Chtm-Biol.
Znteructions, 11 (1975) 235-243
0 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
MERCURY
INHIBITION
ROLLIN H. THAYER**
235
OF FATTY ACID SYNTHESIS IN CHICKS*
AND W. E.
DONALDSON
DeparttneiH of Poultry Science, North Carolina State University, Raleigh, N.C., 27607 (U.S.A.)
(Received October 29th. 1974) (Revision received February IOth, 1975)
SUMMARY
Male chicks were fed a commercial ration and were given drinking water which contained 0, 50, 100, 150, 200 or 300 pg of mercury/ml as mercuric chloride from hatching to 3 weeks of age. In one experiment, the mercuric chloride was administered by injection into the abdominal cavity rather than in the drinking water. At 3 weeks the chicks were killed, and the livers were removed and weighed. The activity of fatty acid synthetase in the 800 x gav supernatant fractions of the liver homogenates and in Vito incorporation of [Wlacetate into liver and carcass fatty acids and respiratory WOa was determined as indicated. Administration of mercury at a treatment level of 300 pg/ml of drinking water depressed growth, feed and water consumption, liver weight, hepatic fatty acid synthetase activity, and in viro incorporation of [W]acetate into liver and carcass fatty acids, and increased the production of respiratory 14COs as compared with controls. In experiments in which graded doses of mercury were administered, body weights, liver weights, and feed and water intakes of the chicks receiving 0, 50 and 100 ,ug of mercury/ml of drinking water were similar to each other, but these parameters were severely depressed by 200 pg of mercury/ml of drinking water. Mercury caused a dose-related decrease of fatty acid synthetase activity. Incorporation of [Wlacetate into carcass fatty acid was depressed by 50 and 200 ,ug of mercury/ml of drinking water; incorporation into liver fatty acids and production of respiratory 1WOs was not affected by mercury. Intra-abdominal injection of 6 mg of mercury/l00 g body weight (as mercuric chloride) into well alimented chicks depressed hepatic fatty acid synthetasc activity at 1 h post-injection. The data are consistent with the hypothesis that a portion of the effects of mercury on fatty acid synthesis are direct rather than a secondary effect of inanition.
* Papar No. 4514 of the Journal Series of the North Carolina Agricultural Experiment Station, Raleigh, N.C. (U.S.A.). ** On leave from the Department of Animal Sciences and Industry, Oklahoma State University, Stillwater, Okla. 74974 (U.S.A.).
236 INTRODUCTION
Intact, normally functioning cellular membranes are vital to all organisms since these membranes serve as barriers to passage of materials into and out of cells and subcellular organelles. There is considerable evidence that mercury interferes with normal membrane functionl-*. Biological membranes are lipoproteins and hence it is conceivable that mercury interacts with one or both of the moieties which constitute these membranes. Mercury is known to bind with the free sulfhydryl groups of proteins to form mercaptides 5-7. It is also possible that mercury interferes with the enzymatic synthesis of the fatty acid precursors of membrane lipids. Mercury compounds have been used to inhibit the activities of a number of enzymes in vitro. Among these are the oxidation of NADM by malate dehydrogenase*, 6-phosphogluconate dehydrogenase and glucosed-phosphate dehydrogenases, pyruvate dehydrogenase 10, D-amino acid oxidase” I and succinic dehydrogenase and cytochrome c oxidasel@. There are few reports of in vivaeffects of mercury on enzyme activities. Miller et aZ.13r;eported the inhibition of kidney alkaline phosphatase by injections of mercuric chioride into chicks, Reductions of liver content of the microsomal cytochromes P-450 and bs, as w&l as the activities of aminopyrine demethylase and U~Pglucuronyltransferase in rats by subcutaneous administration of methylmercury hydroxide were reported by Lucier et al.la. The activity of the latter enzyme required higher mercury levels before inhibition was obsec’red. The purpose of this investigation WPSto determine the effects of exposure of chicks to mercuric chloride administered in the drinking water or by injection on the hepatic activity of fatty acid synthetase, and on the metabolism of ff%]acetate in intact chicks. MATERIALS
