Germ cell studies in mice after prolonged exposure to nitrous oxide

TOXICOLOGY AND APPLIED PHARMACOLOGY67, 370-375 (1983)

Germ Cell Studies in Mice after Prolonged Exposure to Nitrous Oxide RICHARD I. MAZZE,*,’ SUSAN A. RICE,* ANDREW J. WYROBEK,~ JAMES S. FELTON,? JAY B. BRODSKY,* *Department Service,

of Anesthesia, Stanford Veterans Administration Division, Lawrence

University Medical Livermore

Received

August

AND JEFFREY

M. BADEN*

School of Medicine, Stanford, California 94305 and Anesthesiology Center, Palo Alto, California 94304, and tBiomedica1 Sciences National Laboratory, Livermore. Cali/brnia 94550

6, 1982;

accepted

November

8. 1982

Germ Cell Studies in Mice after Prolonged Exposure to Nitrous Oxide. MAZZE, R. I., RICE, S. A., WYROBEK, A. J., FELTON, J. S., BRODKSY, J. B., AND BADEN, J. M. (1983). Toxicol. Appl. Pharmacol. 67, 370-375. Male and female Swiss Webster (SW) mice, age 13 to 14 weeks, were exposed by inhalation for 4 hr per day, 5 days per week, for 14 weeks, to either room air, 0.5% nitrous oxide, 5.0% nitrous oxide, or 50% nitrous oxide. Murine germ cells were examined for evidence of injury after this exposure. A group of male mice were treated with methyl methanesulfonate (MMS) as a positive control for sperm abnormalities while a group of female mice were treated with 3-methylcholanthrene (3-MC) as a positive control for oocyte destruction. There were no significant differences among the four inhalation exposure groups in testesweight, percentage of abnormally shaped sperm, sperm count, or histologic appearance of the testes; the mean percentage (&SE) of abnormal sperm ranged from 8.9 & 2.4 (5.0% nitrous oxide) to 13.5 f 0.5 (50% nitrous oxide) with a concurrent control value of 10.4 + 2.3%. In the positive control experiment, 25.2 rf: 4.1% of sperm from mice treated with MMS were abnormal compared with 2.5 + 0.3% of sperm from mice treated with saline (p < OOOl), indicating that sperm of SW mice are sensitive to chemical damage. There was no significant difference between the mean number of oocytes in mice treated with 50% nitrous oxide (33.3 f 14.4) and in control mice (29.8 ? 8.0). In the positive control experiment, mice treated with 3-MC had significantly fewer (p < 0.001) primordial oocytes, 67.2 + 19.5 compared with control mice, 222.4 + 21.9, indicating that this strain is sensitive to chemical damage of the ovary. Thus, murine germ cells showed no evidence of toxic effects due to prolonged exposure to nitrous oxide.

Epidemiologic studies have reported signifi- tionally, several studies have reported incant increases in the rates of infertility, spon- creased incidences of spontaneous abortion taneous abortion, congenital anomalies of off- and congenital anomalies among the offspring, and malignancies among operating spring of occupationally exposed men whose room workers chronically exposed to waste female partners were not exposed (ASA, 1974; anesthetic gases (ASA, 1974; Knill-Jones et Knill-Jones et al., 1975; Askrog and Harvald, al., 1972, 1975; Askrog and Harvald, 1970). 1970; Cohen et al., 1980). Thus, there is amSurveys of dentists and their chairside assis- ple reason to examine the effects of exposure tants exposed to nitrous oxide have been sim- of male and female germ cells to anesthetics, ilarly interpreted (Cohen et al., 1980). Addisince damage to these cells could be the common denominator in the observed injuries. Nitrous oxide was the drug used in this study ’ To whom all correspondence should be sent: Anesbecause it is the most commonly used inhathesiology Service ( 112A), Veterans Administration Medlation anesthetic in clinical practice today. ical Center, 3801 Miranda Ave., Palo Alto. Calif. 94304. 0041-008X/83 Copyright All

rights

0 of

1983

$3.00 by

reproduction

Academic in any

370 Press. form

Inc. resewed.

