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Tissue distribution of 2,3,7,8-TCDD in bullfrogs obtained from a 2,3,7,8-TCDD-contaminated area
Chemosphere, Vol.15, No.2, pp 121-126, Printed in Great Britain
1986
OO45-6535/86 $3.OO + .00 O1986 Pergamon Press Ltd.
TISSUE DISTRIBUTION OF 2,3,7,8-TCDD IN BULLFROGS OBTAINED FROMA 2,3,7,8oTCDD-CONTAMINATED AREA Walter A. Korfmacher , Eugene B. Hansen,Jr., Kenneth L. Rowland Department of Health and Human Services Food and Drug Administration National Center for Toxicological Research Division of Chemistry Jefferson, Arkansas 72079 USA ABSTRACT Adult bullfrogs
(Rana catesbetana) were collected
Arkansas, a known area of 2,3,7,8-TCOD contamination.
along the Rockey Branch Creek in Various tissues from the frogs were
analyzed for 2,3,7,8-TCDO. Levels of 2,3,7,8-TCDD ranging from 87 ppt for a muscle sample to 68,000 ppt for a fat sample were measured. INTRODUCTION Due to the high t o x i c i t y of 2,3,7,8-tetrachlorodibenzo-~-dioxin (2,3,7,8-TCDD)
and i t s
release into the environment, there continues to be an interest in the analysis of environmental samples for 2,3,7,8-TCDD (1,2).
Whtle i t is generally assumed that 2,3,7,8-TCDO concentrates in
the f a t and l i v e r of animals, very few tissue d i s t r i b u t i o n studies of environmentally contaminated animals have been reported. Most of the tissue distribution studies have been laboratory feeding studies, and t h e i r relationship to environmental (3-7). We report catesbeiana).
here the analysis of 2,3,7,8-TCDD in
samples remains unclear
various parts of the bullfrog
(Rana
The frogs were collected from the Rockey Branch Creek which flows through an
industrial site in central Arkansas that is known to be contaminated by 2,3,7,8-TCDD and empties into the Bayou Meto. The industrial s i t e was formerly used for production of 2,4,5-T and 2,4-O and currently, several thousand barrels of 2,3,7,8-TCDD-contaminated waste remain on the s i t e . The recently validated method of Korfmacher et al. (2) for the isemer-spectfic analysis of 2,3,7,8-TCDD in fat samples was used for the analysis of the frog samples. This method uses the technique of fused s i l i c a GC combined with negative ionization atmospheric pressure ionization mass spectrometry (NIAPIMS) for the ouantitation step. EXPERIMENTAL Samples. The frogs were collected in 1984 and 1985 from the Rockey Branch Creek in central Arkansas below the industrial site and above i t s junction with the Bayou Meto. The frogs were stored at -20"C prior to dissection. actually analyzed.
The tissue portions were also stored at -20"C until
For a recovery experiment, frog legs were obtained from a local store and
one sample was spiked at a level of 100 ppt with 2,3,7,8-TCDD-14C and carried through the extraction and cleanup. Extraction and Cleanup. The extraction and cleanup of the samples has been described elsewhere (2). T y p i c a l l y 10 g of sample were weighed i n t o a f l a s k , spiked with
121
122
2,3,7,8-TCDD-13C, and then refluxed with KOH/ETOH solution for 2.5 hours. The solution was then extracted with hexane, washed with sulfuric acid, neutralized and passed through an alumina column.
The sample was then carried through s i l i c a HPLC using hexane as the eluent.
Final
cleanup was performed by a reverse-phase (ODS) HPLC step using acetonitrile as the eluent.
The
sample was then taken to dryness and then taken up into 30 pL of benzene for analysis by GC-NIAPIMS. Apparatus.
The atmospheric pressure ionization mass spectrometer
(Extranuclear
Laboratories, Inc.) has been described previously (8,9). For the current analysis, a 50-m SP2100 fused s i l i c a capillary column (0.2 mm i . d . , Hewlett-Packard, #19091-60050) connected directly to the APIMS source was employed.