AND METHODS
Male chicks were maintained in electrically heated batteries from day-old to 3 weeks of age. A commercially prepared chick starter diet was available a& ~~~~~u~ throughout each ex~riment. In Ex~rimen~ 1 through 4, mercury was administered ia deionized drinking water as mercuric chloride. A stock solution of mercuric chloride (1.84 - lo-sM) was prepared weekly and stored in a polyethylene container. This solution was diluted daily to provide mercury levels of 50, 100, 150, 200 or 300 pg of mercury/ml of drinking water as specified for each experiment. in each experiment, a control group was maintained in which the drinking water contained no mercury. In all treatments, the drinking water was available ad ~j~~f~~and was provided in Pyrex fountains. Subsequent to the ~ornpl~tiol~ of these experiments, we became aware of the report of Greenwood and Clarkson’s concerning losses of mercury from submolar solutions. Based upon that report, our best estimate of possible mercury loss in the present experiments is from < 5 % to 15 %, with the lower value most plausible. Individual body weights were recorded at weekly intervals beginning when the
237 chicks were day-old. Feed and water consumption were measured at weekly intervals. Mortality was recorded daily. In all experiments, additional parameters were measured with 6 chicks selected at random from each treatment. In Experiments i and 2, the incorporation of [rJC]acetate into respiratory COz and into long-chain fatty acids in the liver and carcass was measured. In these experiments, 2 ,&i of sodium [I-i%]acetate were injected into the abdominal cavity. The chicks were placed immediately in a glass respiratory chamber and expired COZ was collected in NaOH during a 3O-min post-injection period. One-half ml samples of the NaOH solution were taken and radioassays were made using a liquid scintillation spectrometer. Total 14COz activity of samples collected from each chick was assayed by the method of HarlanIs. The chicks were then killed by cervical dislocation, the livers were removed immediately, weighed, and placed in cold 0.25 M sucrose. Each liver was minced, rinsed, and homogenized in a Potter-Elvehjem tissue grinder with a Teflon pestle. The homogenates were centrifuged for 10 min at 800 x gav. The pellet was discarded and the supernatant fractions were used to assay for fatty acid synthetase by a previously described method17. All steps were carried out at O-5”. All assays were with freshly prepared fractions except where noted. Prior to centrifugation, 10 % ofthe liver homogenate was set aside for extraction and radioassay of fatty acids. After liver removal, the skin, head, shanks and feet were removed from each carcass. The remaining carcass was then homogenized for 5 min in a Waring Blender with an equal weight of distilled water. Samples equal to 4% of this homogenate were taken for extraction and radioassay of fatty acids. The extraction and assay procedures have been described previouslyl*. TABLE I EFFECTS OF MERCURY EN THE DRINKMG
3
WATER ON MORTALITY,
Es-perhncnt No.
Number of chicks
Percent nrortaiity
-
1 2
Treat
fmnt
f&in drinking water ylglml)
Body weighta (g)
Liver weight” (g)
351 & 6
11.0 f 0.3
--
16
0
16
37.5
12 12
0 0 0
12
17
12 12 12 12
0 0 0 0
12
3
BODY WEIGHT AND LIVER WEIGHT AT
WEEKS OF AGE
* Mean i SE. b Significantly different from control, P < 0.01.