EFFECT OF NrO ON MURINE

METHODS Sixty male and sixty female, 3-to 4-week-old SW mice2 were housed according to sex, four to a cage. They were marked with metal ear tags and quarantined for IO weeks. Room temperature was maintained at 2 I + I ‘C, and artificial light was provided from 7 a.m. to 8 p.m. each day. No germicides or pesticides were used in the animal quarters. Mice were fed a standard laboratory animal dietr and tap water, ad libiwn, except during treatment periods, and were bedded on ground corncob.’ All mice were examined daily for signs of ill health and were weighed weekly. At age I3 to 14 weeks, mice were randomly assigned to four groups, each consisting of 15 males and 15 females. Groups were exposed for 4 hr per day, 5 days per week, for 14 weeks, to either room air, 0.5% nitrous oxide, 5.0% nitrous oxide, or 50% nitrous oxide; oxygen concentration for all treatments was maintained at 2 1 to 25%. The highest dose was previously determined to be the maximum tolerated dose for this mouse strain (Mazze ei al., 1982); the lowest dose was in the range of exposure levels which have been reported in dental operatories but is considerably higher than the average levels present in most operating rooms (Whitcher, 1980). Control and nitrous oxide exposures were carried out in two gas-tight Plexiglass chambers, each with a capacity of 1000 liters. Two exposures were started daily, at 8 AM and 2 PM, with the frequency of morning and afternoon exposures the same for a11groups. Cages were placed randomly in the chambers, and all mice in a treatment group were exposed simultaneously. Atier chambers were brought to the desired nitrous oxide concentrations (a maximum of 15 min was required for the 50% group and less for the other groups), total gas flow in the chambers was maintained at IS to 20 liters/ min with a combination of fresh and recirculated gases. Medical quality gaseswere used for all exposures. Nitrous oxide and oxygen concentrations in each chamber were continuously monitored with a Miran 1A infared analyzer and an Instrumentation Laboratory 40 1 oxygen analyzer, respectively. Concentrations always were maintained within 5% of the desired values. Carbon dioxide concentration was monitored intermittently with a Beckman LB-2 carbon dioxide analyzer and did not exceed 0.3% during an exposure. Temperature in the chambers was continuously monitored and ranged from 2 1 to 24‘C. At the end of an exposure, nitrous oxide was exhausted from the chambers to the outside of the building away from any air intake vent. After 15 mitt, mice were re-

’ Hla:(SW)BR; Hilltop Lab Animals, Inc., Scottsdale, Pa. ’ Wayne Lab Blox, Allied Mills, Inc., Chicago, Ill. ’ Bed-O’Cobs: Anderson’s Cob Division, Maumee, Ohio.

