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Cytogenetic analysis of lymphocytes from rats following formaldehyde inhalation
241
Toxicology Letters, 21 (1984) 241-246 Elsevier TOXLett 1203
CYTOGENETIC ANALYSIS OF LYMPHOCYTES FOLLOWING FORMALDEHYDE INHALATION (Formaldehyde;
A.D. KLIGERMAN,
SCE; rat lymphocyte;
chromosome
FROM RATS
breakage)
M.C. PHELPS and G.L. EREXSON
Chemical Industry Institute of Toxicology, P.O. Box 12137, Research Triangle Park, NC27709 (U.S.A.) (Received November 23rd, 1983) (Accepted December 3Oth, 1983)
SUMMARY Male and female Fischer-344 rats were exposed to target concentrations of 0.5, 6, or 15 ppm formaldehyde by inhalation for 6 h/day for 5 days. Blood was removed by cardiac puncture within 1 h following termination of exposures and cultured in the presence of 5bromodeoxyuridine (BrdU) (4 PM) for analyses of sister-chromatid exchange (SCE) and chromosome breakage. Formaldehyde did not cause a statistically significant increase in either SCE frequency or in the number of metaphases displaying chromosome aberrations.
INTRODUCTION
Formaldehyde is a widely used chemical recently shown to cause squamous cell carcinoma in the nasal turbinates of rats following exposure by inhalation to 6 and 15 ppm [ 11. Formaldehyde is also a genotoxicant causing dominant lethal mutations in Drosophila [2,3], forward [4] and reverse [5,6] mutations in Salmonella typhimurium, forward mutations in human lymphoblasts [7], chromosome aberrations in human fibroblasts [8], SCE in CHO cells and human lymphocytes in culture [9], and initiation of transformation in C3H/lOTM cells [lo]. Few studies have assessed the in vivo genotoxic potential of formaldehyde. Epstein et al. [I I] reported that i.p. injection of formaldehyde (16 to 40 mg/kg) did not induce dominant lethal mutations in the mouse. However, Fontignie-Houbrechts [12] reported an increase in dominant lethal mutations in mice from litters sired by males exposed i.p. to 50
Abbreviation:
BrdU, 5-bromodeoxyuridine;
SCE, sister-chromatid
0378-4274/84/$ 03.00 0 Elsevier Science Publishers B.V.
exchange.
242
mg formaldehyde/kg but found no effects of formaldehyde on the chromosomes of primary spermatocytes of mice. A major route of human exposure to formaldehyde is by inhalation. Because of the lack of information available on the genotoxic effects of in vivo formaldehyde exposure, we designed an experiment to investigate the cytogenetic effects of formaldehyde on lymphocytes of rats exposed by inhalation. MATERIALS AND METHODS
Animals
CDF (F-344)/CrlBR rats (180-220 g) were obtained from Charles River Breeding Laboratories, Inc. (Kingston, NY) and housed and caged as described previously [13]. Food and water were withheld during exposures. Exposures
Exposures were conducted in an 8-m3 stainless steel and glass inhalation chamber with a 2000 liter/min air flow. Temperature and humidity were monitored each hour during the exposures. Formaldehyde was generated and monitored as described previously [14]. Three animals of each sex were exposed for 6 h/day for 5 days to target concentrations of 0.5, 6, and 15 ppm formaldehyde. 15 ppm formaldehyde was chosen as the peak concentration in this study for several reasons. Previous studies [15] demonstrated that exposure to 39 ppm formaldehyde killed 11 of 20 F-344 rats during a 2-week exposure. Furthermore, 15 ppm formaldehyde is highly carcinogenic to rats in a 2-year bioassay [16]; it induces hyperplasia and nasal epithelial degeneration after only one 6-h exposure; and causes severe ulcerative rhinitis [17], mucostasis [ 181, and ciliastasis [ 181 after a 5-day exposure. Thus, a 15-ppm formaldehyde exposure for 5 days seriously impairs the defense mechanisms of the nasal cavity, a factor thought to be responsible for the non-linearity of the formaldehyde tumor response in rats [18]. Animals exposed only to room air served as concurrent controls for each separate exposure. Blood culture, microscopy,