None 300
90 f
14b
3.5 f 0.6b
None
331 f
9
11.9 f 0.8
50 100
327 f 323 f
12
10.6 rt 0.4
2aO
12 202 + Ilb
10.7 j, 0.s 6.4 & 0.4b
None 50 100 200
343 rt 336 f 308 f 164 f
11 14 12 14b
12.6 f 12.0 & 10.7 & 6.3 f
1.1 0.6 0.6 O.gb
TABLE iI EFFECTS OF MERCURY
IN THE DRINKING WATER ON FEED INTAKE, WATER INTAKE AND EST&IATED MERCURY
INTAKE DURING 3-WEEK EXPERIMENTS*
Experiment Number of chicks1 No. treatment
1
16
Feed intake (g]chickldayl
None
1 2 3 1 2 3
13 29 41 6 6 8
38 64 96 8 13 18
2.4 3.9 5.4
! 2 3 1 2 3 1 2 3 1 2 3
12 26 42 11 25 42 10 25 41 8 14 24
24 57 99 22 53 88 18 56 76 16 39 42
-
1 2 3 1 2 3 1 2 3
11 30 43 11 29 41 11 29 38 8 15 19
26 64 118 26 58 105 27 55 92 23 40 45
300
2
12
None
50
100
200
3
12
Water intake Estimated” (mllchickjaiay) Hg intake (t4birdlday.J
Wee& Treatment (Hg in drinking water, &ml)
None
50
100
200 : 3
1.1 2.7 4.4 1.8 5.6 7.6 3.2 7.8 8.4 1.3 2.9 5.3 2.7 5.5 9.2 4.6 8.0 9.0
U Each value in the table is the mean for that treatment group. b The estimation assumes no losses of Hg during storage (see text and ref. 15).
In Experiments 1, 3 and 5, fatty acid synthetase activity was determined in the 800 x gav supernatant fraction of livers. In Experiment 5, 12 untreated 3-weekold chicks were starved for 1 h, and then permitted access to feed for 2 h. Each chick was palpated to be sure that its digestive tract (crop) was full. Six chicks were injected with mercury in the form of mercuric chloride at a level of 6 mg of mercury/l00 g of body weight. The injection was made into the abdominal cavity and the chicks were killed 1 h post-injection. Six noninjected chicks served as controls. Fatty acid synthetase activity is expressed in Units where 1 Unit = 1 pmole malonyl-CoA incorporated into long-chain fatty acids/min at 25”. Protein content
239 TABLE III EFFECTS OF 0 AND INTAKE,
MERCURY
PERIMENT
300 ,ug MERCURY/d
OF DRINKING WATER ON BODY WEIGHT, FEED INTAKE,
INTAKE AND LIVER WElGHT WITH TREATMENTS REVERSED AT
2
WATER
WEEKS OF AGE (EX-
4)”
wg in ~~~k~~ water f pgjml) O-2 wk
2-3 wk
None None 300 300
None 300 300 None
2wk 2-3 wk &dy weight goin (gl (g)
3rd wk feed intake (gfchick)
3& wk ~r~in~red water intake 3rd wk (ml/chick) mercury intake img/chick)
236 225 75 74
310 104 81 138
612 133 140 356
-I- 147b 9 + 40 + 96
0 40 42 0
Li%P
weight Igt
12.@ 7.5 4.2 6.2
B Each value in the table is the mean for that treatment group. b Analysis of variance (2 x 2 factorial) indicated: significant mercury effect P < 0.01. significant reversal effect P c 0.01 non-significant interaction between the 0-2 week treatment and no treatment groups. c Analysis of variance (2 x 2 factorial) indicated: significant mercury effect P < 0.01 significant reversa1effect P < 0.05 significant interaction P < 0.01 between the 0-2 week treatment and no treatment groups.
of the liver fractions was determined by the biuret method of Cleland and SlaterlQ. Statistical significance was determined by analysis of variance or t test. The ex~rimental desigqwas considered to be completely randomized, and within treatment variance was used as the error term. RESULTSAND DISCUSSION
Efict ~~~?ercu~y on g~~~~~ ~erf~r~ff~ce. Data on body weight, liver weight, feed consumption, water intake, mercury intake and mortality for Experiments 1, 2 and 3 are summarized in Tables I and II. A mercury level of 300 pg/ml of drinking water depressed body and liver weights as compared with controls (P c 0.01) in Experiment 1. Feed and water intakes were also depressed by mercury. In experiment 2 and 3, growth was depressed by 200 pg mercury~ml, but body weight was not markedly affected by 50 and 100 pg mercury/ml. Liver weight and feed and wuter intakes paralleled changes in body weight. Mortality was observed with 300 pg mercury/ml in Experiment 1 and 200 ,ug of mercury/ml in Experiment 2. There was no mortality in Experiment 3. An evaluation of the mortality and body weight data in Experiment 1 showed an interesting pattern. Approximately one-third of the chicks which received 300 pg of mercury/ml of drinking water seemed to be unable to make the necessary physiological adjustment and died during the first week. Another one-third grew little, if any, during the 3week feeding period. The remaining one-third grew at a rate about 33% as fast as
240 TABLE IV EFFECT OF MERCURY in vivo [%]ACETATE
IN THE DRINKING WATER ON HEPATlC FATTY ACID SYNTHETASE ACTIVITY AND INCORPORATION INTO RESPIRATORY COa, LIVER FATTY ACIDS, AND CARCASS
3 WJZKSOFAGE"
FAlTYACIDSAT
~~~~~~ Experiment No.
