ENVIRONMENTAL
RESEARCH
SO2
and
9, 84-93
( 1975)
Its Metabolite: Egg
Effects
on Mammalian
Chromosomes
G. M. JAGIELLO, J. S. LIN, AND M. B. DUCAYEN Departments of Obstetrics and Gynecology and Human Genetics and Development, International Institute for the Study of Human Reproduction, Columbia University, College of Physicians and Surgeons, New York, New York 10032
and
Received March 6, 1974 The ubiquity of sulfur dioxide as an air pollutant combined with reports of effects of the gas and its blood metabolite on DNA and chromosomes, prompted a study of possible mutagen action on mammalian oocytes. Utilizing in vitro and in vivo systems, mouse, ewe, and cow oocytes were examined for cytologic evidence of structural or numerical damage in meiosis. Fragmentation with rearrangement seen at M’ and M’ and anaphase lagging seen in vitro in each species are classically known to be etiologic in chromosome abnormalities and must be considered, especially in contaminated areas with foci of fetal loss involving ewe and cow.
Sulfur dioxide has repeatedly been shown to be a significant contaminating constituent of air, particularly in areas near smelters, coal-fired power plants, burning wood, copper, lead and zinc refinery operations, and refuse incineration (Waldbott, 1973; Cooper, 1965; Levine, 1960; U. S. Department of Health, Education and Welfare, 1967). Evidence has accrued which demonstrated a depression of DNA synthesis and chromosome abnormalities in human lymphocytes by SO, (Schneider and Calkins, 1970), as well as growth inhibition of established cell lines, including HeLa (Thompson and Pace, 1962), by sulfur dioxide and its sodium salts. In addition, reaction of sodium bisulfite (NAHSO,) with pyrimidines and a mutagenic action on phage and E. coli have been reported (Chandler, 1972). Since Lin (1956) has observed a detectable concentration of Z5Sin the cytoplasm of mouse oocytes following intraperitoneal injection during hormonally induced superovulation, it seemed reasonable to explore the possible mutagenic action of a sodium salt of sulfur oxides on oocytes of female mammalian meiotic systems. The ability of many mammalian oocytes to spontaneously complete normal meiotic maturation in vitro through first and second metaphase following removal from the ovary was used for cytogenetic examination seeking induced heritable effects on these germ cells. MATERIALS
AND
METHODS
It has been reported in the rat that inhaled SO, appeared rapidly in the blood and was converted to the K and Na salts (K, + Na,SO,) (Sidorenko, 1957). A similar finding has been reported in the guinea pig (Lee, 1966). For these reasons, the Na salt was used in both the in Vitro and in viva experiments. NaZS03, analytic grade (98%) minimum assay’ was used. Preliminary tests, made with ‘Arsenic
(As)
0.0001%;
chloride
(Cl)
0.008%; f ree acid 84
to pass test;
free
alkali
(as NazCoJ)
so,
In
Vitro
McCoy’s 5a + 40% FCS + Na2S03 I%/ cd
0 10 25 t50 100 150 250 350 500 750 1000 10,000
EFFECTS
AND
OF SODIUM
MAMMALIAN
EGG
TABLE (NaZ&)
SULFITE
85
CHROMOSOMES
1A ON MOUSE
MExosIs-(Metaphase’)
OOCYTB
Anaphase’ Tot,al no. oocytes
No. division
“%1 division
Metaphaselfl N
A
87 G 3.5 36 34 3:3 28
75 8671-----Gl(-J) g----24 69 10 -15 42 9 34 100 26 21 (j419-----18 64 ,fj ---
33 52 2.5 19 18
20 22 0 0 0
0 N = normal; and E: = atretic
61 9 42 (j 0 0 o-------
-
B
-
-
C
Metaphaseta I)
P:
14 6-8 -
_
-
-
-
---
A = fragment,ed and/or rearranged; (an advanced stage of “C”).
