.22 ± 0.18%) and no significant difference in the trend test. The mean percentage of MN-RET in the positive control (cyclophosphamide, 2 × 10 mg/kg) group was 0.63 ± 0.18%, which was statistically significantly higher than the negative control value. Under the conditions of this study, FA inhalation did not increase the frequency of MN in peripheral blood of rats. 4. Discussion Our results show that inhalation of formaldehyde does not induce genotoxic effects in peripheral blood of rats. The comet assay, the SCE test and the MNT were applied because these genetic endpoints have been shown to be sensitive indicators of FA genotoxicity in vitro and also revealed some positive results in human bio-monitoring studies. The alkaline comet assay with whole rat blood was performed according to the standard protocol and also in combination with gamma-irradiation. The standard protocol detects DNA strand breaks and alkali-labile sites (ALS) which lead to increased DNA migration. It can also detect cross-links (DNA–DNA and DNA–protein) which lead to reduced baseline DNA migration. However since the baseline DNA migration in rat leukocytes under the conditions used here is rather low (about 2% DNA in tail) the sensitivity of this protocol for the detection of cross-links is limited. The use of increased electrophoresis time was avoided because it leads to high variability between slides at different positions in the gel box (Speit et al., unpublished results). Therefore, we performed the additional modification with irradiation, which has been shown to be a very sensitive approach for the detection of cross-links and, in particular, of FA-induced DPX [5,7,28]. Inhalation of FA did not induce DNA migration in peripheral blood of rats, indicating that FA did not induce DNA strand breaks and ALS. It is still a matter of discussion, whether FA is able to induce DNA strand breaks
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Table 5 Frequency of SCE in peripheral blood of rats exposed to formaldehyde (FA) by inhalation. Dose [ppm]
Seq. no.a
Mean of 30 metaphases
MMS
3 5 16 20 37 39
10.70 10.37 8.80 8.90
0
23 24 29 36 52 54
4.63 4.47 3.70 4.60 4.50 5.57
4.58 (0.60)
0.5
2 4 19 31 40 48
4.13 5.27 5.20 4.73 5.63 4.70
4.94 (0.53)
0.27
1
1 18 44 45 47 53
4.77 5.20 4.60 4.90 4.63 4.43
4.76 (0.27)
0.59
2
8 11 22 26 28 43
4.50 4.37 4.87 5.10 5.20 5.47
4.92 (0.42)
0.30
6
6 9 13 14 27 34
4.84 (0.40)
0.47
10
7 15 21 30 32 38
4.57 3.73 4.80
4.77 (0.92)
0.60
10 12 17 25 41 50
5.50 4.33 5.47 5.43 4.03 5.37
5.02 (0.66)
0.18
15
a b
Group mean (±S.D.)b
P-value (t-test)
9.69 (0.98)
0.0004
4.37 4.67 5.10 5.23
5.97
Sequence number of the blood sample. Standard deviation.
but a recent comprehensive investigation clearly showed that DNA strand breaks do not play a significant role in FA-induced genotoxicity and DPX are the most relevant type of DNA damage induced by FA in mammalian cells [6]. Irradiation of blood samples immediately before the performance of the comet assay caused a clear increase in DNA migration but there was no reduction of this effect in rats exposed to FA as a consequence of FA-induced DPX. These comet assay results clearly indicate that FA did not induce DPX in peripheral blood of rats that can be detected by the comet assay. Previous studies have shown that DPX occur in the nasal mucosa of rats and in the upper respiratory tracts of monkeys exposed to FA [9,10], but increased amounts of DPX were not found in tissues other than the respiratory tract. No induction of DPX was measured in the bone marrow of rats exposed to airborne FA at concentrations up to 15 ppm and in the bone marrow of GSH-depleted rats at concentrations as high as 10 ppm FA [12,29]. Formation of DPX was also not
found in the bone marrow of Rhesus monkeys exposed to 6 ppm FA [30]. Our results support the previous experimental animal studies which used other methods for the detection of DPX and which all failed to demonstrate distant-site genotoxicity of inhaled FA. However, one recently published study measured comet-assay effects in lymphocytes and liver cells of rats exposed to FA (5 and 10 ppm) by inhalation for 2 weeks [31]. The authors reported a very similar dose-related increase in DNA migration in both cell types. This study was commented on and its reliability was questioned [32]. Besides the fact that systemic effects of FA are unlikely, it was surprising that increased DNA migration was measured in FA-exposed rats and similar effects were observed in liver cells and lymphocytes despite administration by inhalation. All in all, the results of this in vivo study are biologically implausible. Therefore, they should not be used for the assessment of the in vivo genotoxicity of FA.
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Table 6 Frequency of micronuclei in peripheral blood of rats exposed to formaldehyde (FA). Dose [ppm]
Seq. no.a
RET [%]b
CP
33 35 42 46 49 51
0.61 0.47 0.33 0.38 0.41
0
23 24 29 36 52 54
1.56 1.28 1.61 1.65 1.51
0.5
2 4 19 31 40 48
1.36 1.52 1.67 1.50 1.52
1
1 18 44 45 47 53
1.14 1.20 1.56 1.48 1.93 1.80
2
8 11 22 26 28 43
1.58 1.59 1.60 1.56 1.56 1.45
6
6 9 13 14 27 34
1.21 1.60 1.57 1.57 1.20 1.46
10
7 15 21 30 32 38
1.36 1.45 1.57 1.83 1.77 1.66
15
10 12 17 25 41 50
1.18 1.82 1.00 1.08 1.53 1.28
a b c d
Group mean (±S.D.)c
0.44 (0.11)
1.52 (0.14)
1.51 (0.11)
0.53 0.16 0.08 0.20 0.14 0.03 0.36 0.17 0.15 0.19
0.63 (0.18)
0.22 (0.18)
0.18 (0.12)
1.52 (0.31)
0.32 (0.23)
1.56 (0.06)
0.13 0.02 0.31 0.27 0.07 0.59
0.23 (0.21)
1.44 (0.18)
0.03 0.19 0.30 0.10 0.01 0.19
0.14 (0.11)
1.61 (0.18)
0.05 0.28 0.15 0.63 0.18 0.06
0.23 (0.21)
1.32 (0.31)
0.26 0.23 0.21 0.16 0.19 0.25
0.22 (0.04)
Sequence number of the blood sample. Percentage of reticulocytes. Standard deviation. Percentage of reticulocytes with micronuclei.
RET [%]a
MN-RET [%]b
Formaldehyde ANOVA Linear regression
0.7116 0.9972
0.4923 0.2747
Cyclophosphamide ANOVA Linear regression
0.0004 n.a.
0.0047 n.a.
a
0.46 0.51 0.92 0.66 0.59
Group mean (±S.D.)
0.19 0.51 0.15 0.64 0.06 0.37
Table 7 Statistical analysis (P-value) of the micronucleus test with peripheral blood.
b
MN-RET [%]d
Percentage of reticulocytes. Percentage of reticulocytes with micronuclei.