A moving needle injector (Chrompack #9001) was
connected to the fused s i l i c a GC column via a silicone rubber adhesive sealant (g).
The GC
carrier gas was helium, set at a flow rate of about 0.5 cm3/min (with an injector head pressure of 50 p . s . i . ) and was made up to a flow of 18 cm3/min into the APIMS by 0.1% oxygen in nitrogen. The APIMS was operated in the negative ion mode. RESULTS AND DISCUSSION The results of the analyses of the bullfrog tissue samples are shown in Table I.
These
results show that all the bullfrogs contained r e l a t i v e l y high levels of 2,3,7,8-TCDD. These data suggested a relationship for the distribution of 2,3,7,8-TCDD in different tissues. Table I I shows the same data normalized to the amount of 2,3,7,8-TCDD in the leg muscle for each frog and includes the average and standard deviation for these ratios.
These results show a
significant range in the 2,3,7,8-TCDD tissue distribution for these frogs,
ranging from a
relative ratio of 1.0 to 661 for leg muscle and the one fat sample that was quantitated, respectively. After extraction and cleanup, the recovery of 2,3,7,8-TCDD-14C in the purchased frog leg sample was found to be 33% which is a level similar to that which has been reported previously for this procedure (2). Fig. 1 shows the GC-NIAPIMS response for a frog ovary sample (frog D). This figure shows the response for 2,3,7,8-TCDD at m/z 176 and m/z 178. Under NIAPIMS conditions, 2,3,7,8-TCDD reacts with 02 via an ion-molecule reaction producing the 4,5-dichloro-1,2-benzoouinone anion (8,9,11).
The peak at m/z 182 is due to the 2,3,7,8-TCDD-13C internal standard.
The results suggest two points.
The f i r s t point is that frogs seem to be able to tolerate
a substantial body burden of 2,3,7,8-TCDD. When captured, these frogs were alive and appeared to be healthy.
There is a significant range in the toxicity of 2,3,7,8-TCDD for various
species. Single-dose oral LDso values for 2,3,7,8-TCDD range from 2 ~g/kg for the guinea pig to 50 ~g/kg for the monkey to 1157 ~g/kg for the hamster (3). Beaty et al. (4) found no significant level of mortality after ( i . p . ) injection of 1000 ~g/kg of 2,3,7,8-TCDD into (Rana catesbeiana) tadpoles or after ( i . p . ) injection of up to 500 ~g/kg into adult (Rana catesbeiana) bullfrogs.
Therefore, our results tend to confirm their findings that 2,3,7,8-TCDD is not
highly toxic to adult bullfrogs; The second point is that tissue distribution of 2,3,7,8-TCDD that was found is of interest because we believe i t is the only report for the tissue distribution of 2,3,7,8-TCDD in I
123
Table I - Results of Analysis of Tissue Samples for 2,3,7,8-TCDDa'b Parameter
Fro9 A
Frog B
Fro9 C
F
F
F
Large
Large
Sexc Size d Muscle
Skin
218 +
60
364 + 66
Oviduct
988 + 448 1100 +
Liver
277 +
60
229 +
41
623 +
105
661 +
134
496 +
57
Fro9 D F
Medium
Medium
Fro 9 F
M
M
Large
87 +
26
103 + 17
637 + 202
217 +
34
637 + 100
1710 + 854
Large 566 + 73
1590 + 424 m
148 + 22
2050 + 197
NA
NA
154 564 1250 + 174
1260 + 122
Ovaries 7460 + 1440 10400 + 5370 2830 + 998
2700 + 340
Fat
Fro 9 E
2390 + 279
3680 +
NO
NO
NO
68000 + 7 1 2 0
1130 + 3280 9800 + 983 NA >14,000 e
NA ---
Levels are p a r t s - p e r - t r i l l i o n (ppt ~ pg/g)
of 2,3,7,8-TCDD; "~ values" are 95%
confidence l i m i t s
some duplicate cases samples were
(see reference 10);
in
analyzed and each r e s u l t is reported in t h i s table. NO = tissue not observed in frog; NA = tissue not available due to sex of frog; ....