GERM

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371

moved from the chambers and returned to the animal quarters. After 14 weeks oftreatment, mice were killed by carbon dioxide inhalation. The caudae epididymides of male mice were removed, and a sperm suspension from each mouse was prepared by mincing both caudae in 2 ml of phosphate buffered physiologic saline and by filtering the resulting mixture through an 80 p stainless-steel mesh to remove tissue fragments. A portion of the filtered suspension was mixed 1O:l with 1% eosin Y in water, and 30 min later duplicate smears from each preparation were made, allowed to airdry, and mounted under a coverslip. Coded slides were examined at a 400-fold magnification with a blue-green filter; 1000 sperm were examined for morphological abnormalities by criteria defined by Wyrobek and Bruce (1975). Results are expressed as the percentage of abnormal sperm per animal. Additionally, with a hemocytometer, the sperm count/epididymis of the filtered buffered saline sperm suspension was determined. After the caudae epididymides were removed, the testes were weighed, then preserved in 10% buffered formalin solution and processed for histologic examination. Tissue embedded in paraffin was cut 4 to 6 Nrn thick and stained with hematoxylin and eosin. Sections were examined with a light microscope without prior knowledge of the treatment group. To determine whether nitrous oxide treatment resulted in oocyte destruction, ovaries of six female mice from the control group and six mice from the 50% nitrous oxide group were examined, and the number of primordial oocytes was determined by the method of Felton et al. ( 1978). In essence, a formalin fixed ovary from each mouse was embedded in pamgin, serially sectioned at 5 p, and stained with Delatields’ hematoxylin and eosin. Slides were coded and primordial oocytes in every 20th section were counted at lOOO-fold magnification. Results are expressed as the total number of oocytes counted. Prior to this experiment, studies of sperm abnormalities and oocyte destruction in mice had not been previously performed in SW mice from Hilltop Lab Animals. Thus, to determine if this strain of mice could respond if appropriately stimulated, they were treated with chemicals known to give positive results in other strains (Wyrobek and Bruce, 1975; Felton et al., 1978). To test for sperm abnormalities, eighteen 14-week-old SW male mice were administered, ip, daily doses of 75 mg/kg of methyl methanesulfonate (MMS) for 5 consecutive days (Wyrobek and Bruce, 1975); 9 control mice were treated with equal volumes of 0.9% saline. Thirty-five days after the first injection, mice were killed by cervical dislocation, their caudae epididymides were removed, and sperm smears were made as described above. To determine if oocyte destruction would occur, five 13day-old SW mice were treated with a single injection of 80 maJkg of 3methylcholanthrene (3-MC) dissolved in corn oil so that each mouse received the same volume ofoil (20 &g body weight) (Felton et al, 1978); five control mice received corn oil only. Fourteen days after treatment, mice were

372

MAZZE

killed by cervical dislocation; their ovaries were removed, prepared, and examined as described above. Several statistical tests were used to make comparisons among groups. Analysis of variance and Student’s f test were used when data were normally distributed (Snedecor and Cochran, 1967). Nonparametric tests (i.e., MannWhitney and Kolmogorov-SmimolT) were used when data were not distributed normally (Siegel, 1965). Proportions were compared by the z test. All tests were one-sided since we were looking for detrimental changes.

ET AL.

newborn, juvenile, and adult SW mice obtained from Simonsen Laboratories. Although the nitrous oxide experiment was not carried out during the period of greatest sensitivity of the oocyte (Day 13) the results of the positive control experiment indicate that the SW strain we used can respond to chemical damage of the ovary. DISCUSSION

RESULTS Treatment with nitrous oxide did not cause excitement or general anesthesia; rather, mice behaved normally during exposures. There were no significant differences among the four exposure groups in testes weight, percentage of abnormally shaped sperm, sperm count (Table 1), or histologic appearance of the testes. The mean percentage (&SE) of abnormal sperm ranged from 8.9 & 2.4 (5.0% nitrous oxide) to 13.5 & 0.5 (50% nitrous oxide), and the sperm count (X 106) ranged from 17.1 ? 1.6 (50% nitrous oxide) to 21.3 & 1.7 (0.5% nitrous oxide). In the positive control experiment both the percentage of abnormal sperm and the sperm count were significantly changed following treatment with MMS (Table 1). Mice treated with MMS had 25.2 k 4.1% abnormal sperm while control mice treated with saline exhibited 2.5 & 0.3% abnormal sperm (p < 0.001). The sperm count (X 106) was significantly decreased from 17.3 & 2.2 in the saline-treated group to 4.7 * 0.7 in the MMS-treated group (p < 0.00 1). These changes from control levels indicate that sperm of SW mice are sensitive to chemical damage. There was no significant difference between the mean number of oocytes of the group treated with 50% nitrous oxide (33.3 * 14.4) and the control group (29.8 + 8.0). In the positive control experiment, mice treated with 3MC had significantly fewer (p < 0.00 1) primordial oocytes, 67.2 & 19.5, compared with 222.4 & 21.9 for control mice. These results are in agreement with those previously reported by Dobson et ul. (1978) who studied