Human bio-monitoring studies of workers exposed to FA performed by one group suggested an induction of DPX in circulating lymphocytes [23]. However, the effect was small, not correlated with the FA concentration and in the range of the control level of a previous study [33]. The authors used the K-SDS assay for the detection of DPX and argue that their method is extremely sensitive for the detection of DPX. However, this argument is not substantiated by the results presented and comparative investigations have shown that the K-SDS assay is not significantly more sensitive than the comet assay [4,34]. Therefore, doubts arose concerning the reliability of these findings [7,11]. Surprisingly, one recent biomonitoring study reported increased DNA migration in the comet assay with blood from FA-exposed subjects [19]. Given the abundance of evidence that FA predominantly induces DPX and DPX lead
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to a reduction in DNA migration in the comet assay, this result is difficult to interpret. However, the assessment of these bio-monitoring studies becomes clearer in the context of SCE test results. SCE are reciprocal exchanges of the DNA molecule in a replicating cell at stalled replication forks [35]. SCE are induced by a wide variety of DNA alterations including FA-induced DPX [4,7]. In cultured peripheral lymphocytes of experimental animals or human subjects exposed to mutagens, SCE are exclusively formed in vitro as a consequence of persisting DNA damage. Thus, increased frequencies of SCE in peripheral lymphocytes after in vivo exposure to FA requires that the lymphocytes carry increased levels of DPX at the time of sampling, that DPX are not completely repaired before the cells enter S-phase (at least 24 h after the start of the cultures) and cause the formation of SCE during replication in the presence of BrdU. Our results clearly show that FA does not induce SCE in peripheral blood of rats after exposure to FA concentrations as high as 15 ppm. In an earlier study, rats were exposed to FA (0.5, 6 and 15 ppm) for 5 days (6 h/day) and the frequencies of SCE were measured in peripheral blood. In accordance with our results, there was no induction of SCE at any of the concentrations tested [14]. Again, despite clearly negative results in in vivo genotoxicity tests under clearly defined exposure conditions and exposure to low and high FA concentrations, some genotoxic effects were published for the SCE test with peripheral lymphocytes of human subjects exposed to FA [16,19–22,24]. However, it is highly unlikely that these effects are actually related to FA exposure, because relevant exposure and induction of DPX in lymphocytes that leads to a positive SCE test cannot be expected [2]. Furthermore, ex vivo experiments with human blood have shown that DPX present in lymphocytes at the start of the culture are efficiently removed during lymphocyte culture before the cells reach the S-phase. Only very high levels of DPX at the beginning of the in vitro cultivation ensure sufficient amounts of persisting DPX until replication [7]. Such DPX levels cannot be reached by environmental and work-place exposures in peripheral lymphocytes, and increased SCE frequencies in subjects exposed to FA are very probably not related to FA exposure. Thus, a positive SCE test with lymphocytes of FAexposed human subjects does not confirm positive results for other endpoints within the same study; on the contrary, it is a strong argument against FA as the relevant exposure. In the light of the positive SCE test results reported within the same study, even the effects on DPX levels [16] and effects in the comet assay [19] have to be interpreted as not being FA-related. The MNT with peripheral blood using flow cytometric analysis is now a well-established in vivo genotoxicity test with higher sensitivity than the traditional bone-marrow MNT [25,36]. Two previous inhalation studies measuring chromosomal aberrations in bone marrow revealed clearly negative results after exposure of rats to 0.5, 3 and 15 ppm for 1 or 8 weeks (5 days/week, 6 h/day) [13] or after exposure of rats to 0.5, 6 and 15 ppm for 5 days (6 h/day) [14]. Our study confirms these negative cytogenetic tests and shows that FA does not exert a systemic clastogenic effect after inhalation for 4 weeks at concentrations as high as 15 ppm. In contrast, some human bio-monitoring studies reported positive effects in the MNT with cultured lymphocytes after exposure to relatively low FA concentrations [17–19,21]. These studies used the MNT with cytochalasin B and scored MN in binuclear cells. It can be assumed that these MN mainly arise during in vitro cell division [37,38] as a consequence of persisting damage. A comparative ex vivo study that investigated the persistence of FA-induced DPX and the formation of SCE and MN in cultured human lymphocytes, came to the conclusion that MN are induced with lower sensitivity than SCE [7]. Therefore, a positive MNT with peripheral lymphocytes cannot readily be explained by an in vivo exposure of lymphocytes to FA. Interestingly, one bio-monitoring study reported increased MN frequencies in the
absence of effects in the SCE test [18]. These results support the view that the cytogenetic effects measured are not FA-related. Another study showed, that increased MN frequencies in FAexposed subjects mainly represent centromeric MN, i.e., are due to aneugenic events [17]. However, FA mainly acts by a clastogenic mode of action [7,8]. Aneugenic effects are usually thresholded and require high substance concentrations in the target cells. Therefore, an aneugenic effect in the MNT in a bio-monitoring study with FA-exposed subjects is even less likely than a clastogenic effect and most likely not directly related to the assumed FA exposure. In summary, our in vivo study with inhalation exposure of rats for 4 weeks to FA at concentrations ranging from 0.5 to 15 ppm performed under GLP conditions, provides important information with regard to potential systemic genotoxic effects of FA. The results clearly exclude any genotoxic effect of FA in the comet assay including DPX formation, the SCE test and the MNT performed with peripheral blood. Thus our results add an important piece of evidence to the already existing experimental data suggesting that inhaled FA does not induce any systemic genotoxic effect in appropriately performed in vivo genotoxicity tests with peripheral blood. Our results are in agreement with earlier results showing the failure of inhaled FA to increase the FA concentration in the blood of rats, monkeys and humans and the lack of detectable DPX in the bone marrow of normal and GSH-depleted (metabolically inhibited) rats [2,3,11]. The results also support the view that genotoxic effects of FA reported in human bio-monitoring studies lack plausibility because positive test results cannot be expected in peripheral lymphocytes after FA inhalation [7,11]. The reported effects are either test artefacts, chance findings or due to another kind of exposure. Consequently, positive genotoxicity tests from human bio-monitoring do not support models for systemic effects of FA (including cancer) in humans exposed by inhalation. Conflict of interest No conflicts of interest. Acknowledgements We wish to thank Mrs. Petra Schütz, Mrs. Irmgard Weber and Mr. Renato Niggli for excellent technical assistance and Prof. J. Högel for helping us with the statistical analysis of the data. The financial support provided by the European Chemical Industry Council (CEFIC) is gratefully acknowledged. References [1] IARC, 2006. Formaldehyde, 2-butoxyethanol and 1-tert-butoxypropan-2-ol, pp. 1–478. [2] D. Pyatt, E. Natelson, R. Golden, Is inhalation exposure to formaldehyde a biologically plausible cause of lymphohematopoietic malignancies? Regul. Toxicol. Pharmacol. 