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Sperm head abnormalities and dominant lethal effects of formaldehyde in albino rats
Mutation Research 389 Ž1997. 141–148
Sperm head abnormalities and dominant lethal effects of formaldehyde in albino rats P.G.C. Odeigah Genetics Laboratory, Department of Biological Sciences, UniÕersity of Lagos, Akoka, Lagos, Nigeria Received 20 March 1996; revised 30 July 1996; accepted 17 September 1996
Abstract The effects of formaldehyde exposure have been investigated in rats by two short-term in vivo mutagenicity tests Žsperm head abnormality and dominant lethal mutation assays.. Five daily interperitonial injections of formaldehyde resulted in a statistically significant increase in induction of sperm head abnormalities at 0.125 to 0.500 mgrkg. The frequency of dominant lethal mutations in female rats sired by males exposed to formaldehyde was significantly higher than the control group. There was also a reduction of fertile matings in females mated 1–7 days after treatment of males with formaldehyde. The genetic and environmental health implications of the results are discussed. Keywords: Mammalian Mutagenicity assay; Formaldehyde exposure; Rat
1. Introduction In recent years, there has been an increasing awareness of the genotoxic potential of a wide variety of drugs and chemicals to which the human population is exposed either environmentally or occupationally. This awareness is paralleled by the recent development of appropriate, sensitive and practical methods for detecting and estimating the effects of these substances. The extensive use of formaldehyde in many domestic products, laboratory and industrial processes has generated considerable interest in its effects. Although the mutagenicity of formaldehyde was reported by Rapoport as early as 1946 w1x, most of the subsequent studies were concentrated in Drosophila w2x. Studies of its mutagenic effects in mammals are few and mostly limited to in vitro experiments. Increases in chromosomal aberration frequencies
have been reported in Chinese hamster ovary cells in culture w3x and in cultured human lymphocytes w4,5x as a result of formaldehyde treatment. In contrast, no such increases were observed in cultured human lymphocytes of pathology workers w6x or by in vivo tests on bone marrow and spermatocytes of mice w3,7x. Increases in the frequencies of siter-chromatid exchange have been observed in cultured human peripheral blood lymphocytes treated with formaldehyde w8x and in the lymphocytes of a small group of anatomy students exposed to formaldehydeembalming solution w9x. The effects of formaldehyde on the DNA repair processes in human fibroblasts w10x and its induction of nasal tumours in rats w11x and mice w12x have also been noted. A large number of the chemical, cytological and histological analyses of the effects of formaldehyde in mammalian systems have been carried out by examination of in vitro tissue culture materials. This
1383-5718r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 1 3 8 3 - 5 7 1 8 Ž 9 6 . 0 0 1 3 6 - X
142
P.G.C. Odeigahr Mutation Research 389 (1997) 141–148
study explores further the effects of formaldehyde exposure in rats using two short-term in vivo mutagenicity assays Žsperm head abnormality and dominant lethal mutation tests..
2. Materials and methods Animal husbandry. Isogenic strains of albino rats Ž Rattus norÕegicus. were obtained from the Department of Biological Sciences, small animal colony, University of Lagos. All animals were housed in suspended mesh-bottomed cages and maintained on food ŽPfizer products, pellets. and water ad libitum. Rats used for the experiment were 12 to 14 weeks old. Male and female rats used for the sperm assay and dominant lethal tests were litter mates. Test substance. Formaldehyde, the test substance, is of reagent grade and was obtained from Sigma Chemical Co. as 37% solution Žstabilised with 10% methanol.. The doses used in the study were prepared on the basis that 1 litre is equivalent to 1.08 kg Ždensity of the liquid..
3. Preliminary acute toxicity studies The mean lethal dose ŽLD50 . of formaldehyde administered interperitonially to the rats was determined before carrying out the sperm head abnormality and dominant lethal mutation assays. Fifty rats were used, and a mean lethal dose of 2 mgrkg body weight was obtained. Symptoms of toxicity of lethal doses include growth retardation, weight loss, lack of appetite and visual impairment Žranging from red spots in the eyes to conjunctivitis and complete blindness.. Three sublethal doses Ž1r4 to 1r16 LD50 dose. were used to test for sperm head abnormalities and dominant lethal mutations. Sperm head abnormality assay. This was carried out according to the method of Topham w13x. Briefly, a batch of 24 rats Ž6 per control or dose of test substance. received 5 daily interperitonial injections of vehicle alone, water Ž5 mlrkgrday. or formaldehyde at 0.125 mgrkg, 0.250 mgrkg and 0.50 mgrkg body weight. The rats were sacrificed 3 weeks after the last injection, and the sperm countrmg of epididymis
determined by haemocytometry as follows: one epididymis from each rat was weighed and minced with fine scissors into 1 mg aliquots in physiological saline. After vigorous pipetting, the suspension was separated from tissue fragments by filtering it through an 80 mm stainless mesh and the yield of sperm per mg of epididymis was determined with a haemocytometer count. A fraction of each suspension was mixed with 1% eosin Y solution Ž10 : 1. for 30 min and air-dried smears were prepared on glass slides. The slides were coded, randomised and examined for head abnormalities. Differences between the control and experimental groups were analysed by the Wilson Rank-Sum test. Dominant lethal assay. The method used was the modified dominant lethal technique of Epstein et al. w14x. Twelve males were used for each treatment dose while 5 were used for the control. Each male rat received 5 daily interperitonial injections of formaldehyde at doses of 0.125 mgrkg, 0.250 mgrkg and 0.6 mgrkg or of vehicle alone as a concurrent control. Each of the 12 males in each treatment dose was caged with 2 untreated virgin females which were replaced weekly for 3 consecutive weeks giving a total of 24 females for the periods 1–7, 8–14 and 15–21 days post injection, respectively. The total number of mated females for the control was 30. Mating was detected by daily examination of females for vaginal plugs or a discharge of the plug. All females were sacrificed 13 days after the midweek of their caging. At autopsy, each female was scored for total implants which comprised of live implants and early fetal deaths. The frequency of induced dominant lethal mutations was calculated according to the method of Ehling et al. w15x. By this method, the dominant lethal mutation index is given by the formula: 1y
Live implants treated Live implants control
= 100
Differences between the control and experimental group were analysed by Student’s t-test.