A
4---------------I------
-
1
N
8 l(j -
-----_
.-
B = clumped;
B
C
-
--
-
-
11
-
---
E
N
Abnorma1
-
-
_ _ _
-
-
-
-
C = fuzzy;
2 _ _ _
1_ 1 _ _ _ _
1 _ _ _ _
I) = pulverized
appropriate lo-fold concentrations of the principal contaminants (As, Pb, Fe) to rule out any possible effects on oocytes in the test system, were negative. In vitro studies of oocyte meiotic maturation were made with mouse, ewe, and cow oocytes. Utilizing techniques for meiotic maturation previously described (Jagiello, 1965; Jagiell o et al., 1974) test concentrations of Na,SO,, were determined by extrapolation from previous reports of Thompson and Pace (1962) and preliminary experiments utilizing the present culture system for minimal effective doses. Freshly made stock solutions of Na,SO:, were diluted to the desired concentrations with the appropriate media for the species. Mouse oocytes were collected from the ovaries of adult, cycling female mice of the random bred Camm” strain using a Zeiss dissecting microscope ( 64 X ) . These were pooled and immediately placed into the test culture system of McCoy’s modified Sa plus 40% fetal calf serum (FCS)” which contained various concentrations of Na,SO, (Tables 1A and 1B). 0 ne series of 14-hour in vitro experiments WCSmade with added Na,SO, and freshly prepared pyrimidine bases (Table 2) to test the effect on meiotic progression reported by Timson (1972) to occur in human lymphocytes in mitosis. Harvests for oocytes at first metaphase ( M’ ) were collected at 5 hours and for second metaphase ( M’) at 15 hours (Fig. I). Incubations were carried out in a 5% CO,/air atmosphere with high humidity. The pH of the culture was 0.15%; heavy metals (as PB) 0.001%; insoluble matter 0.0058; mineral 98% ( Mallinckrodt ). ’ Camm Research Institute, Wayne, New Jersey. 3 Grand Island Biological Co., Grand Island, New York.
iron
(Fe)
0.001%;
assay
(Na,SO.,)
86
JAGIELLO,
LIN
AND
TABLE In
Vitro
EFFECTS
UF SODIUM
1B
(Na$308)
SULFITE
DUCAYEN
ON
~‘IOUSE
OOCYTE:
~\IEIosI~-~~~~TAPHAsE~)
McCoy’s 5a +
405; FCS + NanbOs I%/
Anaphasel Total
Cm"
oo:;;es
0 5 50 100 150 200 250 350 500 1000 10,000
92 20 23 15 55 52 35 32 32 26 16
No. dis:i 76 20 18 1j 27 2.5 21 4 14 0 0
y;, di& 83 100 79 100 49 48 60 44 44 0 0
Metaphasela N
A
11 _ I------
iMetapha.se*a
B
C
I>
_
_
-
E _
-----,i
-
-
-
-
-
13
-
-
-
-
-
4----8----l ----s------1
------
1
Abnorma1
N
A
B
C
D
E
N
65
-
-
-
-
-
1g-----
_ -
_ -
18------lo-----
-
_ -
12-----
-
19----12------4----g
6------z ------
2 11
-
_ _
-_
-
_
QSee foot,note to Table I A.