and data analyses
Within 1 h after termination of exposures, blood was removed from the animals by cardiac puncture and cultured as described previously [ 191except that 4 FM BrdU was added at 20 h of culture. Total culture time was 54 h. For each animal 1000 nuclei, 100 metaphases, and 20 second-division metaphases were scored for mitotic activity, cell cycle kinetics, and SCE, respectively. 50 first-division metaphases were scored from each high exposure (14.8 ppm) and each control animal for chromosome aberration analysis. For statistical evaluation of SCE data, a one-way
243
analysis of variance was performed, Linear regression analysis was used to determine if there were relationships between dose and mitotic activity [20]. Student’s ttest was used to evaluate the chromosome aberration data [20]. The level of significance chosen was 0.05. Histograms of the data were plotted for detection of cells with high numbers of SCEs [21]. RESULTS AND DISCUSSION
Formaldehyde did not cause an increase in SCE frequency or chromosome aberrations at any of the concentrations examined (Table I). Histograms of the distribution of metaphases with SCEs show that there was no increase in the number of metaphases displaying high numbers of SCEs (i.e., 1 18 SCEs) at any of the concentrations examined (Fig. 1). Although there appears to be a slight inhibition of cell cycle progression in lymphocytes from formaldehyde-treated animals compared to concurrent controls (Table II), the variabilities in cell cycle kinetics among the controls precludes a meaningful interpretation of this result. Formaldehyde had no significant doserelated effect on mitotic activity. Our previous studies have shown that mutagens, such as ethylene oxide [13], 7,12-dimethylbenz[a]anthracene [22], and ethyl methanesulfonate [23], when administered by inhalation, gavage, or i.p. injection, respectively, can lead to increases in SCE frequency in lymphocytes in the peripheral blood of rats. The negative findings with formaldehyde in vivo are in contrast to several positive in vitro mutagenicity studies, but this may be explained by the pharmacokinetics and deposition of in-
TABLE I EFFECTS OF IN VIVO EXPOSURE TO FORMALDEHYDE OF CULTURED RAT LYMPHOCYTESasb Concentration 0 0.47 * 0.04 5.88 + 0.08 14.78 rt 0.27
(ppm)
ON CYTOGENETIC PARAMETERS
Number of SCE/metaphase animals
Chromosome aberrations’ (%)
Mitotic index (%)
6 6 6 6
2.0 f 1.3 n.d. n.d. 2.0 f 2.3d
2.8 4.3 3.3 2.0
9.5 9.7 8.5 9.9
+ f f *
1.0 0.6 1.0 0.5d
* t + +
2.0 0.9 1.7 1.6
a Each datum is mean f S.D. among animals; results from males and females were pooled (no significant sex-related differences); n.d., not done. b Animals exposed for 5 days, 6 h/day (temp.: 71.4 f 0.8”F; rel. hum. 59 f 3%). ’ Percentage of cells with one or more chromosome aberrations. No cell had more than one aberration. The aberration found in the controls consisted of 1 dicentric, 3 chromatid deletions, I paired fragment, and 1 isochromatid break. In the 15 ppm formaldehyde exposed animals, there were 2 chromatid deletions and 2 paired fragments. d 4 animals scored for chromosome breakage and S for SCE analyses and cell cycle kinetics due to low mitotic indices; data for controls are pooled.
244 35
35
CONTROL
r
6ppm
l-
FORMALDEHYDE
r
30 $
25
=
20
w = lb
IO
-;s
15 0 : -
5
0
35
I5 ppm
FORMALDEHYDE
30 =
NUMBER
OF
ScEs/
25
f
METAPHASE
Fig, 1. SCE frequency distribution of cultured lymph~ytes from peripheral blood of control and formaldehyde-exposed rats. Each histogram was pfotted from 120 metaphases (6 animals, 3 males and 3 females, 20 metaphases/animal) except at 15 ppm which is plotted from 100 metaphases from 5 animals owing to a low mitotic index in blood from one female rat.