Sample not analyzed.
F = Female; M = Male. Large = 450 - 800 g t o t a l weight; medium = 200 - 450 g t o t a l weight of frog. Result was outside the calibration range of the instrument, due to the r e l a t i v e l y low 2,3,7,8-TCDD-13C spike for this sample.
G) >
¢) n"
° l 4~
1
rE) t'-
Ol
58
m/z 176
I'--"
I
I
I
I
I
"~
I'---'I
....
I------I
10 Retention Time, min.
I
....
"I"--°-1
"'-'
2 , 3 , 7 , 8 - T C D D J S C =.--
~:';,~;,..,~,~,,_"~,,~,llu~
2,3,7,8-TCDD
16
-'1
-
"1-"
~t.t.,L~,/ll
Figure 1. GC-NIAPIMSresponse for a frog ovary sample found to contain 2700+ 340 ppt 2,3,7,8-TCDD
1 .....
m/z 182
m/z 178
~ ~ . ,
I----"
0
70-
100
4==
I'-"
125
Table I I - Relative Ratios of Tissue Levels of 2,3,7,8-TCDDa
Parameters
Averages +_ SDb
Frog A
B
C
D
E
F
F
F
F
F
M
M
Muscle
1.0
1.0
1.0
Skin
1.7
2.5
2.5
6.2
Oviduct
4.8
2.0
1.7
19.9
NA
NA
Liver
11.0
14.6
14.3
12.2
17,8
17.3
13.
~ 2.0
15.
+ 2.7
Ovaries
34.2
41.2
32.5
26.2
NA
NA
36.
~4.6
34.
+6.2
NO
NO
Sex
Fat
NO
1.0
661.
A-C
A-F
1.0
1.0
1.0
1.0
2.6
---
2.2+0.5
3.1 + 1.8
2.8+1.7
7.1 + 8.6
>20
---
661.
a The 2,3,7,8-TC0D muscle level was defined as 1.0 for each frog. b
Standard deviation.
environmentally-contaminated
bullfrogs.
In a laboratory
feeding
study on guinea pigs,
Gaslewlcz and N@al (5) reported relative ratios of 2,3,7,8-TCDD ranging from 1.0 for bone marrow to 16 for liver.
Olson et al. (6) reported a tissue distribution study for a hamster
injected with 2,3,7,8-TCDD which ranged from a relative ratio for 2,3,7,8-TCDD of 1.0 for skin to 29 for the liver.
In another laboratory study by Gasiewicz et al. (7), mice were injected
with 2,3,7,8-TCDD and for the {C57) mice they found a relative 2,3,7,8-TCD0 ratio range of 1.0 for the heart muscle to 17 for adipose tissue to 38 for liver. Gaslewlcz et al. {7) reported similar results for two other strains of mice. In a recent report, by J. J. Ryan et al. (12), the tissue distribution of "total dioxins" {tetra- to octa-chlorodlbenzo-p-dloxins) humans was studied.
in two
In each case, the lowest level of total dioxins was the kidney and the
highest was in fat tissue.
Their results suggest that most of-the concentration variation was
due to the percent lipid in the tissues analyzed.
Similarly, it is likely that the percent
lipid in the frog tissues shown in Table I would account for much of concentration differences found in these tissues. Thus, the results obtained from the analysis of 2,3,7,8-TCDD - contaminated frog tissues agree reasonably well for the muscle, skin and fat data that is available from laboratory animal studies of other animals (5-7).
The one fat sample that was quantltated was found to
have the highest relative ratio of 2,3,7,8-TCD0 for the tissues that were studied. did not have significant levels of fat that was available for analysis.