51 (2008) 119–133. [3] R. Golden, D. Pyatt, P.G. Shields, Formaldehyde as a potential human leukemogen: an assessment of biological plausibility, Crit. Rev. Toxicol. 36 (2006) 135–153. [4] O. Merk, G. Speit, Significance of formaldehyde-induced DNA–protein crosslinks for mutagenesis, Environ. Mol. Mutagen. 32 (1998) 260–268. [5] G. Speit, O. Schmid, S. Neuss, P. Schutz, Genotoxic effects of formaldehyde in the human lung cell line A549 and in primary human nasal epithelial cells, Environ. Mol. Mutagen. 49 (2008) 300–307. [6] G. Speit, P. Schutz, J. Hogel, O. Schmid, Characterization of the genotoxic potential of formaldehyde in V79 cells, Mutagenesis 22 (2007) 387–394. [7] O. Schmid, G. Speit, Genotoxic effects induced by formaldehyde in human blood and implications for the interpretation of biomonitoring studies, Mutagenesis 22 (2007) 69–74. [8] G. Speit, O. Merk, Evaluation of mutagenic effects of formaldehyde in vitro: detection of crosslinks and mutations in mouse lymphoma cells, Mutagenesis 17 (2002) 183–187. [9] M. Casanova, K.T. Morgan, W.H. Steinhagen, J.I. Everitt, J.A. Popp, H.D. Heck, Covalent binding of inhaled formaldehyde to DNA in the respiratory tract of
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Mutation Research/Genetic Toxicology and Environmental Mutagenesis journal homepage: www.elsevier.com/locate/gentox Community address: www.elsevier.com/locate/mutres
Inhalation of formaldehyde does not induce systemic genotoxic effects in rats Günter Speit a,∗ , Jasmin Zeller a , Oliver Schmid a , Azeddine Elhajouji b , Lan Ma-Hock c , Simone Neuss a a
Universität Ulm, Institut für Humangenetik, D-89069 Ulm, Germany Novartis Pharma AG, Preclinical Safety, CH-4002 Basel, Switzerland c BASF SE, Experimental Toxicology and Ecology, D-67065 Ludwigshafen, Germany b
a r t i c l e
i n f o
Article history: Received 27 March 2009 Received in revised form 14 May 2009 Accepted 18 May 2009 Available online 17 June 2009 Keywords: Comet assay DNA–protein cross-links Sister chromatid exchange test Micronucleus test Peripheral blood
a b s t r a c t Male Fischer-344 rats were exposed to formaldehyde (FA) by inhalation for 4 weeks (6 h/day, 5 days/week). Groups of six rats each were exposed to the target concentrations of 0, 0.5, 1, 2, 6, 10 and 15 ppm. Potential systemic genotoxic effects were investigated as part of a comprehensive study on local and systemic toxic and genotoxic effects. For this purpose, peripheral blood samples were obtained by puncturing the retroorbital venous plexus at the end of the exposure period. Blood sampling was carried out in a randomized sequence and samples were coded by sequence number to ensure blind evaluation. Blood samples were used for the comet assay, the sister chromatid exchange test (SCE test) and the micronucleus test (MNT). DNA migration in the comet assay was measured both directly and after irradiation of the blood samples with 2 Gy gamma-radiation. The latter modification of the comet assay was included to increase its sensitivity for the detection of DNA–protein cross-links (DPX). The following positive control groups were included: one group (six animals) was treated with 50 mg/kg methyl methanesulfonate (MMS) once by gavage 4 h before blood sampling. Another group (six animals) was treated twice orally with 10 mg/kg cyclophosphamide (CP) with an interval of 24 h. The last application of CP was 24 h before blood sampling. For the comet assay, four slides were analysed from each blood sample, two without and two with irradiation. From each slide, 50 randomly selected cells were measured by image analysis, and tail intensity (% tail DNA) and tail moment were evaluated. For the SCE test, blood was cultured for 56 h in the presence of BrdU (10 g/ml for the last 35 h) and SCE were counted in 30 second-division metaphases per sample. The MNT with peripheral blood was performed according to the instructions for the micronucleus analysis kit MICROFLOW (Litron Laboratories). Approximately 20,000 cells per sample were analysed by flow cytometry and the percentage of reticulocytes with micronuclei (MN) was determined. The positive control substances induced a significant effect in the genotoxicity tests and thus demonstrated the sensitivity of the test systems. FA did not induce any significant effect in any of the genotoxicity tests performed. It can be concluded that inhalation of FA in a 28-day study with FA concentrations up to 15 ppm does not lead to systemic genotoxic effects in the blood of rats. © 2009 Elsevier B.V. All rights reserved.
1. Introduction The possibility that inhaled formaldehyde (FA) may induce systemic (distant-site) toxicity and lympho-hematopoietic malignancies has been raised repeatedly over the years and is still a matter of debate [1–3]. With regard to risk assessment, it is of particular interest whether FA is able to induce systemic genotoxic and mutagenic effects. There is no doubt that FA induces a variety of genotoxic and mutagenic effects in directly exposed proliferating mammalian cells in vitro [4–8] and also induces DNA alterations (DNA–protein cross-links, DPX) in the nasal mucosa of rats and the upper respiratory tract of monkeys exposed to FA by inhala-
∗ Corresponding author. Tel.: +49 731 500 65440; fax: +49 731 500 65402. E-mail address: [email protected] (G. Speit). 1383-5718/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2009.05.020
tion [9,10]. However, a large body of evidence suggests that FA is not delivered to distant sites and, therefore, should not be able to induce systemic (geno-)toxic effects [2,3,11]. Systemic genotoxic effects of FA have been studied in the context of human bio-monitoring and in animal experiments. Contradictory results – positive and negative – were reported for humans exposed to FA by inhalation in human bio-monitoring studies. Some studies reported genotoxic effects in peripheral blood, including increased frequencies of DPX, sister chromatid exchange (SCE) and micronuclei (MN). However, in most of the studies, the descriptions of exposure of the human subjects to FA are quite vague and information on potential co-exposure and other confounding factors is incomplete. A critical evaluation of these studies is hampered by these shortcomings. Based on toxicokinetic data, results from ex vivo studies and theoretical considerations, the reliability of these results has been questioned [2,7].
G. Speit et al. / Mutation Research 677 (2009) 76–85
77
Table 1 DNA migration (tail moment) in peripheral blood of rats exposed to formaldehyde (FA) by inhalation measured by the standard alkaline comet assay. Dose [ppm]
Seq. no.a
Without irradiation
MMS
3 5 16 20 37 39
15.38 19.65 25.06 20.16
19.24 16.31 23.44 19.31
17.31 17.98 24.25 19.74
0
23 24 29 36 52 54
0.17 0.16 0.17 0.27 0.08 0.37
0.17 0.15 0.22 0.26 0.08 0.19
0.17 0.16 0.20 0.27 0.08 0.28
0.5
2 4 19 31 40 48
0.25 0.20 0.15 0.39
0.20 0.16 0.17 0.43
0.23 0.18 0.16 0.41
1
1 18 44 45 47 53
0.37 0.14 0.38 0.14 0.21
0.15 0.11 0.40 0.13 0.20
0.26 0.13 0.39 0.14 0.21
2
8 11 22 26 28 43
0.21 0.17 0.15 0.17 0.14 0.17
0.17 0.12 0.13 0.23 0.11 0.16
6
6 9 13 14 27 34
0.18 0.12 0.20 0.12 0.12 0.09
10
7 15 21 30 32 38
15
10 12 17 25 41 50
a b
Mean
Group mean (±S.D.)b
P-value (t-test)
19.82 (3.13)
0.0011
0.19 (0.07)
0.24 (0.11)
0.28
0.22 (0.11)
0.48
0.19 0.15 0.14 0.20 0.13 0.17
0.16 (0.03)
0.49
0.12 0.11 0.18 0.12 0.10 0.10
0.15 0.12 0.19 0.12 0.11 0.10
0.13 (0.03)
0.17
0.10 0.12 0.08 0.48 0.11
0.12 0.27 0.11 0.22 0.10
0.11 0.20 0.10 0.35 0.11
0.17 (0.11)
0.67
0.12 0.15 0.15 0.24 0.17 0.20
0.13 0.19 0.25 0.16 0.18 0.13
0.13 0.17 0.20 0.20 0.18 0.17
0.17 (0.03)
0.67
Sequence number of the blood sample. Standard deviation.