haled formaldehyde. Formaldehyde is a highly reactive gas that readily binds to proteins and polysaccharides [24,25]. It is likely that most of the inhaled formaldehyde would react with the mucus and nasal epithelium before reaching the lungs or be rapidly metabolized by the erythrocytes in the blood 1261.Concordantly, it has been TABLE 11 CELL CYCLE KINETICS FOR LYMPHOCYTES CONCURRENT CONTROLS
OF FORMALDEHYDE-EXPOSED
RATS AND
Number of 1st division
2nd division
3rd and 4th division
animais
metaphases (Vo)
metaphases (%)
metaphases (To)
0.04
2 6
14.5 f 0.7 18.0 + 3.1
52.0 -f 4.2 40.7 Sk 4.3
33.5 f 41.3 f
0
2
22.0 f
41.0 * 0.0
37.0 * 2.8
5.88 + 0.08
6
32.0 t 0.5
45.2 f
3.7
22.5 f
6.1
0 14.78 * 0.27
2 5
39.5 f 9.2 48.4 rt 8.3
45.0 f 8.5 37.8 It 8.9
15.5 f 13.8 i
0.7 4.8
Concentration 0 0.47 f
(ppm)
2.8
4.9 5.4
245
found that blood from rats exposed by inhalation to 14.2 ppm formaldehyde for 2 h showed no increase in measurable amounts of free or reversibly bound formaldehyde (H. Heck and M. Casanova-Schmitz, unpublished results). Two independent investigations support our findings. Fleig et al. [27] found no increase in chromosome breakage in lymphocytes of workers manufacturing formaldehyde, and Ward et al. [28] reported no increase in SCE frequency or chromosome breakage in lymphocytes of hospital personnel performing autopsy services. In summary, our results do not dispute the fact that formaldehyde is a genotoxicant; however, they suggest that cytogenetic analysis of lymphocytes following in vivo exposure to formaldehyde will likely yield negative results. ACKNOWLEDGEMENTS
The authors would like to thank Dr. T. Starr for aid with the statistical analysis and Mr. J. Wilmer, Dr. J.- Swenberg, and Dr. B. Butterworth for valuable suggestions.
REFERENCES 1 J.A. Swenberg, W.D. Kerns, R.I. Mitchell, E.J. Gralla and K.L. Pavkov, Induction of squamous cell carcinoma of the rat nasal cavity by inhalation exposure to formaldehyde vapor, Cancer Res., 40 (1980) 3398-4402. 2 I.A. Rapoport, Carbonyl compounds and the chemical mechanism of mutation, C. R. Acad. Sci. (URSS), 54 (1946) 65-67. 3 J. Szabad, I. Sobs, G. Polgir and G. Hejja, Testing the mutagenicity of malondialdehyde and formaldehyde by the Drosophila mosaic and sex-linked recessive lethal tests, Mutation Res., 113 (1983) 117-133. 4 P. Temcharoen and W.G. Thilly, Toxic and mutagenic effects of formaldehyde in SulmoneNu typhimurium, Mutation Res., 119 (1983) 89-93. 5 C.J. Boreiko, D.B. Couch and J.A. Swenberg, Mutagenic and carcinogenic effects of formaldehyde, in R.R. Tice, D.L. Costa and K.M. Schaich (Eds.), Genotoxic Effects of Airborne Agents, Plenum, New York, 1982, pp. 353-367. 6 T.H. Connor, M.D. Barrie, J.C. Theiss, T.S. Matney and J.B. Ward, Jr., Mutagenicity of formalin in the Ames assay, Mutation Res., 119 (1983) 145-149. 7 V.S. Goldmacher and W.G. Thilly, Formaldehyde is mutagenic for cultured human cells, Mutation Res., 116 (1983) 417-422. 8 S. Levy, S. Nocentini and C. Billardon, Induction of cytogenic effects in human fibroblast cultures after exposure to formaldehyde or X-rays, Mutation Res., 119 (1983) 309-317. 9 G. Obe and B. Beek, Mutagenic activity of aldehydes, Drug Alcohol Depend., 4 (1979) 91-94. 10 D.L. Ragan and C.J. Boreiko, Initiation of C3H/lOT% cell transformation by formaldehyde, Cancer Lett., 13 (1981) 325-331. 11 S.S. Epstein, E. Arnold, J. Andrea, W. Bass and Y. Bishop, Detection of chemical mutagens by the dominant lethal assay in the mouse, Toxicol. Appl. Pharmacol., 23 (1972) 288-325. 12 N. Fontignie-Houbrechts, Genetic effects of formaldehyde in the mouse, Mutation Res., 88 (1981) 109-I 14.