maintained at 7.2 throughout the culture period. Ewe and cow oocytes were obtained from anestrous adult females of mixed breed. Ovaries were collected at a local slaughterhouse immediately following exsanguination, washed with penicillin/streptomycin and transported quickly to the laboratory in a sterile container. Oocytes were removed with sterile precautions in the same fashion as were the mouse oocytes, pooled and rapidly placed in Diploid medium” with 12%sheep serum’ and appropriate concentrations of Na$03 (Tables 3 and 4). Harvests of M’ and M” for both species were collected at 28 hours (Fig. 1). A modification of the method of Tarkowski (1966) was used for the preparation of harvests for cytologic examination of bivalents of M’ and chromosomes of M* of all species. These were stained with 1%toluidine blue in resin and well-spread, intact cells were scored for number and structure using a Zeiss photomicroscope ( 1250~ ). Structural damage was scored as A = fragmented and/or rearranged; B = clumped but with sufficient structural integrity to be recognizable as to meiotic stage; C = fuzzy, indefinite outlines to either bivalents or chromosomes but with a normal number of each; D = pulverized or shattered into innumerable parts and reminiscent of structural damage seenwith virus infections in mitotically dividing cells; and E = atretic, an advanced stage of “c” (Fig. 2a-e). In addition, observations were made for possible spindle abnormalities such as multipolarity, lagging of chromosomes during anaphase’ (Fig. 2f) and suppression of polar body formation expressed as a mixed M2/PB1 complement. Photomicrographs were taken and examined for verification of microscopic observations. Statistical analysis of effects of Na,SO, on division rates was carried out using the chi-square method. ’ Colorado Serum Co., Denver, Colorado.
so,
AND
MAMSlALIAN
EGG
CHROMOSOMES
87
88
JAGIELLO,
LIN
AND
DUCAYEN
* b3 P P rb FIG. 1. chromosomes
Normal at M2:
appearance (b) mouse;
of oocytes (d) cow; (f)
at M’: (a) ewe. 1188~
mouse;
(c)
cow;
(e)
f ewe;
and
Diploid + SS 1270 + Na303 &cm3
Plnaphasel Total no. (IOcytes
No. division
:;, divisiou
N
50 100
28 16 18
19 14 13
68 87
1 _----_
250
20
II
r,s
-
350
23
13
500 1250
47 33
YG 24
56 76 72
2----2 2 1 s -
0
a Pee footnote b Lag.
Metaph‘tielLL
72
tu Table
-4
B
>letaph;~ Chromosome Conference, p. 17. U. S. Dept. Health, Education & Welfare (1967). Air Quality Criteria for Sulfur Oxides, pp. 76-79, Washington, D. C. WALDBOTT, G. L. (1973). Health effects of Environmental Pollutants. p. 80. C. V. Mosby, Co., St. Louis, MO.
RESEARCH
SO2
and
9, 84-93
( 1975)
Its Metabolite: Egg
Effects
on Mammalian
Chromosomes
G. M. JAGIELLO, J. S. LIN, AND M. B. DUCAYEN Departments of Obstetrics and Gynecology and Human Genetics and Development, International Institute for the Study of Human Reproduction, Columbia University, College of Physicians and Surgeons, New York, New York 10032
and
Received March 6, 1974 The ubiquity of sulfur dioxide as an air pollutant combined with reports of effects of the gas and its blood metabolite on DNA and chromosomes, prompted a study of possible mutagen action on mammalian oocytes. Utilizing in vitro and in vivo systems, mouse, ewe, and cow oocytes were examined for cytologic evidence of structural or numerical damage in meiosis. Fragmentation with rearrangement seen at M’ and M’ and anaphase lagging seen in vitro in each species are classically known to be etiologic in chromosome abnormalities and must be considered, especially in contaminated areas with foci of fetal loss involving ewe and cow.