4. Results The effects of formaldehyde treatment on frequency of sperm head abnormalities, 3 weeks after
P.G.C. Odeigahr Mutation Research 389 (1997) 141–148
the last injection are shown on Table 1. One thousand spermatozoa from each of 6 rats in each treatment group were scored under blind code for the
143
presence of sperm head abnormalities. A frequency of 1.50% sperm head abnormalities was recorded in the sperm suspensions of control rats which received
Fig. 1. Types of sperm head abnormalities found in normal and formaldehyde-treated rats. ŽA. Normal sperm with a definite head shape especially accented by a marked hook. ŽB–G. Abnormal sperm heads as follows: ŽB. pin-head; the head is small and pin-head shaped, no hook. ŽC. short hook; the hook on the head is shorter than normal. ŽD. long hook; head hook is elongated more than normal. ŽE. hook at wrong angle; hook on head wrongly situated. ŽF. unusual sperm head; head hook looks nubbed but with a spherical spot at the tip. ŽG. Wide acrosome; sperm acrosome is wider than normal. ŽA–C =200; D–G =400..
P.G.C. Odeigahr Mutation Research 389 (1997) 141–148
144
Table 1 Incidence and types of sperm-head abnormalities in formaldehyde-treated rats Treatment
a
Žmgrkg. Control Ždist H 2 O. Formaldehyde 0.125 0.250 0.500
Number of abnormal sperm-heads of each type Pinhead
b
Hook at wrong angle
Total
Short hook
Long hook
Unusual head
Wide acrosome
No.
0
58
32
0
0
0
90
0 38 28
85 145 205
53 156 185
0 22 36
0 16 12
46 59 48
184 436 514
FreqŽ%. "SEM c 1.50 " 0.11
3.09 7.27 8.57
)
" 0.16 " 0.30 )) " 0.33 ))
a
Five daily IP injections: rats killed 3 weeks from last injection. 1000 sperm scored from each of six rats in each treatment group. c Standard error of the mean for 6 rats. ) Significantly increased over control, p - 0.05, Wilcoxon’s Rank-Sum test. )) Significantly increased over control, p - 0.001, Wilcoxon’s Rank-Sum test. b
injections of distilled water alone. Formaldehyde treatment resulted in a statistically significant increase in the induction of sperm head abnormalities. There was also a positive dose-response effect over the entire dose range tested. As shown in Table 1, the percentage sperm head abnormalities for the untreated rat Žcontrol. was 1.50%, while the corresponding values for rats treated with 0.125, 0.250 and 0.50 mgrkg b.wt formaldehyde were 3.07, 7.27 and 8.57%, respectively. The types of sperm head abnormalities observed are shown in Fig. 1. Six different types of sperm head abnormalities were recorded. Of these, short hook Žhook shorter than normal. and long hook Žhook elongated more than normal. were the most common Žsee Table 1.. The sperm count determined by haemocytometry was inversely related to the dose of formaldehyde used for treatment. Five haemocytometer counts were taken from each of 6 rats in the control and each treatment group. For the control, the mean sperm countrmg of epididymis for each of the 6 rats were 45 950, 46 060, 46 150, 46 200, 46 440 and 46 700. The computed mean of the means of the 6 rats was thus 46 250 " 250 ŽFig. 2.. Thus the approximate number of spermrmg of epididymis in the normal untreated male rat is 46 250. For rats treated with 0.125 mgrkg of formaldehyde, the mean of the means for the 6 rats was 27 500 " 102rmg of epididymis while the 6 individual values were 27 360, 27 370, 27 510, 27 540, 27 600 and 27 620. The cor-
responding values for rats treated with 0.250 and 0.500 mgrkg were 13 250 " 74rmg of epididymis Žindividual values; 13 130, 13 220, 13 250, 13 300 and 13 370. and 6500 " 44rmg of epididymis Žindividual values: 6440, 6460, 6490, 6500, 6550, 6560., respectively. Although there was a slight individual variation in sperm count among the rats in each group, there was a significant general decrease in sperm count with increase of dosage of formaldehyde treatment ŽFig. 2.. The frequency of dominant lethal mutations in female rats sired by males exposed to formaldehyde is compiled in Table 2.
Fig. 2. Effect of concentration of formaldehyde treatment on sperm count in the male rat. Results from 6 experiments Ž6 rats. were pooled and each point represents the mean of 5 haemocytometer readings in an individual rat. Errors are too small to be plotted, and are given in text.
0.250
0.50
Formaldehyde
Formaldehyde
1–7 8–14 15–21 1–7 8–14 15–21 1–7 8–14 15–21
0–21
Time of mating post injection Ždays.
12
12
12
5
No. of males used
24 24 24 24 24 24 24 24 24
30
Females mated
75.0 Ž18. 79.17 Ž19. 91.67 Ž22. 33.33 Ž8. 50.0 Ž12. 87.5 Ž21. 25.0 Ž6. 29.17 Ž7. 83.33 Ž20.
96.67rŽ29.
Ž%. ŽNo. of fertile females.
Overall fertile matings a
7.18"0.3 Ž18. 7.38"0.5 Ž19. 7.68"0.2 Ž22. 5.75"0.3 Ž8. 6.60"0.2 Ž12. 7.25"0.4 Ž21. 5.05".03 Ž6. 5.27".01 Ž7. 7.08".04 Ž20.
7.86"0.2rŽ29.
Žmean"SE. ŽNo. of females with implants.
Implants per female a
b
Figures are based on fertile or ‘pregnant’ females, the respective numbers of which are indicated in parentheses. Dominant lethal mutation index is: Ž Live implants experiment group per female. 1y =100 Ž Live implants of control group per female. .
a
0.125
0
Žmgrkg.