There are only a relatively small number of studies available that investigated systemic genotoxicity of FA in experimental animals. Three inhalation studies with FA doses up to 10 or 15 ppm did not find an induction of DPX and cytogenetic effects (chromosomal aberrations, SCE) in peripheral blood or bone-marrow cells [12–14]. One study reported increased frequencies of chromosomal aberrations in bone-marrow cells after exposure of rats to FA (0.42–1.25 ppm) for 4 months [15]. However, this study suffers from severe methodological shortcomings and is not sufficiently reliable. Therefore, to clarify the uncertainties with regard to systemic genotoxic effects, we now performed an inhalation study with rats under GLP conditions and measured genotoxic effects in the comet assay, the SCE test and the MNT with peripheral blood. These investigations are part of a comprehensive study on local and systemic toxic and genotoxic effects after inhalation of FA.
We used Fischer-344 rats because FA induced squamous cell carcinoma in Fischer-344 rats after exposure to 6 and 15 ppm for 24 months, whereas 2 ppm did not induce tumours. We tested a broad range of concentrations (i.e., from 0.5 to 15 ppm) to include the carcinogenic concentrations but also lower, non-carcinogenic concentrations because induction of genotoxic effects might already occur under non-carcinogenic conditions. The genetic endpoints DPX/comet assay, SCE and MN were chosen because FA very efficiently induced these endpoints in in vitro studies with mammalian cells [4–7] and positive effects were reported for these genetic endpoints in several human bio-monitoring studies [16–24]. Our results clearly show that inhalation of FA for 4 weeks at concentrations between 0.5 and 15 ppm does not induce genotoxic effects in peripheral blood of rats. This comprehensive study supports previous investigations suggesting that (geno-)toxic effects of FA at distant sites are highly unlikely and confirms the view that
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G. Speit et al. / Mutation Research 677 (2009) 76–85
Table 2 DNA-migration (tail moment) in peripheral blood of rats exposed to formaldehyde (FA) by inhalation measured by the alkaline comet assay with irradiation. Dose [ppm]
Seq. no.a
With irradiation
Mean
Group mean (±S.D.)b
P-value (t-test)
MMS
3 5 16 20 37 39
28.92 27.48 28.07 30.90 24.28 26.90
28.69 23.28 28.60 30.50 29.64 24.84
28.81 25.38 28.34 30.70 26.96 25.87
27.68 (2.00)
0.0001
0
23 24 29 36 52 54
2.55 2.58 1.82 3.22 2.42 3.13
2.23 2.51 2.25 3.04 2.30 2.28
2.39 2.55 2.04 3.13 2.36 2.71
2.53 (0.34)
0.5
2 4 19 31 40 48
3.07 3.57 2.10 2.32 3.23 3.39
2.97 3.26 3.06 2.76 3.42 3.26
3.02 3.42 2.58 2.54 3.33 3.33
3.03 (0.39)
0.19
1
1 18 44 45 47 53
3.93 2.06 1.21 3.15 1.51 2.30
3.68 2.45 1.19 3.71 1.35 2.78
3.81 2.26 1.20 3.43 1.43 2.54
2.44 (1.04)
0.83
2
8 11 22 26 28 43
2.87 0.91 2.41 2.24 1.85 3.75
2.41 1.14 2.85 2.71 1.94 3.14
2.64 1.03 2.63 2.48 1.90 3.45
2.35 (0.82)
0.65
6
6 9 13 14 27 34
2.36 1.00 2.61 2.44 2.39 2.45
1.37 1.02 2.33 2.48 2.64 2.14
1.87 1.01 2.47 2.46 2.52 2.30
2.10 (0.59)
0.26
10
7 15 21 30 32 38
2.86 2.07 2.75 1.96 2.86 2.34
2.37 2.07 2.64 2.17 2.55 2.45
2.62 2.07 2.70 2.07 2.71 2.40
2.42 (0.30)
0.79
15
10 12 17 25 41 50
0.84 1.24 2.18 2.14 2.71 1.90
1.02 1.39 2.74 2.82 2.83 1.91
0.93 1.32 2.46 2.48 2.77 1.91
1.98 (0.73)
0.15
a b
Sequence number of the blood sample. Standard deviation.
positive results obtained in human bio-monitoring studies are not related to exposure to FA.
The animals were approximately 9 weeks old at the beginning of the experiment. The study was approved by the local authorizing agency for animal experiments (Landesuntersuchungsanstalt Koblenz, Germany).
2. Materials and methods 2.1. Animals
2.2. Inhalation procedure and analysis
Male Fischer-344 (F-344/DuCrl) rats, specific pathogen-free, approximately 7 weeks old, were purchased from Charles River Laboratories (Research Models and Services GmbH; Sulzfeld, Germany). Animals were maintained in an AAALAC-accredited facility. All procedures for animal care and exposure were conducted under the rule of the German Animal Welfare Act (1998). The animals were housed socially (3–8 animals per cage) in H-Temp (PSU) cages (floor area 610 mm × 435 mm × 215 mm; TECNIPLAST, Germany) in accommodation maintained at 20–24 ◦ C, with a relative humidity of 30–70% and a light/dark cycle of 12 h (beginning 6:00 AM). The rats were allowed to acclimatize to these conditions for approximately 2 weeks before the start of the study. In home cages the animals were allowed free access to rat laboratory diet (Provimi Kliba SA, Basel, Switzerland) and water, but not during the exposure.
Animals were randomly divided in seven groups of six rats/group. The rats were exposed (whole-body) to 0, 0.5, 1, 2, 6, 10 or 15 ppm formaldehyde for 4 weeks (5 days/week, 6 h/day). The exposures were conducted in 1.4-m3 stainless-steel and glass inhalation chambers, where the animals were maintained individually in wire cages (DK III, BECKER & Co., Castrop-Rauxel, Germany). To generate the test atmosphere, constant amounts of formalin were supplied to tempered vaporizers by means of metering pumps. The vapors were mixed with streams of conditioned air and introduced into the chamber air supply and were removed later by an exhaust air system. The air change rate was 20 times per hour. Formaldehyde concentrations were measured using a calibrated Fourier-transform infrared spectrophotometer Gasmet DX4000 (Ansyco, Karlsruhe, Germany). Formaldehyde concentrations in the analytical chambers were measured during the 4 weeks of exposure and averaged
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79
Table 3 DNA migration (tail intensity) in peripheral blood of rats exposed to formaldehyde (FA) by inhalation measured by the standard alkaline comet assay. Dose [ppm]
Seq. no.a
Without irradiation
MMS
3 5 16 20 37 39
44.89 51.64 59.12 55.94
51.43 47.24 58.45 53.54
48.16 49.44 58.79 54.74
0
23 24 29 36 52 54
1.65 1.80 2.24 2.40 1.76 2.67
2.15 1.99 2.50 2.28 1.80 1.94
1.90 1.90 2.37 2.34 1.78 2.31
0.5
2 4 19 31 40 48
2.41 2.24 1.91 3.17
2.15 1.75 1.92 2.98
2.28 2.00 1.92 3.08
1
1 18 44 45 47 53
2.28 1.90 2.95 1.86 2.37
1.83 1.66 2.65 1.76 2.03
2.06 1.78 2.80 1.81 2.20
2
8 11 22 26 28 43
2.39 2.47 1.79 1.72 2.16 1.81
1.90 2.09 1.69 2.58 1.63 1.68
6
6 9 13 14 27 34
1.96 2.14 2.33 1.57 1.72 1.33
10
7 15 21 30 32 38
15
10 12 17 25 41 50
a b
Mean
Group mean (±S.D.)b
P-value (t-test)
52.78 (4.91)
0.0002
2.10 (0.27)
2.32 (0.53)
0.39
2.13 (0.41)
0.90
2.15 2.28 1.74 2.15 1.90 1.75
1.99 (0.23)
0.64
1.48 2.10 1.95 1.67 1.52 1.56
1.72 2.12 2.14 1.62 1.62 1.45
1.78 (0.29)
0.16
1.48 1.61 1.31 3.69 1.65
1.48 2.29 1.65 2.12 1.54
1.48 1.95 1.48 2.91 1.60
1.88 (0.60)
0.36
2.09 2.51 1.84 2.29 1.87 2.21
2.28 3.24 2.71 1.66 1.93 1.70
2.19 2.88 2.28 1.98 1.90 1.96
2.19 (0.36)
0.67
Sequence number of the blood sample. Standard deviation.