Sulfur dioxide has repeatedly been shown to be a significant contaminating constituent of air, particularly in areas near smelters, coal-fired power plants, burning wood, copper, lead and zinc refinery operations, and refuse incineration (Waldbott, 1973; Cooper, 1965; Levine, 1960; U. S. Department of Health, Education and Welfare, 1967). Evidence has accrued which demonstrated a depression of DNA synthesis and chromosome abnormalities in human lymphocytes by SO, (Schneider and Calkins, 1970), as well as growth inhibition of established cell lines, including HeLa (Thompson and Pace, 1962), by sulfur dioxide and its sodium salts. In addition, reaction of sodium bisulfite (NAHSO,) with pyrimidines and a mutagenic action on phage and E. coli have been reported (Chandler, 1972). Since Lin (1956) has observed a detectable concentration of Z5Sin the cytoplasm of mouse oocytes following intraperitoneal injection during hormonally induced superovulation, it seemed reasonable to explore the possible mutagenic action of a sodium salt of sulfur oxides on oocytes of female mammalian meiotic systems. The ability of many mammalian oocytes to spontaneously complete normal meiotic maturation in vitro through first and second metaphase following removal from the ovary was used for cytogenetic examination seeking induced heritable effects on these germ cells. MATERIALS
AND
METHODS
It has been reported in the rat that inhaled SO, appeared rapidly in the blood and was converted to the K and Na salts (K, + Na,SO,) (Sidorenko, 1957). A similar finding has been reported in the guinea pig (Lee, 1966). For these reasons, the Na salt was used in both the in Vitro and in viva experiments. NaZS03, analytic grade (98%) minimum assay’ was used. Preliminary tests, made with ‘Arsenic
(As)
0.0001%;
chloride
(Cl)
0.008%; f ree acid 84
to pass test;
free
alkali
(as NazCoJ)
so,
In
Vitro
McCoy’s 5a + 40% FCS + Na2S03 I%/ cd
0 10 25 t50 100 150 250 350 500 750 1000 10,000
EFFECTS
AND
OF SODIUM
MAMMALIAN
EGG
TABLE (NaZ&)
SULFITE
85
CHROMOSOMES
1A ON MOUSE
MExosIs-(Metaphase’)
OOCYTB
Anaphase’ Tot,al no. oocytes
No. division
“%1 division
Metaphaselfl N
A
87 G 3.5 36 34 3:3 28
75 8671-----Gl(-J) g----24 69 10 -15 42 9 34 100 26 21 (j419-----18 64 ,fj ---
33 52 2.5 19 18
20 22 0 0 0
0 N = normal; and E: = atretic
61 9 42 (j 0 0 o-------
-
B
-
-
C
Metaphaseta I)
P:
14 6-8 -
_
-
-
-
---
A = fragment,ed and/or rearranged; (an advanced stage of “C”).
A
4---------------I------
-
1
N
8 l(j -
-----_
.-
B = clumped;
B
C
-
--
-
-
11
-
---
E
N
Abnorma1
-
-
_ _ _
-
-
-
-
C = fuzzy;
2 _ _ _
1_ 1 _ _ _ _
1 _ _ _ _
I) = pulverized
appropriate lo-fold concentrations of the principal contaminants (As, Pb, Fe) to rule out any possible effects on oocytes in the test system, were negative. In vitro studies of oocyte meiotic maturation were made with mouse, ewe, and cow oocytes. Utilizing techniques for meiotic maturation previously described (Jagiello, 1965; Jagiell o et al., 1974) test concentrations of Na,SO,, were determined by extrapolation from previous reports of Thompson and Pace (1962) and preliminary experiments utilizing the present culture system for minimal effective doses. Freshly made stock solutions of Na,SO:, were diluted to the desired concentrations with the appropriate media for the species. Mouse oocytes were collected from the ovaries of adult, cycling female mice of the random bred Camm” strain using a Zeiss dissecting microscope ( 64 X ) . These were pooled and immediately placed into the test culture system of McCoy’s modified Sa plus 40% fetal calf serum (FCS)” which contained various concentrations of Na,SO, (Tables 1A and 1B). 0 ne series of 14-hour in vitro experiments WCSmade with added Na,SO, and freshly prepared pyrimidine bases (Table 2) to test the effect on meiotic progression reported by Timson (1972) to occur in human lymphocytes in mitosis. Harvests for oocytes at first metaphase ( M’ ) were collected at 5 hours and for second metaphase ( M’) at 15 hours (Fig. I). Incubations were carried out in a 5% CO,/air atmosphere with high humidity. The pH of the culture was 0.15%; heavy metals (as PB) 0.001%; insoluble matter 0.0058; mineral 98% ( Mallinckrodt ). ’ Camm Research Institute, Wayne, New Jersey. 3 Grand Island Biological Co., Grand Island, New York.