246 13 A.D. Kligerman, G.L. Erexson, M.C. Phelps and J.L. Wilmer, Sister chromatid exchange induction in peripheral blood lymphocytes of rats exposed to ethylene oxide by inhalation, Mutation Res., 120 (1983) 37-44. 14 J.C.F. Chang, W.H. Steinhagen and C.S. Barrow, Effect of single and repeated formaldehyde exposure on minute volume of B6C3Fl mice and F-344 rats, Toxicol. Appl. Pharmacol., 61 (1981) 451-459. 15 Chemical Industry Institute of Toxicology, A Ninety Day Inhalation Study in F-344 Albino Rats and B6C3Fl Mice Exposed to Atmospheric Formaldehyde Vapor, Chemical Industry Institute of Toxicology, Research Triangle Park, NC, 1977, 68 pp. 16 W.D. Kerns, K.L. Pavkov, D.J. Donofrio, E.J. Gralla and J.A. Swenberg, Carcinogenicity of formaldehyde in rats and mice after long-term inhalation exposure, Cancer Res., 43 (1983) 4382-4392. 17 J.C.F. Chang, E.A. Gross, J.A. Swenberg and C.S. Barrow, Nasal cavity deposition, histopathology, and cell proliferation after single or repeated formaldehyde exposures in B6C3Fl and F-344 rats, Toxicol. Appl. Pharmacol., 68 (1983) 161-176. 18 J.A. Swenberg, C.S. Barrow, C.J. Boreiko, H. d’A. Heck, R.J. Levine, K.T. Morgan and T.B. Starr, Non-linear biological responses to formaldehyde and their implications for carcinogenic risk assessment, Carcinogenesis, 4 (1983) 945-952. 19 A.D. Kligerman, G.L. Erexson, J.L. Wilmer and M.C. Phelps, Analysis of cytogenetic damage in rat lymphocytes following in vivo exposure to nitrobenzene, Toxicol. Lett., 18 (1983) 219-226. 20 G.W. Snedecor and W.G. Cochran, Statistical Methods, The Iowa State University Press, Ames, IA, 1967, 593 pp. 21 A.V. Carrano and D.H. Moore II, The rationale and methodology for quantifying sister chromatid exchange in humans, in J.A. Heddle (Ed.), Mutagenicity: New Horizons in Genetic Toxicology, Academic Press, New York, 1982, pp. 267-304. 22 A.D. Kligerman, J.L. Wilmer and G.L. Erexson, The use of rat and mouse lymphocytes to study cytogenetic damage after in vivo exposure to genotoxic agents, in B.A. Bridges, B.E. Butterworth, I.B. Weinstein (Eds.), Banbury Report 13: Indicators of Genotoxic Exposure, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1981, pp. 277-291. 23 A.D. Kligerman, J.L. Wilmer and G.L. Erexson, Characterization of a rat lymphocyte system for assessing sister chromatid exchange after in vivo exposure to genotoxic agents, Environ. Mutagen., 3 (1981) 531-543. 24 D. French and J.T. Edsall, The reactions of formaldehyde with amino acids and proteins, Adv. Prot. Chem., 2 (1945) 277-335. 25 Y. Kihara, M. Kasuya and K. Tanaka, Reaction of aldehydes on starch, Denpun Kogyo Gakkaishi, 10 (1982) l-6. 26 G. Malorny, N. Rietbrock and M. Schneider, Die Oxydation des Formaldehyds zu Ameisenslure im Blut, ein Beitrag rum Stoffwechsel des Formaldehyds, Arch. Exp. Pathol. Pharmakol., 250 (1965) 419-436. 27 I. Fleig, N. Petri, W.G. Stocker and A.M. Theiss, Cytogenetic analyses of blood lymphocytes of workers exposed to formaldehyde in formaldehyde manufacturing and processing, J. Occup. Med., 24 (1982) 1009-1012. 28 J.B. Ward, Jr., M.S. Legator, L.W. Chang and M.A. Periera, Evaluation of occupational exposure to formaldehyde using a battery of tests for genetic exposure, Environ. Mutagen., 5 (1983) 433.