Frogs A-C
126
Frogs A-C were gravid female frogs caught at approximately the same time which did not have significant levels of fat bodies available for analysis. They all had distended ovaries and oviducts which contained large numbers of eggs. This may be the reason that they had very l i t t l e observable fat tissue. Frog D, on the other hand, had relatively small ovaries and oviducts and had a sufficient amount of fat tissue for analysis. Table I I shows the average relative tissue ratios for frogs A-C as a well matched subgroup separately from the averages for all six frogs. The results for all six bullfrogs show a clear pattern for bullfrog tissues in which the muscle contains the least amount of 2,3,7,8-TCDD per gram and the ovaries or fat contains the most 2,3,7,8-TCDD per gram. While the data set is limited, the results also suggest that the ratio of liver to muscle and skin to muscle was similar for male and female frogs. Additionally, the data set suggests that the relative level of 2,3,7,8-TCDD in the ovaries of female frogs was higher than the level in the liver for those frogs. Therefore, these results tend to support the hypothesis that frogs are much less sensitive to 2,3,7,8-TCDD than are guinea pigs (4). In addition, these results suggest that female frogs store a significant amount of their 2,3,7,8-TCDD body burden in their ovaries and oviducts, a finding that has not previously been reported. ACKNOWLEDGEMENT: The authors thank Lyle Davis for typing the manuscript and Daniel L. Davis (Jacksonville, Arkansas) for helping to collect the bullfrogs. REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
T. Cairns, L. Fishbein, and R. K. H'itchm, Biomed. Mass Spectrom., 7_., 484 (1980). W.A. Korfmacher, G. F. Moler, R. R. Delongchamp, R. K. Mltchum, and R. L. Harless, Chemosphere, 13, 669 (1984). R . A . Neal, J. R. Olson, T. A. Gasiewicz, and L. E. Geiger, Dru9 Metab. Rev., 13, 355 (1982). P.W. Beatty, M. A. Holscher, and R. A. Neal, Bull. Environ. Contam. Toxic., 16, 578 (1976). T . A . Gasiewicz and R. A. Neal, Toxicol. Appl. Pharmacol., 51, 329 (1979). J.R. Olson, T. A. Gasiewicz, and R. A. Neal, Toxicol. Appl. Pharmacol., 56, 78 (1980). T . A . Gasiewicz, L. E. Geiger, G. Rucci, and R. A. Neal, Dru9 Metab. Dispo. 11, 397 (1983). R.K. Mitchum, G. F. Moler, and W. A. Korfmacher, Anal. Chem., 52, 2278 (1980). R.K. Mitchum, W. A. Korfmacher, G. F. Moler, and D. L. Stalling, Anal. Chem., 54, 719 (1982). G. F. Moler, R. R. Delongchamp, W. A. Korfmacher, B. A. Pearce, and R. K. Mitchum, Anal. Chem., 55, 835 (1983). R. K. Mitchum, W. A. Korfmacher, and J. R. Althaus, Or9, Mass Spectrom. 19, 63 (1984). J. J. Ryan, A. Schecter, R. Lizotte, W. F. Sun, and L. Miller, Chemosphere, 14, 929 (1985). ~Received in Germany 28 October 1985- accepted
3 December
1985)
1986
OO45-6535/86 $3.OO + .00 O1986 Pergamon Press Ltd.
TISSUE DISTRIBUTION OF 2,3,7,8-TCDD IN BULLFROGS OBTAINED FROMA 2,3,7,8oTCDD-CONTAMINATED AREA Walter A. Korfmacher , Eugene B. Hansen,Jr., Kenneth L. Rowland Department of Health and Human Services Food and Drug Administration National Center for Toxicological Research Division of Chemistry Jefferson, Arkansas 72079 USA ABSTRACT Adult bullfrogs
(Rana catesbetana) were collected
Arkansas, a known area of 2,3,7,8-TCOD contamination.
along the Rockey Branch Creek in Various tissues from the frogs were
analyzed for 2,3,7,8-TCDO. Levels of 2,3,7,8-TCDD ranging from 87 ppt for a muscle sample to 68,000 ppt for a fat sample were measured. INTRODUCTION Due to the high t o x i c i t y of 2,3,7,8-tetrachlorodibenzo-~-dioxin (2,3,7,8-TCDD)
and i t s
release into the environment, there continues to be an interest in the analysis of environmental samples for 2,3,7,8-TCDD (1,2).