Number Treatment (Hg in chicks drinking water, PglmU
Fatty acid synthetase activity (mUnits/mg protein)
[14ClAcetate incorporafion Respirutory COa ( % of dose)
Long-chain fatty acids ( ohof available dose)e Liver
Carcass
1
6 6
None 300
0.54 f 0.07” 0.20 f 0.051
15.32 f 1.09 26.56 + 1.67’
3.79 f 0.43 1.81 f 0.31’
9.62 f 1.17 2.27 f 0.68’
2
6 6 6 6
None 50 100 200
-
17.34 rt 2.35 15.03 f 1.30 16.60 rt: 1.58 16.74 + 2.50
3.13 3.86 3.65 3.40
3
6 6 6 6
None 50 100 200
4.22 3.90 2.86 2.37
-
-
-
-
-
a b c d e * g
of
zk 0.51~~’ + 0.31 i 0.24s f 0.11’
f f C f
0.32 0.53 0.74 0.51
6.44 3.27 4.61 2.81
f f It f
0.76 0.57’ 1.26 0.57’
Values in the tables are means f standard errors. Liver fraction frozen prior to assay. Enzyme activity determined with freshly prepared liver fraction. Significant linear effect, P < 0.01 Available dose = total dpm in dose - dpm in respiratory COs. Significantly different from control P < 0.01. Significantly different from control P < 0.05.
the chicks not receiving mercury. The basis for this differential response is unknown. In Experiment 4 chicks were administered either 0 or 300 yg of mercury/ml of drinking water for 2 weeks. During the third week, one-half of the chicks in each group were continued on the same treatment while the treatment was reversed for the remaining chicks. The results are shown in Table III. Chicks maintained on 0 pg of mercury/ml of drinking water grew normally while those shifted from 300 pg to 0 pg of mercury/ml gained considerably less weight. Conversely, chicks maintained on 300 yg of mercury/ml grew while those shifted from 0 to 300 yg of mercury/ml lost weight. Feed intake and liver weights paralleled body weight. The data clearly indicate that chicks shifted to 300 ,ug were not as tolerant of mercury as those maintained on 300 ,ug throughout. This effect may be related to the differential response mentioned above (Experiment 1). Efic? oSmercury op1Zipid metabolism. The effects of mercury in the drinking water on hepatic fatty acid synthetase activity of randomly selected chicks from Experiments 1 and 3 are shown in Table IV. In both experiments, the addition of mercury to the drinking water depressed enzyme activity. The mercury effect was dose-related. It should be mentioned here that freezing of liver fractions prior to assay (Experiment 1) reduces fatty acid synthetase activity as compared with freshly
241 TABLE V HEPATIC FATTY ACID SYN’KHFXASf3 ACTIVITY WITH INTRA-ABDOMINAL JNJECTJON OF MERCURY AT
3 WEEKS
OF AGE (EXP~ZRIMENT5p
None Injected
338 =t 25 315 & 24
13.11 f 1.24 13.96 4 1.38
7.66 f 0.85 3.42 IJ; 0.530
a V&m in the table are means f stantierrors. b Activity detemrined on unfro?sn liver frrrction. C SingScantly different from non-injected, P < 0.01.