Dose
Distilled water Žcontrol. Formaldehyde
Treatment
Table 2 Dominant lethal mutations after exposure of rats to formaldehyde
5.95".2 6.30".5 6.89".3 2.05".3 3.91".2 6.63".3 1.10".5 1.50".6 5.79".4
7.43"0.3
Žmean"SE.
Live embryos per female a
1.23".05 1.08".03 0.79".05 3.70".04 2.69".02 0.62".05 3.95".22 3.77".28 1.29".17
0.43"0.8
Dead implants per female a
19.92 15.21 7.27 72.41 47.38 10.77 85.20 79.81 22.07
0
Dominant lethal mutation index b
P.G.C. Odeigahr Mutation Research 389 (1997) 141–148 145
146
P.G.C. Odeigahr Mutation Research 389 (1997) 141–148
The time interval and mating schedule used, were chosen to represent samples of different postmeiotic stages of spermatogenesis. Apart from the effect of the treatment, infertility of mates should occur in the control and experimental groups with the same frequency. So all females in both control and experimental groups were used in the calculation of the % fertile matings in the respective groups. All females that had implants were regarded as ‘pregnant’ or fertile and the number of implants, live embryos and dead implants per female were based on ‘pregnant’ or fertile females. The difference between implants per fertile female and live embryos per fertile female represents post implantation death or dead implants per female. Among control females, 0.43 dead implantsrfemale was obtained. A lower frequency of fertile matings was recorded in females mated 1–7 and 8–14 days after treatment of males with formaldehyde. The lowest percentage of fertile matings Ž25%. was recorded in females mated 1–7 days after treatment of males with the highest dose of formaldehyde used Ž0.50 mgrkg.. However, frequency of fertile matings in females mated 15–21 days after treatment of males at 0.125 mgrkg, 0.250 mgrkg and 0.50 mgrkg, respectively, was not significantly different from control ŽTable 2.. Also, there was no significant difference in the number of implants per female between the control and treated groups in females mated 15–21 days after treatment of males with the different doses of formaldehyde. However, there was a statistically significant increase in the number of dead implants in females mated 1–7 days after treatment of males with formaldehyde. The highest number of dead implants per female Ž3.95 " 0.22. was recorded in females mated 1–7 days after treatment of males with the highest concentration used. Another significant observation was that the increase in post-implantation death was associated with a corresponding decrease in the number of live embryos in the experimental females. The dominant lethal mutation index was calculated using the formula shown in Table 2. For each dose of treatment there was a significant increase in the dominant mutation index particularly for matings 1 week post-injection of formaldehyde. The overall results suggest a positive dose-response relationship between treatment and induction of dominant lethal mutations.
5. Discussion In the present study, a significantly increased frequency of sperm head abnormalities was observed in the spermatozoa of treated rats. Increases in the incidence of abnormal sperm have been reported after treatment of male mice with irradiation w16,17x, lead acetate and ethyl methane sulphonate w17x and alcohol w18x. It has been reported that high temperatures, extreme nutritional deficiencies and some diseases can cause sperm abnormalities in a wide range of species including mice and men w18x. Feeding male Wistar rats on a diet containing 36% of total calories as ethanol for 41 days caused similar sperm abnormalities as those reported in other mammalian species w19x. In the above study, the mean frequency of sperm head abnormalities in the control groups was 1.20%. The lowest value was 1.0% and the highest 1.60% w19x. The frequency in the control groups reported here is within this range. However, since none of these factors are applicable in the present study, it is concluded that the exposure to formaldehyde is responsible for the significantly increased incidence of sperm head abnormalities. Although formaldehyde is known to produce DNA protein cross-links in a cell, the precise mechanism by which formaldehyde causes sperm head abnormalities is not yet fully established. In general, damage to the sperm cell by substances may occur by either physiological, cytotoxic or genetic mechanisms. In addition there are 2 mechanisms by which chemicals might indirectly affect sperm cell function and morphology: firstly, exposure to chemicals could produce pituitary-hypothalamic or sex hormonal effects which in turn could affect spermatogenesis and secondly exposure could cause abnormalities in seminal fluid, resulting in functional or structural impairment of sperm w17,20x. Bruce and Heddle w21x investigated the mutagenic activity of 61 chemicals by the Ames Salmonella, Mouse micronucleus and sperm abnormality assays. They found positive results in the sperm abnormality assay for 32 of these chemicals Žincluding formaldehyde. and suggested that the morphological abnormalities may have been caused by alterations Ždeletions, point mutations or a combination of both. in testicular DNA which in turn disrupts the process of differentiation of spermatozoa.
P.G.C. Odeigahr Mutation Research 389 (1997) 141–148
The scoring of sperm head abnormalities in the rat is technically easy but the interpretation of results can be confounded by a number of factors such as variable periods of treatment prior to sample collection, frequency of sampling and method of collection or preparation of the sperm smears. Multiple samples from the sample individual may show wide ranges in sperm abnormalities even in the control rats. In spite of this variability there are many clear examples of chemically induced increases in sperm abnormalities, including the one reported in this study. The decrease in frequency of fertile matings and implantations per female from the control value, observed in experimental females mated first week after treatment could have been caused by increased pre-implantation loss due to increased frequency of sperm head abnormalities or decreased fertilisation due to low sperm count. The presence of formaldehyde in the semen may also cause pre-implantation loss by impairing sperm motility or indirectly affect vaginal mucosa and placental development. The results of the dominant lethal mutation assay suggest a considerable loss during pre-natal life which could, to a great extent, be the result of induction of dominant lethals by formaldehyde. The results also suggest that different stages of germ cell maturation respond differently. Similar effects of chemical agents on pre-natal mortality have been reported with guinea pigs, mice and rats w22x. The time schedule employed in the mating scheme is similar to that of mice where 1–7 day post-injection sperm are regarded as spermatozoa of epididymis, 8–14 day post-injection sperm are testicular sperm, and late spermatid and 15–21 day post-injection sperm are early spermatids w15x. Although the duration of the stages of spermatogenesis has not been fully worked out in the rat, the similarities of the results and other reproductive similarities between the mouse and rat suggest that in this study although the effect of formaldehyde is still strong at other stages, it is probably more pronounced at the epididymal spermatozoal stages. It has been argued that dominant lethal mutations may not impose any genetic hazard on man, since they merely result in abortions. It has, however, been shown that formaldehyde produces a wider range of genetic effects besides dominant lethals and sperm head abnormalities w4,5,8x.