Whtle i t is generally assumed that 2,3,7,8-TCDO concentrates in
the f a t and l i v e r of animals, very few tissue d i s t r i b u t i o n studies of environmentally contaminated animals have been reported. Most of the tissue distribution studies have been laboratory feeding studies, and t h e i r relationship to environmental (3-7). We report catesbeiana).
here the analysis of 2,3,7,8-TCDD in
samples remains unclear
various parts of the bullfrog
(Rana
The frogs were collected from the Rockey Branch Creek which flows through an
industrial site in central Arkansas that is known to be contaminated by 2,3,7,8-TCDD and empties into the Bayou Meto. The industrial s i t e was formerly used for production of 2,4,5-T and 2,4-O and currently, several thousand barrels of 2,3,7,8-TCDD-contaminated waste remain on the s i t e . The recently validated method of Korfmacher et al. (2) for the isemer-spectfic analysis of 2,3,7,8-TCDD in fat samples was used for the analysis of the frog samples. This method uses the technique of fused s i l i c a GC combined with negative ionization atmospheric pressure ionization mass spectrometry (NIAPIMS) for the ouantitation step. EXPERIMENTAL Samples. The frogs were collected in 1984 and 1985 from the Rockey Branch Creek in central Arkansas below the industrial site and above i t s junction with the Bayou Meto. The frogs were stored at -20"C prior to dissection. actually analyzed.
The tissue portions were also stored at -20"C until
For a recovery experiment, frog legs were obtained from a local store and
one sample was spiked at a level of 100 ppt with 2,3,7,8-TCDD-14C and carried through the extraction and cleanup. Extraction and Cleanup. The extraction and cleanup of the samples has been described elsewhere (2). T y p i c a l l y 10 g of sample were weighed i n t o a f l a s k , spiked with
121
122
2,3,7,8-TCDD-13C, and then refluxed with KOH/ETOH solution for 2.5 hours. The solution was then extracted with hexane, washed with sulfuric acid, neutralized and passed through an alumina column.
The sample was then carried through s i l i c a HPLC using hexane as the eluent.
Final
cleanup was performed by a reverse-phase (ODS) HPLC step using acetonitrile as the eluent.
The
sample was then taken to dryness and then taken up into 30 pL of benzene for analysis by GC-NIAPIMS. Apparatus.
The atmospheric pressure ionization mass spectrometer
(Extranuclear
Laboratories, Inc.) has been described previously (8,9). For the current analysis, a 50-m SP2100 fused s i l i c a capillary column (0.2 mm i . d . , Hewlett-Packard, #19091-60050) connected directly to the APIMS source was employed.
A moving needle injector (Chrompack #9001) was
connected to the fused s i l i c a GC column via a silicone rubber adhesive sealant (g).
The GC
carrier gas was helium, set at a flow rate of about 0.5 cm3/min (with an injector head pressure of 50 p . s . i . ) and was made up to a flow of 18 cm3/min into the APIMS by 0.1% oxygen in nitrogen. The APIMS was operated in the negative ion mode. RESULTS AND DISCUSSION The results of the analyses of the bullfrog tissue samples are shown in Table I.
These
results show that all the bullfrogs contained r e l a t i v e l y high levels of 2,3,7,8-TCDD. These data suggested a relationship for the distribution of 2,3,7,8-TCDD in different tissues. Table I I shows the same data normalized to the amount of 2,3,7,8-TCDD in the leg muscle for each frog and includes the average and standard deviation for these ratios.