Toxicology Letters, 21 (1984) 241-246 Elsevier TOXLett 1203
CYTOGENETIC ANALYSIS OF LYMPHOCYTES FOLLOWING FORMALDEHYDE INHALATION (Formaldehyde;
A.D. KLIGERMAN,
SCE; rat lymphocyte;
chromosome
FROM RATS
breakage)
M.C. PHELPS and G.L. EREXSON
Chemical Industry Institute of Toxicology, P.O. Box 12137, Research Triangle Park, NC27709 (U.S.A.) (Received November 23rd, 1983) (Accepted December 3Oth, 1983)
SUMMARY Male and female Fischer-344 rats were exposed to target concentrations of 0.5, 6, or 15 ppm formaldehyde by inhalation for 6 h/day for 5 days. Blood was removed by cardiac puncture within 1 h following termination of exposures and cultured in the presence of 5bromodeoxyuridine (BrdU) (4 PM) for analyses of sister-chromatid exchange (SCE) and chromosome breakage. Formaldehyde did not cause a statistically significant increase in either SCE frequency or in the number of metaphases displaying chromosome aberrations.
INTRODUCTION
Formaldehyde is a widely used chemical recently shown to cause squamous cell carcinoma in the nasal turbinates of rats following exposure by inhalation to 6 and 15 ppm [ 11. Formaldehyde is also a genotoxicant causing dominant lethal mutations in Drosophila [2,3], forward [4] and reverse [5,6] mutations in Salmonella typhimurium, forward mutations in human lymphoblasts [7], chromosome aberrations in human fibroblasts [8], SCE in CHO cells and human lymphocytes in culture [9], and initiation of transformation in C3H/lOTM cells [lo]. Few studies have assessed the in vivo genotoxic potential of formaldehyde. Epstein et al. [I I] reported that i.p. injection of formaldehyde (16 to 40 mg/kg) did not induce dominant lethal mutations in the mouse. However, Fontignie-Houbrechts [12] reported an increase in dominant lethal mutations in mice from litters sired by males exposed i.p. to 50
Abbreviation:
BrdU, 5-bromodeoxyuridine;
SCE, sister-chromatid
0378-4274/84/$ 03.00 0 Elsevier Science Publishers B.V.
exchange.
242
mg formaldehyde/kg but found no effects of formaldehyde on the chromosomes of primary spermatocytes of mice. A major route of human exposure to formaldehyde is by inhalation. Because of the lack of information available on the genotoxic effects of in vivo formaldehyde exposure, we designed an experiment to investigate the cytogenetic effects of formaldehyde on lymphocytes of rats exposed by inhalation. MATERIALS AND METHODS
Animals
CDF (F-344)/CrlBR rats (180-220 g) were obtained from Charles River Breeding Laboratories, Inc. (Kingston, NY) and housed and caged as described previously [13]. Food and water were withheld during exposures. Exposures
Exposures were conducted in an 8-m3 stainless steel and glass inhalation chamber with a 2000 liter/min air flow. Temperature and humidity were monitored each hour during the exposures. Formaldehyde was generated and monitored as described previously [14]. Three animals of each sex were exposed for 6 h/day for 5 days to target concentrations of 0.5, 6, and 15 ppm formaldehyde. 15 ppm formaldehyde was chosen as the peak concentration in this study for several reasons. Previous studies [15] demonstrated that exposure to 39 ppm formaldehyde killed 11 of 20 F-344 rats during a 2-week exposure. Furthermore, 15 ppm formaldehyde is highly carcinogenic to rats in a 2-year bioassay [16]; it induces hyperplasia and nasal epithelial degeneration after only one 6-h exposure; and causes severe ulcerative rhinitis [17], mucostasis [ 181, and ciliastasis [ 181 after a 5-day exposure. Thus, a 15-ppm formaldehyde exposure for 5 days seriously impairs the defense mechanisms of the nasal cavity, a factor thought to be responsible for the non-linearity of the formaldehyde tumor response in rats [18]. Animals exposed only to room air served as concurrent controls for each separate exposure. Blood culture, microscopy,