147
The most common route by which humans are exposed to formaldehyde is by inhalation while most studies of the effects of formaldehyde exposure in animal analogues is by injection. The target tissues at risk in both routes are likely to differ. To date, only a handful of human occupational exposures have been evaluated for potential effects on the female or male reproductive systems. Epidemiological investigations in female workers occupationally exposed to formaldehyde reported increases in menstrual disorders and spontaneous abortions w23x. No such increases were observed in other studies, so the results are still conflicting. With respect to the male germ cell, one study suggests a link between poor sperm quality and embryonic failure. Fathers of 201 spontaneous abortions showed significantly higher sperm abnormalities and lower sperm counts than did fathers of 116 normal pregnancies. No data on paternal occupation or chemical exposures were given w20x.
Acknowledgements I am grateful to Dr. ŽMrs. Agiri for assistance, and the University of Lagos for some financial support.
References w1x Rapoport, I.A. Ž1946. Carbonyl compounds and the chemical mechanism of mutation. Comp. Rend. Acad. Sci. ŽURSS., 54, 65–67. w2x Auerbach, C., M. Moutschen-Danmen and J. Moutschen Ž1977. Genetic and cytogenetical effect of formaldehyde and related compounds. Mutation Res., 39, 317–367. w3x Natarajan, A.T., F. Darroudi, C.J.M. Bussman and A.C. van Kestern-van Leeuwen Ž1983. Evaluation of the mutagenicity of formaldehyde in mammalian cytogenetic assays in vivo and in vitro. Mutation Res., 122, 355–360. w4x Miretskaya, L.M. and P.Y. Shvartsman Ž1982. Studies of chromosome aberrations in human lymphocytes under the influence of formaldehyde: I. Formaldehyde treatment of lymphocytes in vitro, Tsitologia, 24, 1056–1060. w5x Bauchinger, M. and E. Schmid Ž1985. Cytogenetic effects in lymphocytes of formaldehyde workers of a paper factory. Mutation Res., 158, 195–199. w6x Thomson, E.J., S. Shackleton and J.M. Harrington Ž1984. Chromosome abberations and sister-chromatid exchange frequencies in pathology staff occupationally exposed to formaldehyde. Mutation Res., 141, 89–93.
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w7x Fontignie-Houbrechts, N. Ž1981. Genetic effects of formaldehyde in the mouse, Mutation Res., 88, 109–114. w8x Kreiger, R.A. and V.F. Garry Ž1983. Formaldehyde-induced cytotoxicity and sister-chromatid exchanges in human lymphocyte cultures. Mutation Res., 120, 51–55. w9x Yager, J.W., K.L. Cohn, R.C. Spear, J.M. Fisher and L. Morse Ž1986. Sister-chromatid exchanges in lymphocytes of anatomy students exposed to formaldehyde-embalming solution, Mutation Res., 174, 135–139. w10x Snyder, R.D. and V.B. Arten Ž1986. Genotoxicity of formaldehyde and an evaluation of its effects on the DNA repair processes in diploid fibroblasts. Mutation Res., 165, 21–30. w11x Swenberg, J.A., D.W. Kerns, R.I. Mitchell, E.J. Gralla and K.L. Pavkov Ž1980. Induction of squamous cell carcinomas of the rat nasal cavity by inhalation exposure to formaldehyde vapour. Cancer Res., 40, 3398–3402. w12x Batelle Columbus Laboratories Ž1981. Final report on a chronic inhalation toxicology study in rats and mice exposed to formaldehyde. CHT, Dec. 31. w13x Topham, J.C. Ž1980. Chemically induced transmissible abnormalities in sperm-head shape. Mutation Res., 70, 109–114. w14x Epstein, D.S., E. Arnold, J. Bass and Y. Bishop Ž1972. Detection of chemical mutagens by the dominant lethal assay in the mouse. Toxicol. Appl. Pharmacol., 23, 288–325. w15x Ehling, U.H., R.B. Cumming and H.V. Malling Ž1968. Induction of dominant lethal mutations by alkylating agents in male mice. Mutation Res., 5, 417–428.
w16x Bruce, W.R., R. Furrer and A.J. Wyrobek Ž1974. Abnormalities in the shape of murine sperm after acute testicular X-irradiation. Mutation Res., 23, 381–386. w17x Wyrobek, A.J. and W.R. Bruce Ž1978. The induction of sperm-shape abnormalities in mice and humans, In: A Hollander and F.J. de Serres ŽEds.., Chemical Mutagens, Vol. 5, Plenum Press, New York, pp. 257–285. w18x Obe, G. and H. Ristow Ž1979. Mutagenic, carcinogenic and teratogenic effects of alcohol. Mutation Res., 65, 229–259. w19x Van Thiel, D.H., J.S. Gavaler, R. Lester and M.D. Goodman Ž1975. Alcohol-induced testicular atrophy. An experimental model for hypogonadism occurring in chronic alcoholic men. Gastroenterology, 69, 326–332. w20x Letz, G. Ž1990. Male reproductive toxicology, In: J. Ladou ŽEd.., Occupational Medicine, Appleton and Lange, Connecticut, pp. 288–296. w21x Bruce, W.R. and J.A. Heddle Ž1979. The mutagenic activity of 61 agents as determined by the micronucleus, Salmonella and sperm abnormality assays. Can. J. Genetic. Cytol., 21, 319–334. w22x Badr, F.M. and R.S. Badr Ž1975. Induction of dominant lethal mutation in mice by ethyl alcohol. Nature, 253, 134– 136. w23x Rudolph, L. and C.S. Forest Ž1990. Female reproductive toxicology, In: J. Ladou ŽEd.., Occupational Medicine, Apleton and Lange, Connecticut, pp. 275–287.