These results show a
significant range in the 2,3,7,8-TCDD tissue distribution for these frogs,
ranging from a
relative ratio of 1.0 to 661 for leg muscle and the one fat sample that was quantitated, respectively. After extraction and cleanup, the recovery of 2,3,7,8-TCDD-14C in the purchased frog leg sample was found to be 33% which is a level similar to that which has been reported previously for this procedure (2). Fig. 1 shows the GC-NIAPIMS response for a frog ovary sample (frog D). This figure shows the response for 2,3,7,8-TCDD at m/z 176 and m/z 178. Under NIAPIMS conditions, 2,3,7,8-TCDD reacts with 02 via an ion-molecule reaction producing the 4,5-dichloro-1,2-benzoouinone anion (8,9,11).
The peak at m/z 182 is due to the 2,3,7,8-TCDD-13C internal standard.
The results suggest two points.
The f i r s t point is that frogs seem to be able to tolerate
a substantial body burden of 2,3,7,8-TCDD. When captured, these frogs were alive and appeared to be healthy.
There is a significant range in the toxicity of 2,3,7,8-TCDD for various
species. Single-dose oral LDso values for 2,3,7,8-TCDD range from 2 ~g/kg for the guinea pig to 50 ~g/kg for the monkey to 1157 ~g/kg for the hamster (3). Beaty et al. (4) found no significant level of mortality after ( i . p . ) injection of 1000 ~g/kg of 2,3,7,8-TCDD into (Rana catesbeiana) tadpoles or after ( i . p . ) injection of up to 500 ~g/kg into adult (Rana catesbeiana) bullfrogs.
Therefore, our results tend to confirm their findings that 2,3,7,8-TCDD is not
highly toxic to adult bullfrogs; The second point is that tissue distribution of 2,3,7,8-TCDD that was found is of interest because we believe i t is the only report for the tissue distribution of 2,3,7,8-TCDD in I
123
Table I - Results of Analysis of Tissue Samples for 2,3,7,8-TCDDa'b Parameter
Fro9 A
Frog B
Fro9 C
F
F
F
Large
Large
Sexc Size d Muscle
Skin
218 +
60
364 + 66
Oviduct
988 + 448 1100 +
Liver
277 +
60
229 +
41
623 +
105
661 +
134
496 +
57
Fro9 D F
Medium
Medium
Fro 9 F
M
M
Large
87 +
26
103 + 17
637 + 202
217 +
34
637 + 100
1710 + 854
Large 566 + 73
1590 + 424 m
148 + 22
2050 + 197
NA
NA
154 564 1250 + 174
1260 + 122
Ovaries 7460 + 1440 10400 + 5370 2830 + 998
2700 + 340
Fat
Fro 9 E
2390 + 279
3680 +
NO
NO
NO
68000 + 7 1 2 0
1130 + 3280 9800 + 983 NA >14,000 e
NA ---
Levels are p a r t s - p e r - t r i l l i o n (ppt ~ pg/g)
of 2,3,7,8-TCDD; "~ values" are 95%
confidence l i m i t s
some duplicate cases samples were
(see reference 10);
in
analyzed and each r e s u l t is reported in t h i s table. NO = tissue not observed in frog; NA = tissue not available due to sex of frog; ....
Sample not analyzed.
F = Female; M = Male. Large = 450 - 800 g t o t a l weight; medium = 200 - 450 g t o t a l weight of frog. Result was outside the calibration range of the instrument, due to the r e l a t i v e l y low 2,3,7,8-TCDD-13C spike for this sample.
G) >
¢) n"
° l 4~
1
rE) t'-
Ol
58
m/z 176
I'--"
I
I
I
I
I
"~
I'---'I
....
I------I
10 Retention Time, min.
I
....
"I"--°-1
"'-'
2 , 3 , 7 , 8 - T C D D J S C =.--
~:';,~;,..,~,~,,_"~,,~,llu~
2,3,7,8-TCDD
16
-'1
-
"1-"
~t.t.,L~,/ll
Figure 1. GC-NIAPIMSresponse for a frog ovary sample found to contain 2700+ 340 ppt 2,3,7,8-TCDD
1 .....
m/z 182
m/z 178
~ ~ . ,
I----"
0
70-
100
4==
I'-"
125
Table I I - Relative Ratios of Tissue Levels of 2,3,7,8-TCDDa
Parameters
Averages +_ SDb
Frog A
B
C
D
E
F
F
F
F
F
M
M
Muscle
1.0
1.0
1.0
Skin
1.7
2.5
2.5
6.2
Oviduct
4.8
2.0
1.7
19.9
NA
NA
Liver
11.0
14.6
14.3
12.2
17,8
17.3
13.