and data analyses
Within 1 h after termination of exposures, blood was removed from the animals by cardiac puncture and cultured as described previously [ 191except that 4 FM BrdU was added at 20 h of culture. Total culture time was 54 h. For each animal 1000 nuclei, 100 metaphases, and 20 second-division metaphases were scored for mitotic activity, cell cycle kinetics, and SCE, respectively. 50 first-division metaphases were scored from each high exposure (14.8 ppm) and each control animal for chromosome aberration analysis. For statistical evaluation of SCE data, a one-way
243
analysis of variance was performed, Linear regression analysis was used to determine if there were relationships between dose and mitotic activity [20]. Student’s ttest was used to evaluate the chromosome aberration data [20]. The level of significance chosen was 0.05. Histograms of the data were plotted for detection of cells with high numbers of SCEs [21]. RESULTS AND DISCUSSION
Formaldehyde did not cause an increase in SCE frequency or chromosome aberrations at any of the concentrations examined (Table I). Histograms of the distribution of metaphases with SCEs show that there was no increase in the number of metaphases displaying high numbers of SCEs (i.e., 1 18 SCEs) at any of the concentrations examined (Fig. 1). Although there appears to be a slight inhibition of cell cycle progression in lymphocytes from formaldehyde-treated animals compared to concurrent controls (Table II), the variabilities in cell cycle kinetics among the controls precludes a meaningful interpretation of this result. Formaldehyde had no significant doserelated effect on mitotic activity. Our previous studies have shown that mutagens, such as ethylene oxide [13], 7,12-dimethylbenz[a]anthracene [22], and ethyl methanesulfonate [23], when administered by inhalation, gavage, or i.p. injection, respectively, can lead to increases in SCE frequency in lymphocytes in the peripheral blood of rats. The negative findings with formaldehyde in vivo are in contrast to several positive in vitro mutagenicity studies, but this may be explained by the pharmacokinetics and deposition of in-
TABLE I EFFECTS OF IN VIVO EXPOSURE TO FORMALDEHYDE OF CULTURED RAT LYMPHOCYTESasb Concentration 0 0.47 * 0.04 5.88 + 0.08 14.78 rt 0.27
(ppm)
ON CYTOGENETIC PARAMETERS
Number of SCE/metaphase animals
Chromosome aberrations’ (%)
Mitotic index (%)
6 6 6 6
2.0 f 1.3 n.d. n.d. 2.0 f 2.3d
2.8 4.3 3.3 2.0
9.5 9.7 8.5 9.9
+ f f *
1.0 0.6 1.0 0.5d
* t + +
2.0 0.9 1.7 1.6
a Each datum is mean f S.D. among animals; results from males and females were pooled (no significant sex-related differences); n.d., not done. b Animals exposed for 5 days, 6 h/day (temp.: 71.4 f 0.8”F; rel. hum. 59 f 3%). ’ Percentage of cells with one or more chromosome aberrations. No cell had more than one aberration. The aberration found in the controls consisted of 1 dicentric, 3 chromatid deletions, I paired fragment, and 1 isochromatid break. In the 15 ppm formaldehyde exposed animals, there were 2 chromatid deletions and 2 paired fragments. d 4 animals scored for chromosome breakage and S for SCE analyses and cell cycle kinetics due to low mitotic indices; data for controls are pooled.
244 35
35
CONTROL
r
6ppm
l-
FORMALDEHYDE
r
30 $
25
=
20
w = lb
IO
-;s
15 0 : -
5
0
35
I5 ppm
FORMALDEHYDE
30 =
NUMBER
OF
ScEs/
25
f
METAPHASE
Fig, 1. SCE frequency distribution of cultured lymph~ytes from peripheral blood of control and formaldehyde-exposed rats. Each histogram was pfotted from 120 metaphases (6 animals, 3 males and 3 females, 20 metaphases/animal) except at 15 ppm which is plotted from 100 metaphases from 5 animals owing to a low mitotic index in blood from one female rat.