Sperm head abnormalities and dominant lethal effects of formaldehyde in albino rats P.G.C. Odeigah Genetics Laboratory, Department of Biological Sciences, UniÕersity of Lagos, Akoka, Lagos, Nigeria Received 20 March 1996; revised 30 July 1996; accepted 17 September 1996
Abstract The effects of formaldehyde exposure have been investigated in rats by two short-term in vivo mutagenicity tests Žsperm head abnormality and dominant lethal mutation assays.. Five daily interperitonial injections of formaldehyde resulted in a statistically significant increase in induction of sperm head abnormalities at 0.125 to 0.500 mgrkg. The frequency of dominant lethal mutations in female rats sired by males exposed to formaldehyde was significantly higher than the control group. There was also a reduction of fertile matings in females mated 1–7 days after treatment of males with formaldehyde. The genetic and environmental health implications of the results are discussed. Keywords: Mammalian Mutagenicity assay; Formaldehyde exposure; Rat
1. Introduction In recent years, there has been an increasing awareness of the genotoxic potential of a wide variety of drugs and chemicals to which the human population is exposed either environmentally or occupationally. This awareness is paralleled by the recent development of appropriate, sensitive and practical methods for detecting and estimating the effects of these substances. The extensive use of formaldehyde in many domestic products, laboratory and industrial processes has generated considerable interest in its effects. Although the mutagenicity of formaldehyde was reported by Rapoport as early as 1946 w1x, most of the subsequent studies were concentrated in Drosophila w2x. Studies of its mutagenic effects in mammals are few and mostly limited to in vitro experiments. Increases in chromosomal aberration frequencies
have been reported in Chinese hamster ovary cells in culture w3x and in cultured human lymphocytes w4,5x as a result of formaldehyde treatment. In contrast, no such increases were observed in cultured human lymphocytes of pathology workers w6x or by in vivo tests on bone marrow and spermatocytes of mice w3,7x. Increases in the frequencies of siter-chromatid exchange have been observed in cultured human peripheral blood lymphocytes treated with formaldehyde w8x and in the lymphocytes of a small group of anatomy students exposed to formaldehydeembalming solution w9x. The effects of formaldehyde on the DNA repair processes in human fibroblasts w10x and its induction of nasal tumours in rats w11x and mice w12x have also been noted. A large number of the chemical, cytological and histological analyses of the effects of formaldehyde in mammalian systems have been carried out by examination of in vitro tissue culture materials. This
1383-5718r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 1 3 8 3 - 5 7 1 8 Ž 9 6 . 0 0 1 3 6 - X
142
P.G.C. Odeigahr Mutation Research 389 (1997) 141–148
study explores further the effects of formaldehyde exposure in rats using two short-term in vivo mutagenicity assays Žsperm head abnormality and dominant lethal mutation tests..
2. Materials and methods Animal husbandry. Isogenic strains of albino rats Ž Rattus norÕegicus. were obtained from the Department of Biological Sciences, small animal colony, University of Lagos. All animals were housed in suspended mesh-bottomed cages and maintained on food ŽPfizer products, pellets. and water ad libitum. Rats used for the experiment were 12 to 14 weeks old. Male and female rats used for the sperm assay and dominant lethal tests were litter mates. Test substance. Formaldehyde, the test substance, is of reagent grade and was obtained from Sigma Chemical Co. as 37% solution Žstabilised with 10% methanol.. The doses used in the study were prepared on the basis that 1 litre is equivalent to 1.08 kg Ždensity of the liquid..
3. Preliminary acute toxicity studies The mean lethal dose ŽLD50 . of formaldehyde administered interperitonially to the rats was determined before carrying out the sperm head abnormality and dominant lethal mutation assays. Fifty rats were used, and a mean lethal dose of 2 mgrkg body weight was obtained. Symptoms of toxicity of lethal doses include growth retardation, weight loss, lack of appetite and visual impairment Žranging from red spots in the eyes to conjunctivitis and complete blindness.. Three sublethal doses Ž1r4 to 1r16 LD50 dose. were used to test for sperm head abnormalities and dominant lethal mutations. Sperm head abnormality assay. This was carried out according to the method of Topham w13x. Briefly, a batch of 24 rats Ž6 per control or dose of test substance. received 5 daily interperitonial injections of vehicle alone, water Ž5 mlrkgrday. or formaldehyde at 0.125 mgrkg, 0.250 mgrkg and 0.50 mgrkg body weight. The rats were sacrificed 3 weeks after the last injection, and the sperm countrmg of epididymis
determined by haemocytometry as follows: one epididymis from each rat was weighed and minced with fine scissors into 1 mg aliquots in physiological saline. After vigorous pipetting, the suspension was separated from tissue fragments by filtering it through an 80 mm stainless mesh and the yield of sperm per mg of epididymis was determined with a haemocytometer count. A fraction of each suspension was mixed with 1% eosin Y solution Ž10 : 1. for 30 min and air-dried smears were prepared on glass slides. The slides were coded, randomised and examined for head abnormalities. Differences between the control and experimental groups were analysed by the Wilson Rank-Sum test. Dominant lethal assay. The method used was the modified dominant lethal technique of Epstein et al. w14x. Twelve males were used for each treatment dose while 5 were used for the control. Each male rat received 5 daily interperitonial injections of formaldehyde at doses of 0.125 mgrkg, 0.250 mgrkg and 0.6 mgrkg or of vehicle alone as a concurrent control. Each of the 12 males in each treatment dose was caged with 2 untreated virgin females which were replaced weekly for 3 consecutive weeks giving a total of 24 females for the periods 1–7, 8–14 and 15–21 days post injection, respectively. The total number of mated females for the control was 30. Mating was detected by daily examination of females for vaginal plugs or a discharge of the plug. All females were sacrificed 13 days after the midweek of their caging. At autopsy, each female was scored for total implants which comprised of live implants and early fetal deaths. The frequency of induced dominant lethal mutations was calculated according to the method of Ehling et al. w15x. By this method, the dominant lethal mutation index is given by the formula: 1y
Live implants treated Live implants control
= 100
Differences between the control and experimental group were analysed by Student’s t-test.