14.96 ± 0.05, 10.00 ± 0.39, 6.12 ± 0.34, 2.02 ± 0.15, 1.00 ± 0.11, 0.51 ± 0.05 and 0 ppm on a daily basis. 2.3. Study design This study was performed to investigate potential local (site of contact) and systemic toxic and genotoxic effects of FA exposure. Only results from genotoxicity tests with peripheral blood are reported here. In addition to the formaldehyde-exposed animals, two groups of positive control animals were included. One group of animals (six males) was treated twice orally, with an interval of 24 h, with 10 mg/kg bw/day cyclophosphamide (CP). This is the standard positive control for the in vivo MNT with peripheral blood. For the other endpoints (SCE test, comet assay), a second group of positive control animals was treated once by gavage with MMS (50 mg/kg bw) 4 h before blood samples were taken. The inhalation study was performed at BASF SE (Department of Experimental Toxicology and Ecology) in accordance with the OECD Principles of Good Laboratory Practice (GLP). During the exposure-free days, a check for moribund or dead animals was carried out twice per day on working days and once per day on weekends. In addition, a clinical inspection of the animals exposed to formaldehyde was performed at least
three times on exposure days, once a day during the acclimatization period, and on the day of necropsy. The positive control animals were examined once a day (except weekends) for clinical abnormalities. Signs and findings were recorded for each animal. Body weight was determined weekly for all animals. At the end of the exposure period, blood sampling was carried out in a randomized sequence and the samples were coded by sequence number. Blood samples were taken by puncturing the retro-orbital venous plexus and collected in tubes containing an anticoagulant (heparin) for the comet assay and the SCE test. These blood samples were stored on ice and immediately carried to the laboratory at Ulm University, where the comet assay and the SCE test were started immediately (i.e. about 4 h after blood sampling). For the MNT (Litron MicroFlow Rat Micronucleus Analysis Kit), approximately 120 l of blood were collected from each animal into a tube containing 300 l anticoagulant solution. Blood samples were fixed in ultra-cold methanol within 2 h according to the manufacturer’s recommendations and stored at −75 ◦ C until they were sent on dry ice to the laboratory at Novartis Pharma, where the test was performed. After blind evaluation of all tests, the complete raw data sets were sent to BASF, where they were decoded. After decoding, final assessment and statistical analysis of the test results was performed at the Ulm University (comet assay and SCE test) and at Novartis Pharma AG (MNT).
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G. Speit et al. / Mutation Research 677 (2009) 76–85
Table 4 DNA migration (tail intensity) in peripheral blood of rats exposed to formaldehyde (FA) by inhalation measured by the alkaline comet assay with irradiation. Dose [ppm]
Seq. no.a
With irradiation
Mean
Group mean (±S.D.)b
P-value (t-test)
MMS
3 5 16 20 37 39
65.84 63.42 66.22 68.91 59.14 70.08
66.65 58.80 68.36 71.57 67.01 65.04
66.25 61.11 67.29 70.24 63.08 67.56
65.92 (3.30)
0.001
0
23 24 29 36 52 54
11.23 11.04 9.77 13.01 10.96 12.96
10.14 10.79 11.74 11.94 9.73 9.99
10.69 10.92 10.76 12.48 10.35 11.48
11.11 (0.77)
0.5
2 4 19 31 40 48
12.60 15.20 10.61 9.65 14.43 13.43
12.20 13.72 11.88 11.54 13.04 13.35
12.40 14.46 11.25 10.60 13.74 13.39
12.64 (1.50)
0.17
1
1 18 44 45 47 53
15.51 9.89 7.54 13.94 8.75 10.36
14.80 10.42 7.20 14.67 8.05 12.24
15.16 10.16 7.37 14.31 8.40 11.30
11.11 (3.13)
0.99
2
8 11 22 26 28 43
13.38 6.84 10.48 10.50 9.48 13.77
11.72 8.46 11.45 12.79 10.21 12.34
12.55 7.65 10.97 11.65 9.85 13.06
10.95 (1.98)
0.89
6
6 9 13 14 27 34
10.10 7.03 11.97 11.59 11.92 11.92
8.96 7.47 11.79 11.72 11.16 10.76
9.53 7.25 11.88 11.66 11.54 11.34
10.53 (1.82)
0.60
10
7 15 21 30 32 38
13.33 10.06 11.78 9.78 12.10 12.31
11.41 8.87 10.55 10.85 12.02 12.66
12.37 9.47 11.17 10.32 12.06 12.49
11.31 (1.22)
0.85
15
10 12 17 25 41 50
6.42 8.44 10.33 10.55 11.34 10.22
7.66 9.64 12.38 12.53 11.68 10.53
7.04 9.04 11.36 11.54 11.51 10.38
10.14 (1.80)
0.38
a b
Sequence number of the blood sample. Standard deviation.
2.4. Comet assay
2.5. Sister chromatid exchange test (SCE test)
The comet assay was performed according to our standard protocol for human blood [7] with 3 l whole rat blood per slide, a 25-min alkali denaturation period, and electrophoresis (0.86 V/cm) during 25 min at pH > 13. Determination of DPX by the reduction of induced DNA migration was accomplished by irradiating blood samples with 2 Gy ␥-rays (Cs-137; Gammacell 2000, Nuclear Data, Frankfurt, Germany) immediately before conducting the comet assay. Images of 50 randomly selected cells stained with ethidium bromide were analysed from each of two parallel slides. Measurements were made by image analysis (Comet Assay II, Perceptive Instruments). For all experiments, we evaluated two image-analysis parameters: tail intensity and tail moment. Tail moment (TM) is calculated according to the formula: TM = (tail intensity/total comet intensity) × (tail centre of gravity − peak position). Statistical analysis was performed for the results of the comet assay using the twotailed Student’s t-test and ANOVA (analysis of variance) followed by a Dunnett test. In addition, the non-parametric Wilcoxon rank test was performed which never led to a divergent result. A statistically significant difference between mean values of the negative control group and the groups exposed to FA or between the negative control group and the positive control group was set at P < 0.05.