haled formaldehyde. Formaldehyde is a highly reactive gas that readily binds to proteins and polysaccharides [24,25]. It is likely that most of the inhaled formaldehyde would react with the mucus and nasal epithelium before reaching the lungs or be rapidly metabolized by the erythrocytes in the blood 1261.Concordantly, it has been TABLE 11 CELL CYCLE KINETICS FOR LYMPHOCYTES CONCURRENT CONTROLS
OF FORMALDEHYDE-EXPOSED
RATS AND
Number of 1st division
2nd division
3rd and 4th division
animais
metaphases (Vo)
metaphases (%)
metaphases (To)
0.04
2 6
14.5 f 0.7 18.0 + 3.1
52.0 -f 4.2 40.7 Sk 4.3
33.5 f 41.3 f
0
2
22.0 f
41.0 * 0.0
37.0 * 2.8
5.88 + 0.08
6
32.0 t 0.5
45.2 f
3.7
22.5 f
6.1
0 14.78 * 0.27
2 5
39.5 f 9.2 48.4 rt 8.3
45.0 f 8.5 37.8 It 8.9
15.5 f 13.8 i
0.7 4.8
Concentration 0 0.47 f
(ppm)
2.8
4.9 5.4
245
found that blood from rats exposed by inhalation to 14.2 ppm formaldehyde for 2 h showed no increase in measurable amounts of free or reversibly bound formaldehyde (H. Heck and M. Casanova-Schmitz, unpublished results). Two independent investigations support our findings. Fleig et al. [27] found no increase in chromosome breakage in lymphocytes of workers manufacturing formaldehyde, and Ward et al. [28] reported no increase in SCE frequency or chromosome breakage in lymphocytes of hospital personnel performing autopsy services. In summary, our results do not dispute the fact that formaldehyde is a genotoxicant; however, they suggest that cytogenetic analysis of lymphocytes following in vivo exposure to formaldehyde will likely yield negative results. ACKNOWLEDGEMENTS
The authors would like to thank Dr. T. Starr for aid with the statistical analysis and Mr. J. Wilmer, Dr. J.- Swenberg, and Dr. B. Butterworth for valuable suggestions.
REFERENCES 1 J.A. Swenberg, W.D. Kerns, R.I. Mitchell, E.J. Gralla and K.L. Pavkov, Induction of squamous cell carcinoma of the rat nasal cavity by inhalation exposure to formaldehyde vapor, Cancer Res., 40 (1980) 3398-4402. 2 I.A. Rapoport, Carbonyl compounds and the chemical mechanism of mutation, C. R. Acad. Sci. (URSS), 54 (1946) 65-67. 3 J. Szabad, I. Sobs, G. Polgir and G. Hejja, Testing the mutagenicity of malondialdehyde and formaldehyde by the Drosophila mosaic and sex-linked recessive lethal tests, Mutation Res., 113 (1983) 117-133. 4 P. Temcharoen and W.G. Thilly, Toxic and mutagenic effects of formaldehyde in SulmoneNu typhimurium, Mutation Res., 119 (1983) 89-93. 5 C.J. Boreiko, D.B. Couch and J.A. Swenberg, Mutagenic and carcinogenic effects of formaldehyde, in R.R. Tice, D.L. Costa and K.M. Schaich (Eds.), Genotoxic Effects of Airborne Agents, Plenum, New York, 1982, pp. 353-367. 6 T.H. Connor, M.D. Barrie, J.C. Theiss, T.S. Matney and J.B. Ward, Jr., Mutagenicity of formalin in the Ames assay, Mutation Res., 119 (1983) 145-149. 7 V.S. Goldmacher and W.G. Thilly, Formaldehyde is mutagenic for cultured human cells, Mutation Res., 116 (1983) 417-422. 8 S. Levy, S. Nocentini and C. Billardon, Induction of cytogenic effects in human fibroblast cultures after exposure to formaldehyde or X-rays, Mutation Res., 119 (1983) 309-317. 9 G. Obe and B. Beek, Mutagenic activity of aldehydes, Drug Alcohol Depend., 4 (1979) 91-94. 10 D.L. Ragan and C.J. Boreiko, Initiation of C3H/lOT% cell transformation by formaldehyde, Cancer Lett., 13 (1981) 325-331. 11 S.S. Epstein, E. Arnold, J. Andrea, W. Bass and Y. Bishop, Detection of chemical mutagens by the dominant lethal assay in the mouse, Toxicol. Appl. Pharmacol., 23 (1972) 288-325. 12 N. Fontignie-Houbrechts, Genetic effects of formaldehyde in the mouse, Mutation Res., 88 (1981) 109-I 14.
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