4. Results The effects of formaldehyde treatment on frequency of sperm head abnormalities, 3 weeks after
P.G.C. Odeigahr Mutation Research 389 (1997) 141–148
the last injection are shown on Table 1. One thousand spermatozoa from each of 6 rats in each treatment group were scored under blind code for the
143
presence of sperm head abnormalities. A frequency of 1.50% sperm head abnormalities was recorded in the sperm suspensions of control rats which received
Fig. 1. Types of sperm head abnormalities found in normal and formaldehyde-treated rats. ŽA. Normal sperm with a definite head shape especially accented by a marked hook. ŽB–G. Abnormal sperm heads as follows: ŽB. pin-head; the head is small and pin-head shaped, no hook. ŽC. short hook; the hook on the head is shorter than normal. ŽD. long hook; head hook is elongated more than normal. ŽE. hook at wrong angle; hook on head wrongly situated. ŽF. unusual sperm head; head hook looks nubbed but with a spherical spot at the tip. ŽG. Wide acrosome; sperm acrosome is wider than normal. ŽA–C =200; D–G =400..
P.G.C. Odeigahr Mutation Research 389 (1997) 141–148
144
Table 1 Incidence and types of sperm-head abnormalities in formaldehyde-treated rats Treatment
a
Žmgrkg. Control Ždist H 2 O. Formaldehyde 0.125 0.250 0.500
Number of abnormal sperm-heads of each type Pinhead
b
Hook at wrong angle
Total
Short hook
Long hook
Unusual head
Wide acrosome
No.
0
58
32
0
0
0
90
0 38 28
85 145 205
53 156 185
0 22 36
0 16 12
46 59 48
184 436 514
FreqŽ%. "SEM c 1.50 " 0.11
3.09 7.27 8.57
)
" 0.16 " 0.30 )) " 0.33 ))
a
Five daily IP injections: rats killed 3 weeks from last injection. 1000 sperm scored from each of six rats in each treatment group. c Standard error of the mean for 6 rats. ) Significantly increased over control, p - 0.05, Wilcoxon’s Rank-Sum test. )) Significantly increased over control, p - 0.001, Wilcoxon’s Rank-Sum test. b
injections of distilled water alone. Formaldehyde treatment resulted in a statistically significant increase in the induction of sperm head abnormalities. There was also a positive dose-response effect over the entire dose range tested. As shown in Table 1, the percentage sperm head abnormalities for the untreated rat Žcontrol. was 1.50%, while the corresponding values for rats treated with 0.125, 0.250 and 0.50 mgrkg b.wt formaldehyde were 3.07, 7.27 and 8.57%, respectively. The types of sperm head abnormalities observed are shown in Fig. 1. Six different types of sperm head abnormalities were recorded. Of these, short hook Žhook shorter than normal. and long hook Žhook elongated more than normal. were the most common Žsee Table 1.. The sperm count determined by haemocytometry was inversely related to the dose of formaldehyde used for treatment. Five haemocytometer counts were taken from each of 6 rats in the control and each treatment group. For the control, the mean sperm countrmg of epididymis for each of the 6 rats were 45 950, 46 060, 46 150, 46 200, 46 440 and 46 700. The computed mean of the means of the 6 rats was thus 46 250 " 250 ŽFig. 2.. Thus the approximate number of spermrmg of epididymis in the normal untreated male rat is 46 250. For rats treated with 0.125 mgrkg of formaldehyde, the mean of the means for the 6 rats was 27 500 " 102rmg of epididymis while the 6 individual values were 27 360, 27 370, 27 510, 27 540, 27 600 and 27 620. The cor-
responding values for rats treated with 0.250 and 0.500 mgrkg were 13 250 " 74rmg of epididymis Žindividual values; 13 130, 13 220, 13 250, 13 300 and 13 370. and 6500 " 44rmg of epididymis Žindividual values: 6440, 6460, 6490, 6500, 6550, 6560., respectively. Although there was a slight individual variation in sperm count among the rats in each group, there was a significant general decrease in sperm count with increase of dosage of formaldehyde treatment ŽFig. 2.. The frequency of dominant lethal mutations in female rats sired by males exposed to formaldehyde is compiled in Table 2.
Fig. 2. Effect of concentration of formaldehyde treatment on sperm count in the male rat. Results from 6 experiments Ž6 rats. were pooled and each point represents the mean of 5 haemocytometer readings in an individual rat. Errors are too small to be plotted, and are given in text.
0.250
0.50
Formaldehyde
Formaldehyde
1–7 8–14 15–21 1–7 8–14 15–21 1–7 8–14 15–21
0–21
Time of mating post injection Ždays.
12
12
12
5
No. of males used
24 24 24 24 24 24 24 24 24
30
Females mated
75.0 Ž18. 79.17 Ž19. 91.67 Ž22. 33.33 Ž8. 50.0 Ž12. 87.5 Ž21. 25.0 Ž6. 29.17 Ž7. 83.33 Ž20.
96.67rŽ29.
Ž%. ŽNo. of fertile females.
Overall fertile matings a
7.18"0.3 Ž18. 7.38"0.5 Ž19. 7.68"0.2 Ž22. 5.75"0.3 Ž8. 6.60"0.2 Ž12. 7.25"0.4 Ž21. 5.05".03 Ž6. 5.27".01 Ž7. 7.08".04 Ž20.
7.86"0.2rŽ29.
Žmean"SE. ŽNo. of females with implants.
Implants per female a
b
Figures are based on fertile or ‘pregnant’ females, the respective numbers of which are indicated in parentheses. Dominant lethal mutation index is: Ž Live implants experiment group per female. 1y =100 Ž Live implants of control group per female. .
a
0.125
0
Žmgrkg.
Dose
Distilled water Žcontrol. Formaldehyde
Treatment
Table 2 Dominant lethal mutations after exposure of rats to formaldehyde
5.95".2 6.30".5 6.89".3 2.05".3 3.91".2 6.63".3 1.10".5 1.50".6 5.79".4
7.43"0.3
Žmean"SE.