SCE tests were performed as previously described for human blood [7] with some modifications. Rat blood samples were carefully washed with 1× PBS. 300 l of the washed blood were then added to 5 ml RPMI 1640 + GlutaMax medium into a T25 cell culture flask. Concanavalin A (20 g/ml) was added and cultures were incubated at 37 ◦ C and 5% CO2 . After 21 h, BrdU (10 g/ml final concentration) was added and cultures were incubated for another 35 h (including exposure to 2 × 10−7 M colcemid for the final 2 h). Chromosome preparation was done following standard procedures. Cells were centrifuged, resuspended in 0.56% KCl for 10 min and fixed three times in methanol:glacial acetic acid (3 + 1). For sister chromatid differentiation, air-dried slides were covered with Sörensen buffer (pH 6.8) and irradiated with an 8-W UV lamp (254 nm) at a distance of 10 cm for 30 min. Subsequently, slides were incubated in 2× SSC for 20 min at 58 ◦ C and then stained with 7% Giemsa in Sörensen buffer. SCE were scored in 30 well-spread and well-stained metaphases per sample. Statistical analysis was performed for the results of the SCE test using the one-tailed Student’s t-test and ANOVA (analysis of variance) followed by a Dunnett test. In addition, the non-parametric Wilcoxon rank test was performed which never led to a divergent result. A statistically significant difference between mean values of the negative
G. Speit et al. / Mutation Research 677 (2009) 76–85 control group and the groups exposed to FA or between the negative control group and the positive control group was set at P < 0.05. 2.6. Peripheral blood micronucleus test (MNT) The MNT with peripheral blood was performed by flow cytometric analysis using the MicroFlow Rat Micronucleus Analysis Kit [25]. On the day of analysis, blood samples were simultaneously treated with 1 mg/ml RNase and anti-CD71-FITC. Fluorescein isothiocyanate-conjugated antibody against the rat transferrin receptor anti-CD71-FITC was used to differentially label and score cells with high transferrin receptor content i.e., the youngest fraction of reticulocytes. The cells were incubated at 4 ◦ C for 30 min and then at 2 ◦ C for 90 min to ensure complete degradation of cellular RNA. The cells were then kept at 4 ◦ C until analysis. Immediately prior to analysis, 1 ml ice-cold propidium iodide staining solution (PI; 1.25 g/ml bicarbonate-buffered saline) was added to a malaria-infected blood sample (biological standard purchased from Litron, NY, USA) for flow cytometer set-up and calibration [26,27]. After the flow cytometer was calibrated, 1 ml cold PI was added to one sample at a time and each was analysed by high-speed flow cytometry using the settings guided by malaria-infected blood standards. The percentage of reticulocytes (RET) among all erythrocytes (RET plus normochromatic erythrocytes, NCE) and the percentage of reticulocytes with MN (MN-RET) were determined. Statistical evaluation was done with SAS V8.2 TS 2M0. ANOVA and linear trend test were performed. Since only males were used, “treatment group” was used as a fixed factor then 1-way ANOVA was performed. For the one-tailed linear trend test, a linear regression with dose as an independent variable was done. The significance level used was ≤0.05.
3. Results 3.1. Clinical examination During the whole study period there were no substance-related clinical signs of toxicity. The body-weight development of the animals exposed to formaldehyde up to 6 ppm was comparable with that of the control animals, so was that of animals receiving CP and MMS. Only the body-weight gain of rats exposed to 10 ppm (day 7 only) and 15 ppm FA was transiently decreased (−16.7% to −32.7%) at the beginning of the exposure period. This effect was no longer statistically significant toward the end of the exposure, although the average body-weight gain of the 15-ppm group was still slightly lower than in the control group. 3.2. Comet assay Tables 1–4 summarize the results for the comet assay with blood from rats exposed to FA by inhalation. Slides from six animals (2 of the positive control group, 2 of the 0.5-ppm group, 1 of the 1-ppm group and 1 of the 10-ppm group) could not be evaluated because of a technical problem during electrophoresis. Tables 1 and 2 comprise the tail-moment results in the absence and presence of gamma irradiation and Tables 3 and 4 those for the tail intensity, respectively. In the absence of irradiation, the effects in the comet assay are either due to DNA strand breaks (increased migration in comparison with the negative control) or cross-links (DPX in the case of FA) indicated by a decreased DNA migration. Because the background levels of DNA migration under standard conditions are low, the detection of DPX is difficult. Therefore, parallel slides were irradiated (2 Gy gamma-radiation) to induce a defined amount of strand breaks and to increase DNA migration. In the presence of DPX, a reduction in gamma ray-induced DNA migration (relative to the negative control after gamma irradiation) is expected. Under standard conditions, the positive control substance MMS caused a clear and statistically significant increase in DNA migration and this effect was enhanced by gamma irradiation. No statistically significant effect in the comet assay was measured for any of the dose groups either in the absence or in the presence of gamma irradiation (ANOVA: tail moment without irradiation: P = 0.2823; tail moment with irradiation: P = 0.1749; tail intensity without irradiation: P = 0.3478; tail intensity with irradiation: P = 0.4137). These results indicate that under the conditions of this study, FA concen-
81
trations up to 15 ppm in a 28-day inhalation study did not induce genotoxic effects in the comet assay. The results excluded a DNA strand breaking effect, the induction of alkali-labile sites (ALS) as well as a cross-linking effect of FA in leukocytes of rats exposed to FA by inhalation. 3.3. SCE test The results for the SCE test with cultured rat lymphocytes are summarized in Table 5. Blood cultures from six animals (2 of the positive control group, 2 of the 6-ppm group and 2 of the 10-ppm group) could not be evaluated due to problems with blood coagulation. The positive control substance MMS clearly induced SCE. The mean SCE frequency (9.7/cell) was about twice the mean value of the negative control group (4.6/cell). There was no statistically significant increase in the SCE frequency in any of the groups exposed to FA (ANOVA: P = 0.8642). Consequently, FA did not induce SCE in cultured lymphocytes of rats exposed by inhalation. 