Male and female murine germ cell populations are relatively easily and inexpensively monitored for deleterious effects. Male gametes may be examined rapidly, reproducibly, and in large numbers (Wyrobek and Bruce, 1978). While the sensitivity of spermatogenic cells to the mutagenic, teratogenic, and carcinogenic effects of many agents does not exactly parallel that of many types of somatic cells, the correlation is reasonably good (Wyrobek and Bruce, 1975, 1978). Well known chemical mutagens such as MMS, metepa, thio-TEPA, mitomycin C, and Myleran, as well as X rays, all produce marked increases in the percentage of abnormally shaped sperm in mice. Teratogens which give rise to increased numbers of abnormally shaped sperm include griseofulvin, Imuran, vinblastine, colchicine, and actinomycin D. Benzo[a]pyrene and 3-MC, which are carcinogens as well as mutagens and teratogens, also cause sperm abnormalities in mice. Sperm abnormalities are sufficiently consistent effects of chemical exposure in mice to permit extrapolation of sperm testing methodology to humans (Wyrobek et al., 198 1, 1983); increases in shape abnormalities of human sperm, as well as reductions in sperm counts, are now used as indicators of chemically induced spermatogenic damage. Although many agents which cause abnormalities in sperm have been identified, it is still not clear how morphological abnormalities arise. Originally, they were thought to be a consequence of chromosomal aberrations, but studies with translocation bearing mice indicate that this aberration is unlikely (Wyrobek et al., 1975)

EFFECT

OF

NzO

ON

ML’RINE

TABLE EFFECT

OF NITROUS

weight k)

Treatment

N

Air 0.5% N20 5.0% N20 50.0% N20

15 14 14 I5

45.2 45.2 45.3 45.0

zk f k z!I

Saline MMS

9 18

43.5 40.4

k 1.7 k 1.2

p<

0.001

as compared

to saline

373

CELLS

I

OXIDE (N20) OR METHYL METHANEWLFONATE ON TESTES AND SPERM (MEAN k SE)

Body

*

CiERM

0.8 I.1 I.0 0.7

Testes

weight w

.255 .2-l-i ,271 .240

* * k T

.Ol2 ,010 ,008 ,008

.186 ?z .OlO .121 k .005*

(MMS)

Mm abnormalities WJ 10.4 9.5 8.9 13.5

2 k k T

2.3 3.0 2.4 2.5

2.5 k 0.3 25.2 k 4.1*

sperm count (X 106) 18.5 21.3 21.1 17.1

k k * k

1.4 1.7 I.9 1.6

11.3 * 2.2 4.1 * 0.7*

control.

and suggest rather, that abnormalities are due to changes in the genes responsible for spermatogenesis (Wyrobek and Bruce, 1975). Regardless of the mechanism, an agent which induces abnormalities in sperm does so by interfering either with the integrity of DNA itself or with the expression of this genetic material. Agents which cause loss of oocytes have been reported to be tumorigenic (Felton er al., 1978), mainly affecting the ovary, although lung and skin also have been involved. In addition to destruction by chemicals, oocytes ,are extremely vulnerable to damage by lowlevel chronic tritium exposure (Dobson et al, 1978). Since the functional destiny of an oocyte is to connect one generation with the next, it is not surprising that significant loss of oocytes leads to sterilization (Dobson et al., 1978). Although, examination of ovaries for evidence of primordial oocyte destruction is not as frequently employed as sperm testing, these studies can yield valuable information regarding the toxicity of some chemical and physical agents. The present study has shown that the germ cells of male and female Hla:(SW)BR mice are sensitive to chemical injury. However, exposure to nitrous oxide levels as high as 500,000 ppm for 4 hr per day for 5 days per week for 14 weeks did not result in a significant increase in the percentage of abnormal

sperm nor was there a decrease in the sperm count; histologic appearance of the testes was unchanged. Destruction of primordial oocytes did not occur. The results of our sperm studies are in agreement with those of Land el al. (1981) who exposed two groups of five (C57Bl X C3H)F, mice to either 8 or 80% nitrous oxide for 4 hr per day for 5 days; they reported no increase in the percentage of abnormal sperm. They did report increased percentages of abnormal sperm after similar exposures to 0.08% chloroform (3.5% abnormal forms), 0.2% trichloroethylene (2.4% abnormal forms), and 1.0% enflurane (2.0% abnormal forms). There were 1.4% abnormal forms in the control group. Although these increases were statistically significant, the absolute values were within or, at most, 0.1% outside of Wyrobek and Bruce’s ( 1975) control range ( 1.2 to 3.4%) for the same strain of mouse. By contrast, the percentage of abnormal sperm after MMS treatment in Wyrobek and Bruce’s (1975) study was 72%. In a subsequent study, Land and Owen (198 1) reported that treatment of mice with halothane at concentrations greater than 0.6% delivered in a mixture of 50% nitrous oxide and 50% oxygen,, resulted in significantly increased percentages of abnormal sperm compared with the same halothane concentrations delivered in air. Again, the maximum difference in the percentage of