Live embryos per female a
1.23".05 1.08".03 0.79".05 3.70".04 2.69".02 0.62".05 3.95".22 3.77".28 1.29".17
0.43"0.8
Dead implants per female a
19.92 15.21 7.27 72.41 47.38 10.77 85.20 79.81 22.07
0
Dominant lethal mutation index b
P.G.C. Odeigahr Mutation Research 389 (1997) 141–148 145
146
P.G.C. Odeigahr Mutation Research 389 (1997) 141–148
The time interval and mating schedule used, were chosen to represent samples of different postmeiotic stages of spermatogenesis. Apart from the effect of the treatment, infertility of mates should occur in the control and experimental groups with the same frequency. So all females in both control and experimental groups were used in the calculation of the % fertile matings in the respective groups. All females that had implants were regarded as ‘pregnant’ or fertile and the number of implants, live embryos and dead implants per female were based on ‘pregnant’ or fertile females. The difference between implants per fertile female and live embryos per fertile female represents post implantation death or dead implants per female. Among control females, 0.43 dead implantsrfemale was obtained. A lower frequency of fertile matings was recorded in females mated 1–7 and 8–14 days after treatment of males with formaldehyde. The lowest percentage of fertile matings Ž25%. was recorded in females mated 1–7 days after treatment of males with the highest dose of formaldehyde used Ž0.50 mgrkg.. However, frequency of fertile matings in females mated 15–21 days after treatment of males at 0.125 mgrkg, 0.250 mgrkg and 0.50 mgrkg, respectively, was not significantly different from control ŽTable 2.. Also, there was no significant difference in the number of implants per female between the control and treated groups in females mated 15–21 days after treatment of males with the different doses of formaldehyde. However, there was a statistically significant increase in the number of dead implants in females mated 1–7 days after treatment of males with formaldehyde. The highest number of dead implants per female Ž3.95 " 0.22. was recorded in females mated 1–7 days after treatment of males with the highest concentration used. Another significant observation was that the increase in post-implantation death was associated with a corresponding decrease in the number of live embryos in the experimental females. The dominant lethal mutation index was calculated using the formula shown in Table 2. For each dose of treatment there was a significant increase in the dominant mutation index particularly for matings 1 week post-injection of formaldehyde. The overall results suggest a positive dose-response relationship between treatment and induction of dominant lethal mutations.
5. Discussion In the present study, a significantly increased frequency of sperm head abnormalities was observed in the spermatozoa of treated rats. Increases in the incidence of abnormal sperm have been reported after treatment of male mice with irradiation w16,17x, lead acetate and ethyl methane sulphonate w17x and alcohol w18x. It has been reported that high temperatures, extreme nutritional deficiencies and some diseases can cause sperm abnormalities in a wide range of species including mice and men w18x. Feeding male Wistar rats on a diet containing 36% of total calories as ethanol for 41 days caused similar sperm abnormalities as those reported in other mammalian species w19x. In the above study, the mean frequency of sperm head abnormalities in the control groups was 1.20%. The lowest value was 1.0% and the highest 1.60% w19x. The frequency in the control groups reported here is within this range. However, since none of these factors are applicable in the present study, it is concluded that the exposure to formaldehyde is responsible for the significantly increased incidence of sperm head abnormalities. Although formaldehyde is known to produce DNA protein cross-links in a cell, the precise mechanism by which formaldehyde causes sperm head abnormalities is not yet fully established. In general, damage to the sperm cell by substances may occur by either physiological, cytotoxic or genetic mechanisms. In addition there are 2 mechanisms by which chemicals might indirectly affect sperm cell function and morphology: firstly, exposure to chemicals could produce pituitary-hypothalamic or sex hormonal effects which in turn could affect spermatogenesis and secondly exposure could cause abnormalities in seminal fluid, resulting in functional or structural impairment of sperm w17,20x. Bruce and Heddle w21x investigated the mutagenic activity of 61 chemicals by the Ames Salmonella, Mouse micronucleus and sperm abnormality assays. They found positive results in the sperm abnormality assay for 32 of these chemicals Žincluding formaldehyde. and suggested that the morphological abnormalities may have been caused by alterations Ždeletions, point mutations or a combination of both. in testicular DNA which in turn disrupts the process of differentiation of spermatozoa.
P.G.C. Odeigahr Mutation Research 389 (1997) 141–148
The scoring of sperm head abnormalities in the rat is technically easy but the interpretation of results can be confounded by a number of factors such as variable periods of treatment prior to sample collection, frequency of sampling and method of collection or preparation of the sperm smears. Multiple samples from the sample individual may show wide ranges in sperm abnormalities even in the control rats. In spite of this variability there are many clear examples of chemically induced increases in sperm abnormalities, including the one reported in this study. The decrease in frequency of fertile matings and implantations per female from the control value, observed in experimental females mated first week after treatment could have been caused by increased pre-implantation loss due to increased frequency of sperm head abnormalities or decreased fertilisation due to low sperm count. The presence of formaldehyde in the semen may also cause pre-implantation loss by impairing sperm motility or indirectly affect vaginal mucosa and placental development. The results of the dominant lethal mutation assay suggest a considerable loss during pre-natal life which could, to a great extent, be the result of induction of dominant lethals by formaldehyde. The results also suggest that different stages of germ cell maturation respond differently. Similar effects of chemical agents on pre-natal mortality have been reported with guinea pigs, mice and rats w22x. The time schedule employed in the mating scheme is similar to that of mice where 1–7 day post-injection sperm are regarded as spermatozoa of epididymis, 8–14 day post-injection sperm are testicular sperm, and late spermatid and 15–21 day post-injection sperm are early spermatids w15x. Although the duration of the stages of spermatogenesis has not been fully worked out in the rat, the similarities of the results and other reproductive similarities between the mouse and rat suggest that in this study although the effect of formaldehyde is still strong at other stages, it is probably more pronounced at the epididymal spermatozoal stages. It has been argued that dominant lethal mutations may not impose any genetic hazard on man, since they merely result in abortions. It has, however, been shown that formaldehyde produces a wider range of genetic effects besides dominant lethals and sperm head abnormalities w4,5,8x.