prepared fractions ~Ex~riment 3). However, the relative ~s~nse of frozen and fresh fractions to prior mercury treatment is unaifected~~. The inh~bjtory effect of mercury on fatty acid synthetase could possibly be an artifact induced by translocation of mercury during tissue preparation from some inert protein to the e&zyme molecule. To rule out this possibility, the in viva incorporation of [l-f4C@e~ate into liver and carcass fatty acids was measured in Experiments I and 2. These results are shown in Table IV also. In Exp~iment 1, 300 pug of mercury/ml of drinking water increased the production of respiratory 14C02 from [1-i*C]acotate and decreased incorporation of acetate into liver and carcass fatty acids. In Experiment 2, up to 200 ,ug of mercury had no effect on production of respiratory 14CQ? from acetate as compared with contruls nor was incorporation of [l-l”C]acetate jnta liver fatty acids affected by mercury. In~rporation of [I-14C]acetate into carcass fatty acids was reduced, in comparison with controls, by 50 and 200 lug, but not by 100 pg of mercury/ml of drinking water. The latter datum was the result of uncharacteristically high incorporations by 2 of the 6 chicks. These results suggest that thee dj~eren~es in fatty acid synthetase activities observed are reflections of in v&o mercury ef&ts and not of translocation of mercury to the enzyme during tissue preparation, O’I-Iea and LeveilW showed that 90-95% of lipogenesis in chicks occurs in liver. Hence, [Wjacetate incorporation into both liver and carcass fatty acids is presumably a fun~ion of the rate of hepatic li~ogenesis. In Experiment 2, rner~~ry reduced acetate incorporation into carcass but not liver fatty acids (Table III). Such a result could be a reflection of reduced hepatic lipogenesis coupled with an impairment by rner~u~ of lipid transport from liver to other body sites. There is no immediately apparent explanation for the differences in respiratory l*C02 production betw~n ~x~riment 1 and 2 except for the differences in levels of mercury admin~stran tion. The data presented above show a definite reduction of fatty acid synthetase activity in chicks by the ingestion of mercury. Starvation also is known to depress the hepatic activity of fatty acid synthetase 2~~23,In view of the reduced growth and
242
feed intake observed with high (100 pg or more of mercury/ml of drinking water) levels of mercury ingestion, it is possible that the results obtained with fatty acid synthetase are a reflection of inanition rather than a direct effect of mercury on the enzyme. To test this possibility, normal chicks were starved for 1 h and then realimented for 2 h. The purpose of the l-h starvation was to insure that the chicks would consume feed when it was made available, and thus would be in a fed state during subsequent treatment. Then one-half of the chicks received an intra-abdominal injection of 6 mg of mercury/100 g of body weight while the remaining one-half received no further treatment. All chicks had access to feed during the post-injection period. The chicks were killed 1 h post-injection and hepatic fatty acid synthetase activity was measured. The results are shown in Table V. Mercury injection caused a marked reduction of fatty acid synthetase activity despite the fact that the mercurytreated chicks were in a fed state (palpable feed in the crop) and had body weights similar to controls. This result is consistent with the hypothesis that mercury directly inhibits fatty acid synthetase, and that the inhibition is not dependent entirely upon prior inanition. We have observed24 that 6-phosphogluconate dehydrogenase activity of chick liver was unaffected by a 3-week period of administration of 150 pg or 300 lug of mercury in the drinking water although fatty acid synthetase activity in the same livers was depressed. This observation is also consistent with the hypothesis of direct inhibition of fatty acid synthetase by mercury rather than inanition effects since dphosphogluconate dehydrogenase activity is correlated to fatty acid synthesis in liver in a variety of physiological states25326. ACKNOWLEDGEMENT
The authors wish to recognize the skillful technical assistance of Mrs. F. D. Suggs and Mr. C. Strickland. The assistance of Mr. Hong-i Yang in making the statistical analyses is gratefully acknowledged. This research was supported in part by the U.S. Public Health Service, grant No. HD-02887, National institutes of Health.