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MAZZE

abnormal sperm they observed was only 1% (2.5% treated vs 1.5% control), so these studies must be interpreted with some caution. The results of our study are at variance with those of Kripke et ul. (1976) who reported atrophy of the seminiferous tubules and decreased testicular weight of LEW/f Mai rats exposed to 20% nitrous oxide and 60% nitrogen, either continuously or for 8 hr a day, for periods of up to 35 days. They also reported a decrease in the number of spermatozoa, and they identified some abnormal forms, although, quantitative determinations were not made. Adverse effects were noted as early as 2 days after beginning the 8 hr exposures and were reversible when nitrous oxide treatment was discontinued. In other studies, Gremigni et ul. ( 1978) reported similar findings to those of Kripke et ul. (1976) in the testes of LEW/ f Mai rats continuously exposed to 20% nitrous oxide for 7 days. Later Coate et uf. ( 1979) reported an increase in chromosomal abnormalities in spermatogonia of Sprague-Dawley rats exposed to the combination of 50 ppm nitrous oxide and 1 ppm halothane for 7 hr per day, 5 days per week, for 52 weeks. The significance of the latter findings are open to question, since no major abortifacient effects or teratologic changes in offspring were observed as a consequence of mating these rats with similarly treated females. No factors are apparent, other than species differences, which can explain the positive findings with rats and the negative findings with mice. In the only study of human sperm from individuals exposed to nitrous oxide, Wyrobek et ul. (I 981) examined semen from 46 anesthesiologists who had worked for a minimum of 1 year in hospitals equipped with modern waste anesthetic gas scavenging devices. Semen samples from 26 beginning anesthesia residents were used as controls. Concentrations of sperm and the percentage of abnormal shanes were determined. There were no differences between the anesthesiologists and the beginning residents. Additionally, semen from 13 of the 26 residents was sampled a second time after I year of expo-

ET AL.

sure to waste anesthetic gases; no significant differences in the sperm were found between the two sampling periods. Nitrous oxide concentrations in the hospital in which the majority of the subjects worked have been measured (Whitcher, 1980) and found to average ~50 ppm with a range of 1 to 500 ppm. Since the concentration of waste halothane is usually about 1/6Oth that of nitrous oxide (NIOSH, 1977) these individuals were probably exposed to waste anesthetic gas levels similar to those tested by Coate et ul. ( 1979). Thus, the negative results of the study by Wyrobek et uf. (1981) as well as those of the present study, although not settling the controversy regarding the possible health hazards associated with exposure to nitrous oxide, should be reassuring for persons working in operating rooms.

ACKNOWLEDGMENTS The authors thank Linda Baker, Mark Krize, Laurie Gordon, and George Watchmaker for their excellent technical assistance.

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WYROBEK, A. J., AND BRUCE, W. R. (1978). The induction of sperm-shape abnormalities in mice and humans. In Chemical A4utagenesis (A. Hollaender and F. J. de Serres, eds.), Vol 5, Chap. 11, pp. 257-285. Plenum, New York. WYROBEK, A. J., GORDON, L. A., BURKHART, J. G., et al. (1983). Chemically induced alterations of spermatogenic function in man as measured by semen analyses parameters: A report for the Genetox program. Mutat. Res., in press. WYROBEK, A, J., HEDDLE, J. A., AND BRUCE, W. R. (1975). Chromosomal abnormalities and the morphology of mouse sperm heads. Canad. J. Genet. Cytol. 17,675-68 I.