147
The most common route by which humans are exposed to formaldehyde is by inhalation while most studies of the effects of formaldehyde exposure in animal analogues is by injection. The target tissues at risk in both routes are likely to differ. To date, only a handful of human occupational exposures have been evaluated for potential effects on the female or male reproductive systems. Epidemiological investigations in female workers occupationally exposed to formaldehyde reported increases in menstrual disorders and spontaneous abortions w23x. No such increases were observed in other studies, so the results are still conflicting. With respect to the male germ cell, one study suggests a link between poor sperm quality and embryonic failure. Fathers of 201 spontaneous abortions showed significantly higher sperm abnormalities and lower sperm counts than did fathers of 116 normal pregnancies. No data on paternal occupation or chemical exposures were given w20x.
Acknowledgements I am grateful to Dr. ŽMrs. Agiri for assistance, and the University of Lagos for some financial support.
References w1x Rapoport, I.A. Ž1946. Carbonyl compounds and the chemical mechanism of mutation. Comp. Rend. Acad. Sci. ŽURSS., 54, 65–67. w2x Auerbach, C., M. Moutschen-Danmen and J. Moutschen Ž1977. Genetic and cytogenetical effect of formaldehyde and related compounds. Mutation Res., 39, 317–367. w3x Natarajan, A.T., F. Darroudi, C.J.M. Bussman and A.C. van Kestern-van Leeuwen Ž1983. Evaluation of the mutagenicity of formaldehyde in mammalian cytogenetic assays in vivo and in vitro. Mutation Res., 122, 355–360. w4x Miretskaya, L.M. and P.Y. Shvartsman Ž1982. Studies of chromosome aberrations in human lymphocytes under the influence of formaldehyde: I. Formaldehyde treatment of lymphocytes in vitro, Tsitologia, 24, 1056–1060. w5x Bauchinger, M. and E. Schmid Ž1985. Cytogenetic effects in lymphocytes of formaldehyde workers of a paper factory. Mutation Res., 158, 195–199. w6x Thomson, E.J., S. Shackleton and J.M. Harrington Ž1984. Chromosome abberations and sister-chromatid exchange frequencies in pathology staff occupationally exposed to formaldehyde. Mutation Res., 141, 89–93.
148
P.G.C. Odeigahr Mutation Research 389 (1997) 141–148
w7x Fontignie-Houbrechts, N. Ž1981. Genetic effects of formaldehyde in the mouse, Mutation Res., 88, 109–114. w8x Kreiger, R.A. and V.F. Garry Ž1983. Formaldehyde-induced cytotoxicity and sister-chromatid exchanges in human lymphocyte cultures. Mutation Res., 120, 51–55. w9x Yager, J.W., K.L. Cohn, R.C. Spear, J.M. Fisher and L. Morse Ž1986. Sister-chromatid exchanges in lymphocytes of anatomy students exposed to formaldehyde-embalming solution, Mutation Res., 174, 135–139. w10x Snyder, R.D. and V.B. Arten Ž1986. Genotoxicity of formaldehyde and an evaluation of its effects on the DNA repair processes in diploid fibroblasts. Mutation Res., 165, 21–30. w11x Swenberg, J.A., D.W. Kerns, R.I. Mitchell, E.J. Gralla and K.L. Pavkov Ž1980. Induction of squamous cell carcinomas of the rat nasal cavity by inhalation exposure to formaldehyde vapour. Cancer Res., 40, 3398–3402. w12x Batelle Columbus Laboratories Ž1981. Final report on a chronic inhalation toxicology study in rats and mice exposed to formaldehyde. CHT, Dec. 31. w13x Topham, J.C. Ž1980. Chemically induced transmissible abnormalities in sperm-head shape. Mutation Res., 70, 109–114. w14x Epstein, D.S., E. Arnold, J. Bass and Y. Bishop Ž1972. Detection of chemical mutagens by the dominant lethal assay in the mouse. Toxicol. Appl. Pharmacol., 23, 288–325. w15x Ehling, U.H., R.B. Cumming and H.V. Malling Ž1968. Induction of dominant lethal mutations by alkylating agents in male mice. Mutation Res., 5, 417–428.
w16x Bruce, W.R., R. Furrer and A.J. Wyrobek Ž1974. Abnormalities in the shape of murine sperm after acute testicular X-irradiation. Mutation Res., 23, 381–386. w17x Wyrobek, A.J. and W.R. Bruce Ž1978. The induction of sperm-shape abnormalities in mice and humans, In: A Hollander and F.J. de Serres ŽEds.., Chemical Mutagens, Vol. 5, Plenum Press, New York, pp. 257–285. w18x Obe, G. and H. Ristow Ž1979. Mutagenic, carcinogenic and teratogenic effects of alcohol. Mutation Res., 65, 229–259. w19x Van Thiel, D.H., J.S. Gavaler, R. Lester and M.D. Goodman Ž1975. Alcohol-induced testicular atrophy. An experimental model for hypogonadism occurring in chronic alcoholic men. Gastroenterology, 69, 326–332. w20x Letz, G. Ž1990. Male reproductive toxicology, In: J. Ladou ŽEd.., Occupational Medicine, Appleton and Lange, Connecticut, pp. 288–296. w21x Bruce, W.R. and J.A. Heddle Ž1979. The mutagenic activity of 61 agents as determined by the micronucleus, Salmonella and sperm abnormality assays. Can. J. Genetic. Cytol., 21, 319–334. w22x Badr, F.M. and R.S. Badr Ž1975. Induction of dominant lethal mutation in mice by ethyl alcohol. Nature, 253, 134– 136. w23x Rudolph, L. and C.S. Forest Ž1990. Female reproductive toxicology, In: J. Ladou ŽEd.., Occupational Medicine, Apleton and Lange, Connecticut, pp. 275–287.