REFERENCES 1 R. A. Moore, S. Goldstein and A. Canowitz, The mitochondria in acute experimental nephrosis due to mercuric chloride, Arch. Pathul., 8 (1929) 930. 2 J. Oliver, Experimental nephritis in the frog, IV. The significance of the functional response to vascular and to parenchymal disturbances in the kidney, .I. Exptl. Med., 55 (1932)295. 3 J. P. Arbuthnott, Haemolytic action of mercurials, fiatwe, 196 (1962) 277. 4 G. Roush Jr. and R. A. Kehoe, Toxicology: inorganic, Ann. Rev. Phannacol., 4 (1964) 247. 5 E. S. G. Barron and G. Kalnitsky, The inhibition of succinoxidase by heavy metals and its reactivation with dithiols, B&hem J., 41 (1947) 346. 6 W. L. Hughes and H. M. Dintzis, Crystallization of the mercury dimers of human and bovine mercaptalbumin, J. Biol. Chem., 239 (1964) 845. 7 Y. Takeda, T. Kunugi, T. Terao and T. Ukita, Mercury compounds in the blood of rats treated with ethylmercuric chloride, Toxicol. Appl. Phurmacol., 13 (1968) 165. 8 H. K. Kuramitsu, Mercury(II) stimulation of malate dehydrogenase activity, J. Biol. Chern., 243 (1968) 1016.
243 9 W. Chefurka,
Oxidative metabolism of carbohydrates in insects, 11. Glucosed-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in the housefly, Enzymology, 18 (1957) 209. 10 H. S. Moyed and D. J. O’Kane, Fractionation of the pyruvate oxidase of Proteus vulgaris, J. Biol. Chem., 195 (1952) 375. 11 W. R. Frissell and L. Hellerman, The sulfhydryl character of n-amino acid oxidase, J. Biol. Chem., 225 (1957) 53. 12 M. A. Smalt, C. W. Kreke and E. S. Cook, Inhibition of enzymes by phenylmercury compounds, J. Riol. Chem., 224 (1957) 999. 13 L. V. Miller, J. A. McIntyre and G. E. Bearse, Kidney alkaline phosphatase in mercuric chloride injected chicks resistant and susceptible to leukosis, Poultry Sci., 48 (1969) 1487. 14 G. Lucier, 0. McDaniel, P. Brubaker and R. Klein, Effects of methylmercury hydroxide on rat liver microsomal enzymes, Chem.-Bid. Interact., 4 (1971/72) 265. 15 M. R. Greenwood and T. W. Clarkson, Storage of mercury at submolar concentrations, Am. Indust. Hyg. Assoc. J., 31 (1970) 250. 16 J. W. Harlan, Liquid scintillation counting of aqueous carbonate solutions, in Atomhght, No. 18, New England Nuclear, Boston, Mass., 1961, pp. 8-l 1. 17 J. V. Mason and W. E. Donaldson, Fatty acid synthesizing systems in chick liver: influences of biotin deficiency and dietary fat, J. Nutrit., 102 (1972) 667. 18 W. E. Donaldson, Adaptation of the chick to dietary energy source, J. Nutrit., 82 ( 1964) 115. 19 K. W. Cleland and E. C. Slater, Respiratory granules of heart muscle, Biochem. J., 53 (1953) 547. 20 W. E. Donaldson, Unpublished observation. 21 E. K. O’Hea and G. A. Leveille, Lipid biosynthesis and transport in the domestic chick (Gallus domesticus), Camp. Biochem. Physiol., 30 (1969) 149. 22 D. M. Gibson and D. D. Hubbard, Incorporation of malonyl CoA into fatty acids by liver in starvation and alloxan-diabetes, Biochem. Biophys. Res. Commute., 3 (1960) 531. 23 P. H. W. Butterworth, R. B. Guchhait, H. Baum, E. B. Olson, S. A. Margolis znd J. W. Porter, Relationship between nutritional status and fatty acid synthesis by microsomal and soluble enzymes of pigeon liver, Arch. B&hem. Biophys., I16 (1966) 453. 24 W. E. Donaldson and J. P. Thaxton, Unpublished observation. 25 J. Tepperman and H. M. Tepperman, Effxts of antecedent food intake pattern on hepatic lipogenesis, Am. J. Physiol., 193 (1958) 55. 26 G. L. Allee, D. R. Romsos, G. A. Leveille and D. H. Baker, Metabolic adaptation induced by meal-eating in the pig, J. Nutrit., 102 (1972) 1115.