3.4. MNT The results for the MNT with peripheral blood are shown in Table 6 and the statistical analysis of the MNT is summarized in Table 7. Samples from three animals, no. 2 (negative control group), no. 8 (0.5-ppm FA group) and no. 50 (cyclophosphamide group) were excluded from the MNT analysis due to poor quality. Biometric analysis revealed no statistically significant difference in the percentages of RET between treated groups and control animals and no significant difference in the trend test. The percentage of MN-RET in the positive control (CP, 2 × 10 mg/kg) group was 0.44 ± 0.11%. Biometric analysis also showed no statistically significant difference between the mean micronucleus frequencies in the treated groups and the negative control group (0.22 ± 0.18%) and no significant difference in the trend test. The mean percentage of MN-RET in the positive control (cyclophosphamide, 2 × 10 mg/kg) group was 0.63 ± 0.18%, which was statistically significantly higher than the negative control value. Under the conditions of this study, FA inhalation did not increase the frequency of MN in peripheral blood of rats. 4. Discussion Our results show that inhalation of formaldehyde does not induce genotoxic effects in peripheral blood of rats. The comet assay, the SCE test and the MNT were applied because these genetic endpoints have been shown to be sensitive indicators of FA genotoxicity in vitro and also revealed some positive results in human bio-monitoring studies. The alkaline comet assay with whole rat blood was performed according to the standard protocol and also in combination with gamma-irradiation. The standard protocol detects DNA strand breaks and alkali-labile sites (ALS) which lead to increased DNA migration. It can also detect cross-links (DNA–DNA and DNA–protein) which lead to reduced baseline DNA migration. However since the baseline DNA migration in rat leukocytes under the conditions used here is rather low (about 2% DNA in tail) the sensitivity of this protocol for the detection of cross-links is limited. The use of increased electrophoresis time was avoided because it leads to high variability between slides at different positions in the gel box (Speit et al., unpublished results). Therefore, we performed the additional modification with irradiation, which has been shown to be a very sensitive approach for the detection of cross-links and, in particular, of FA-induced DPX [5,7,28]. Inhalation of FA did not induce DNA migration in peripheral blood of rats, indicating that FA did not induce DNA strand breaks and ALS. It is still a matter of discussion, whether FA is able to induce DNA strand breaks
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Table 5 Frequency of SCE in peripheral blood of rats exposed to formaldehyde (FA) by inhalation. Dose [ppm]
Seq. no.a
Mean of 30 metaphases
MMS
3 5 16 20 37 39
10.70 10.37 8.80 8.90
0
23 24 29 36 52 54
4.63 4.47 3.70 4.60 4.50 5.57
4.58 (0.60)
0.5
2 4 19 31 40 48
4.13 5.27 5.20 4.73 5.63 4.70
4.94 (0.53)
0.27
1
1 18 44 45 47 53
4.77 5.20 4.60 4.90 4.63 4.43
4.76 (0.27)
0.59
2
8 11 22 26 28 43
4.50 4.37 4.87 5.10 5.20 5.47
4.92 (0.42)
0.30
6
6 9 13 14 27 34
4.84 (0.40)
0.47
10
7 15 21 30 32 38
4.57 3.73 4.80
4.77 (0.92)
0.60
10 12 17 25 41 50
5.50 4.33 5.47 5.43 4.03 5.37
5.02 (0.66)
0.18
15
a b
Group mean (±S.D.)b
P-value (t-test)
9.69 (0.98)
0.0004
4.37 4.67 5.10 5.23
5.97
Sequence number of the blood sample. Standard deviation.
but a recent comprehensive investigation clearly showed that DNA strand breaks do not play a significant role in FA-induced genotoxicity and DPX are the most relevant type of DNA damage induced by FA in mammalian cells [6]. Irradiation of blood samples immediately before the performance of the comet assay caused a clear increase in DNA migration but there was no reduction of this effect in rats exposed to FA as a consequence of FA-induced DPX. These comet assay results clearly indicate that FA did not induce DPX in peripheral blood of rats that can be detected by the comet assay. Previous studies have shown that DPX occur in the nasal mucosa of rats and in the upper respiratory tracts of monkeys exposed to FA [9,10], but increased amounts of DPX were not found in tissues other than the respiratory tract. No induction of DPX was measured in the bone marrow of rats exposed to airborne FA at concentrations up to 15 ppm and in the bone marrow of GSH-depleted rats at concentrations as high as 10 ppm FA [12,29]. Formation of DPX was also not
found in the bone marrow of Rhesus monkeys exposed to 6 ppm FA [30]. Our results support the previous experimental animal studies which used other methods for the detection of DPX and which all failed to demonstrate distant-site genotoxicity of inhaled FA. However, one recently published study measured comet-assay effects in lymphocytes and liver cells of rats exposed to FA (5 and 10 ppm) by inhalation for 2 weeks [31]. The authors reported a very similar dose-related increase in DNA migration in both cell types. This study was commented on and its reliability was questioned [32]. Besides the fact that systemic effects of FA are unlikely, it was surprising that increased DNA migration was measured in FA-exposed rats and similar effects were observed in liver cells and lymphocytes despite administration by inhalation. All in all, the results of this in vivo study are biologically implausible. Therefore, they should not be used for the assessment of the in vivo genotoxicity of FA.
G. Speit et al. / Mutation Research 677 (2009) 76–85
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Table 6 Frequency of micronuclei in peripheral blood of rats exposed to formaldehyde (FA). Dose [ppm]
Seq. no.a
RET [%]b
CP
33 35 42 46 49 51
0.61 0.47 0.33 0.38 0.41
0
23 24 29 36 52 54
1.56 1.28 1.61 1.65 1.51
0.5
2 4 19 31 40 48
1.36 1.52 1.67 1.50 1.52
1
1 18 44 45 47 53
1.14 1.20 1.56 1.48 1.93 1.80
2
8 11 22 26 28 43
1.58 1.59 1.60 1.56 1.56 1.45
6
6 9 13 14 27 34
1.21 1.60 1.57 1.57 1.20 1.46
10
7 15 21 30 32 38
1.36 1.45 1.57 1.83 1.77 1.66
15
10 12 17 25 41 50
1.18 1.82 1.00 1.08 1.53 1.28
a b c d
Group mean (±S.D.)c
0.44 (0.11)
1.52 (0.14)
1.51 (0.11)
0.53 0.16 0.08 0.20 0.14 0.03 0.36 0.17 0.15 0.19
0.63 (0.18)
0.22 (0.18)
0.18 (0.12)
1.52 (0.31)
0.32 (0.23)
1.56 (0.06)
0.13 0.02 0.31 0.27 0.07 0.59
0.23 (0.21)
1.44 (0.18)
0.03 0.19 0.30 0.10 0.01 0.19
0.14 (0.11)
1.61 (0.18)
0.05 0.28 0.15 0.63 0.18 0.06
0.23 (0.21)
1.32 (0.31)
0.26 0.23 0.21 0.16 0.19 0.25
0.22 (0.04)
Sequence number of the blood sample. Percentage of reticulocytes. Standard deviation. Percentage of reticulocytes with micronuclei.
RET [%]a
MN-RET [%]b
Formaldehyde ANOVA Linear regression
0.7116 0.9972
0.4923 0.2747
Cyclophosphamide ANOVA Linear regression
0.0004 n.a.
0.0047 n.a.
a
0.46 0.51 0.92 0.66 0.59
Group mean (±S.D.)
0.19 0.51 0.15 0.64 0.06 0.37
Table 7 Statistical analysis (P-value) of the micronucleus test with peripheral blood.
b
MN-RET [%]d
Percentage of reticulocytes. Percentage of reticulocytes with micronuclei.