iron
(Fe)
0.001%;
assay
(Na,SO.,)
86
JAGIELLO,
LIN
AND
TABLE In
Vitro
EFFECTS
UF SODIUM
1B
(Na$308)
SULFITE
DUCAYEN
ON
~‘IOUSE
OOCYTE:
~\IEIosI~-~~~~TAPHAsE~)
McCoy’s 5a +
405; FCS + NanbOs I%/
Anaphasel Total
Cm"
oo:;;es
0 5 50 100 150 200 250 350 500 1000 10,000
92 20 23 15 55 52 35 32 32 26 16
No. dis:i 76 20 18 1j 27 2.5 21 4 14 0 0
y;, di& 83 100 79 100 49 48 60 44 44 0 0
Metaphasela N
A
11 _ I------
iMetapha.se*a
B
C
I>
_
_
-
E _
-----,i
-
-
-
-
-
13
-
-
-
-
-
4----8----l ----s------1
------
1
Abnorma1
N
A
B
C
D
E
N
65
-
-
-
-
-
1g-----
_ -
_ -
18------lo-----
-
_ -
12-----
-
19----12------4----g
6------z ------
2 11
-
_ _
-_
-
_
QSee foot,note to Table I A.
maintained at 7.2 throughout the culture period. Ewe and cow oocytes were obtained from anestrous adult females of mixed breed. Ovaries were collected at a local slaughterhouse immediately following exsanguination, washed with penicillin/streptomycin and transported quickly to the laboratory in a sterile container. Oocytes were removed with sterile precautions in the same fashion as were the mouse oocytes, pooled and rapidly placed in Diploid medium” with 12%sheep serum’ and appropriate concentrations of Na$03 (Tables 3 and 4). Harvests of M’ and M” for both species were collected at 28 hours (Fig. 1). A modification of the method of Tarkowski (1966) was used for the preparation of harvests for cytologic examination of bivalents of M’ and chromosomes of M* of all species. These were stained with 1%toluidine blue in resin and well-spread, intact cells were scored for number and structure using a Zeiss photomicroscope ( 1250~ ). Structural damage was scored as A = fragmented and/or rearranged; B = clumped but with sufficient structural integrity to be recognizable as to meiotic stage; C = fuzzy, indefinite outlines to either bivalents or chromosomes but with a normal number of each; D = pulverized or shattered into innumerable parts and reminiscent of structural damage seenwith virus infections in mitotically dividing cells; and E = atretic, an advanced stage of “c” (Fig. 2a-e). In addition, observations were made for possible spindle abnormalities such as multipolarity, lagging of chromosomes during anaphase’ (Fig. 2f) and suppression of polar body formation expressed as a mixed M2/PB1 complement. Photomicrographs were taken and examined for verification of microscopic observations. Statistical analysis of effects of Na,SO, on division rates was carried out using the chi-square method. ’ Colorado Serum Co., Denver, Colorado.
so,
AND
MAMSlALIAN
EGG
CHROMOSOMES
87
88
JAGIELLO,
LIN
AND
DUCAYEN
* b3 P P rb FIG. 1. chromosomes
Normal at M2:
appearance (b) mouse;
of oocytes (d) cow; (f)
at M’: (a) ewe. 1188~
mouse;
(c)
cow;
(e)
f ewe;
and
Diploid + SS 1270 + Na303 &cm3
Plnaphasel Total no. (IOcytes
No. division
:;, divisiou
N
50 100
28 16 18
19 14 13
68 87
1 _----_
250
20
II
r,s
-
350
23
13
500 1250
47 33
YG 24
56 76 72
2----2 2 1 s -
0
a Pee footnote b Lag.
Metaph‘tielLL
72
tu Table
-4
B
>letaph;~ Chromosome Conference, p. 17. U. S. Dept. Health, Education & Welfare (1967). Air Quality Criteria for Sulfur Oxides, pp. 76-79, Washington, D. C. WALDBOTT, G. L. (1973). Health effects of Environmental Pollutants. p. 80. C. V. Mosby, Co., St. Louis, MO.