~ 2.0
15.
+ 2.7
Ovaries
34.2
41.2
32.5
26.2
NA
NA
36.
~4.6
34.
+6.2
NO
NO
Sex
Fat
NO
1.0
661.
A-C
A-F
1.0
1.0
1.0
1.0
2.6
---
2.2+0.5
3.1 + 1.8
2.8+1.7
7.1 + 8.6
>20
---
661.
a The 2,3,7,8-TC0D muscle level was defined as 1.0 for each frog. b
Standard deviation.
environmentally-contaminated
bullfrogs.
In a laboratory
feeding
study on guinea pigs,
Gaslewlcz and N@al (5) reported relative ratios of 2,3,7,8-TCDD ranging from 1.0 for bone marrow to 16 for liver.
Olson et al. (6) reported a tissue distribution study for a hamster
injected with 2,3,7,8-TCDD which ranged from a relative ratio for 2,3,7,8-TCDD of 1.0 for skin to 29 for the liver.
In another laboratory study by Gasiewicz et al. (7), mice were injected
with 2,3,7,8-TCDD and for the {C57) mice they found a relative 2,3,7,8-TCD0 ratio range of 1.0 for the heart muscle to 17 for adipose tissue to 38 for liver. Gaslewlcz et al. {7) reported similar results for two other strains of mice. In a recent report, by J. J. Ryan et al. (12), the tissue distribution of "total dioxins" {tetra- to octa-chlorodlbenzo-p-dloxins) humans was studied.
in two
In each case, the lowest level of total dioxins was the kidney and the
highest was in fat tissue.
Their results suggest that most of-the concentration variation was
due to the percent lipid in the tissues analyzed.
Similarly, it is likely that the percent
lipid in the frog tissues shown in Table I would account for much of concentration differences found in these tissues. Thus, the results obtained from the analysis of 2,3,7,8-TCDD - contaminated frog tissues agree reasonably well for the muscle, skin and fat data that is available from laboratory animal studies of other animals (5-7).
The one fat sample that was quantltated was found to
have the highest relative ratio of 2,3,7,8-TCD0 for the tissues that were studied. did not have significant levels of fat that was available for analysis.
Frogs A-C
126
Frogs A-C were gravid female frogs caught at approximately the same time which did not have significant levels of fat bodies available for analysis. They all had distended ovaries and oviducts which contained large numbers of eggs. This may be the reason that they had very l i t t l e observable fat tissue. Frog D, on the other hand, had relatively small ovaries and oviducts and had a sufficient amount of fat tissue for analysis. Table I I shows the average relative tissue ratios for frogs A-C as a well matched subgroup separately from the averages for all six frogs. The results for all six bullfrogs show a clear pattern for bullfrog tissues in which the muscle contains the least amount of 2,3,7,8-TCDD per gram and the ovaries or fat contains the most 2,3,7,8-TCDD per gram. While the data set is limited, the results also suggest that the ratio of liver to muscle and skin to muscle was similar for male and female frogs. Additionally, the data set suggests that the relative level of 2,3,7,8-TCDD in the ovaries of female frogs was higher than the level in the liver for those frogs. Therefore, these results tend to support the hypothesis that frogs are much less sensitive to 2,3,7,8-TCDD than are guinea pigs (4). In addition, these results suggest that female frogs store a significant amount of their 2,3,7,8-TCDD body burden in their ovaries and oviducts, a finding that has not previously been reported. ACKNOWLEDGEMENT: The authors thank Lyle Davis for typing the manuscript and Daniel L. Davis (Jacksonville, Arkansas) for helping to collect the bullfrogs. REFERENCES
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3 December
1985)