Human bio-monitoring studies of workers exposed to FA performed by one group suggested an induction of DPX in circulating lymphocytes [23]. However, the effect was small, not correlated with the FA concentration and in the range of the control level of a previous study [33]. The authors used the K-SDS assay for the detection of DPX and argue that their method is extremely sensitive for the detection of DPX. However, this argument is not substantiated by the results presented and comparative investigations have shown that the K-SDS assay is not significantly more sensitive than the comet assay [4,34]. Therefore, doubts arose concerning the reliability of these findings [7,11]. Surprisingly, one recent biomonitoring study reported increased DNA migration in the comet assay with blood from FA-exposed subjects [19]. Given the abundance of evidence that FA predominantly induces DPX and DPX lead
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to a reduction in DNA migration in the comet assay, this result is difficult to interpret. However, the assessment of these bio-monitoring studies becomes clearer in the context of SCE test results. SCE are reciprocal exchanges of the DNA molecule in a replicating cell at stalled replication forks [35]. SCE are induced by a wide variety of DNA alterations including FA-induced DPX [4,7]. In cultured peripheral lymphocytes of experimental animals or human subjects exposed to mutagens, SCE are exclusively formed in vitro as a consequence of persisting DNA damage. Thus, increased frequencies of SCE in peripheral lymphocytes after in vivo exposure to FA requires that the lymphocytes carry increased levels of DPX at the time of sampling, that DPX are not completely repaired before the cells enter S-phase (at least 24 h after the start of the cultures) and cause the formation of SCE during replication in the presence of BrdU. Our results clearly show that FA does not induce SCE in peripheral blood of rats after exposure to FA concentrations as high as 15 ppm. In an earlier study, rats were exposed to FA (0.5, 6 and 15 ppm) for 5 days (6 h/day) and the frequencies of SCE were measured in peripheral blood. In accordance with our results, there was no induction of SCE at any of the concentrations tested [14]. Again, despite clearly negative results in in vivo genotoxicity tests under clearly defined exposure conditions and exposure to low and high FA concentrations, some genotoxic effects were published for the SCE test with peripheral lymphocytes of human subjects exposed to FA [16,19–22,24]. However, it is highly unlikely that these effects are actually related to FA exposure, because relevant exposure and induction of DPX in lymphocytes that leads to a positive SCE test cannot be expected [2]. Furthermore, ex vivo experiments with human blood have shown that DPX present in lymphocytes at the start of the culture are efficiently removed during lymphocyte culture before the cells reach the S-phase. Only very high levels of DPX at the beginning of the in vitro cultivation ensure sufficient amounts of persisting DPX until replication [7]. Such DPX levels cannot be reached by environmental and work-place exposures in peripheral lymphocytes, and increased SCE frequencies in subjects exposed to FA are very probably not related to FA exposure. Thus, a positive SCE test with lymphocytes of FAexposed human subjects does not confirm positive results for other endpoints within the same study; on the contrary, it is a strong argument against FA as the relevant exposure. In the light of the positive SCE test results reported within the same study, even the effects on DPX levels [16] and effects in the comet assay [19] have to be interpreted as not being FA-related. The MNT with peripheral blood using flow cytometric analysis is now a well-established in vivo genotoxicity test with higher sensitivity than the traditional bone-marrow MNT [25,36]. Two previous inhalation studies measuring chromosomal aberrations in bone marrow revealed clearly negative results after exposure of rats to 0.5, 3 and 15 ppm for 1 or 8 weeks (5 days/week, 6 h/day) [13] or after exposure of rats to 0.5, 6 and 15 ppm for 5 days (6 h/day) [14]. Our study confirms these negative cytogenetic tests and shows that FA does not exert a systemic clastogenic effect after inhalation for 4 weeks at concentrations as high as 15 ppm. In contrast, some human bio-monitoring studies reported positive effects in the MNT with cultured lymphocytes after exposure to relatively low FA concentrations [17–19,21]. These studies used the MNT with cytochalasin B and scored MN in binuclear cells. It can be assumed that these MN mainly arise during in vitro cell division [37,38] as a consequence of persisting damage. A comparative ex vivo study that investigated the persistence of FA-induced DPX and the formation of SCE and MN in cultured human lymphocytes, came to the conclusion that MN are induced with lower sensitivity than SCE [7]. Therefore, a positive MNT with peripheral lymphocytes cannot readily be explained by an in vivo exposure of lymphocytes to FA. Interestingly, one bio-monitoring study reported increased MN frequencies in the
absence of effects in the SCE test [18]. These results support the view that the cytogenetic effects measured are not FA-related. Another study showed, that increased MN frequencies in FAexposed subjects mainly represent centromeric MN, i.e., are due to aneugenic events [17]. However, FA mainly acts by a clastogenic mode of action [7,8]. Aneugenic effects are usually thresholded and require high substance concentrations in the target cells. Therefore, an aneugenic effect in the MNT in a bio-monitoring study with FA-exposed subjects is even less likely than a clastogenic effect and most likely not directly related to the assumed FA exposure. In summary, our in vivo study with inhalation exposure of rats for 4 weeks to FA at concentrations ranging from 0.5 to 15 ppm performed under GLP conditions, provides important information with regard to potential systemic genotoxic effects of FA. The results clearly exclude any genotoxic effect of FA in the comet assay including DPX formation, the SCE test and the MNT performed with peripheral blood. Thus our results add an important piece of evidence to the already existing experimental data suggesting that inhaled FA does not induce any systemic genotoxic effect in appropriately performed in vivo genotoxicity tests with peripheral blood. Our results are in agreement with earlier results showing the failure of inhaled FA to increase the FA concentration in the blood of rats, monkeys and humans and the lack of detectable DPX in the bone marrow of normal and GSH-depleted (metabolically inhibited) rats [2,3,11]. The results also support the view that genotoxic effects of FA reported in human bio-monitoring studies lack plausibility because positive test results cannot be expected in peripheral lymphocytes after FA inhalation [7,11]. The reported effects are either test artefacts, chance findings or due to another kind of exposure. Consequently, positive genotoxicity tests from human bio-monitoring do not support models for systemic effects of FA (including cancer) in humans exposed by inhalation. Conflict of interest No conflicts of interest. Acknowledgements We wish to thank Mrs. Petra Schütz, Mrs. Irmgard Weber and Mr. Renato Niggli for excellent technical assistance and Prof. J. Högel for helping us with the statistical analysis of the data. The financial support provided by the European Chemical Industry Council (CEFIC) is gratefully acknowledged. References [1] IARC, 2006. Formaldehyde, 2-butoxyethanol and 1-tert-butoxypropan-2-ol, pp. 1–478. [2] D. Pyatt, E. Natelson, R. Golden, Is inhalation exposure to formaldehyde a biologically plausible cause of lymphohematopoietic malignancies? Regul. Toxicol. Pharmacol. 51 (2008) 119–133. [3] R. Golden, D. Pyatt, P.G. Shields, Formaldehyde as a potential human leukemogen: an assessment of biological plausibility, Crit. Rev. Toxicol. 36 (2006) 135–153. [4] O. Merk, G. Speit, Significance of formaldehyde-induced DNA–protein crosslinks for mutagenesis, Environ. Mol. Mutagen. 32 (1998) 260–268. [5] G. Speit, O. Schmid, S. Neuss, P. Schutz, Genotoxic effects of formaldehyde in the human lung cell line A549 and in primary human nasal epithelial cells, Environ. Mol. Mutagen. 49 (2008) 300–307. [6] G. Speit, P. Schutz, J. Hogel, O. Schmid, Characterization of the genotoxic potential of formaldehyde in V79 cells, Mutagenesis 22 (2007) 387–394. [7] O. Schmid, G. Speit, Genotoxic effects induced by formaldehyde in human blood and implications for the interpretation of biomonitoring studies, Mutagenesis 22 (2007) 69–74. [8] G. Speit, O. Merk, Evaluation of mutagenic effects of formaldehyde in vitro: detection of crosslinks and mutations in mouse lymphoma cells, Mutagenesis 17 (2002) 183–187. [9] M. Casanova, K.T. Morgan, W.H. Steinhagen, J.I. Everitt, J.A. Popp, H.D. Heck, Covalent binding of inhaled formaldehyde to DNA in the respiratory tract of
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