Znteructions, 11 (1975) 235-243
0 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
MERCURY
INHIBITION
ROLLIN H. THAYER**
235
OF FATTY ACID SYNTHESIS IN CHICKS*
AND W. E.
DONALDSON
DeparttneiH of Poultry Science, North Carolina State University, Raleigh, N.C., 27607 (U.S.A.)
(Received October 29th. 1974) (Revision received February IOth, 1975)
SUMMARY
Male chicks were fed a commercial ration and were given drinking water which contained 0, 50, 100, 150, 200 or 300 pg of mercury/ml as mercuric chloride from hatching to 3 weeks of age. In one experiment, the mercuric chloride was administered by injection into the abdominal cavity rather than in the drinking water. At 3 weeks the chicks were killed, and the livers were removed and weighed. The activity of fatty acid synthetase in the 800 x gav supernatant fractions of the liver homogenates and in Vito incorporation of [Wlacetate into liver and carcass fatty acids and respiratory WOa was determined as indicated. Administration of mercury at a treatment level of 300 pg/ml of drinking water depressed growth, feed and water consumption, liver weight, hepatic fatty acid synthetase activity, and in viro incorporation of [W]acetate into liver and carcass fatty acids, and increased the production of respiratory 14COs as compared with controls. In experiments in which graded doses of mercury were administered, body weights, liver weights, and feed and water intakes of the chicks receiving 0, 50 and 100 ,ug of mercury/ml of drinking water were similar to each other, but these parameters were severely depressed by 200 pg of mercury/ml of drinking water. Mercury caused a dose-related decrease of fatty acid synthetase activity. Incorporation of [Wlacetate into carcass fatty acid was depressed by 50 and 200 ,ug of mercury/ml of drinking water; incorporation into liver fatty acids and production of respiratory 1WOs was not affected by mercury. Intra-abdominal injection of 6 mg of mercury/l00 g body weight (as mercuric chloride) into well alimented chicks depressed hepatic fatty acid synthetasc activity at 1 h post-injection. The data are consistent with the hypothesis that a portion of the effects of mercury on fatty acid synthesis are direct rather than a secondary effect of inanition.
* Papar No. 4514 of the Journal Series of the North Carolina Agricultural Experiment Station, Raleigh, N.C. (U.S.A.). ** On leave from the Department of Animal Sciences and Industry, Oklahoma State University, Stillwater, Okla. 74974 (U.S.A.).
236 INTRODUCTION
Intact, normally functioning cellular membranes are vital to all organisms since these membranes serve as barriers to passage of materials into and out of cells and subcellular organelles. There is considerable evidence that mercury interferes with normal membrane functionl-*. Biological membranes are lipoproteins and hence it is conceivable that mercury interacts with one or both of the moieties which constitute these membranes. Mercury is known to bind with the free sulfhydryl groups of proteins to form mercaptides 5-7. It is also possible that mercury interferes with the enzymatic synthesis of the fatty acid precursors of membrane lipids. Mercury compounds have been used to inhibit the activities of a number of enzymes in vitro. Among these are the oxidation of NADM by malate dehydrogenase*, 6-phosphogluconate dehydrogenase and glucosed-phosphate dehydrogenases, pyruvate dehydrogenase 10, D-amino acid oxidase” I and succinic dehydrogenase and cytochrome c oxidasel@. There are few reports of in vivaeffects of mercury on enzyme activities. Miller et aZ.13r;eported the inhibition of kidney alkaline phosphatase by injections of mercuric chioride into chicks, Reductions of liver content of the microsomal cytochromes P-450 and bs, as w&l as the activities of aminopyrine demethylase and U~Pglucuronyltransferase in rats by subcutaneous administration of methylmercury hydroxide were reported by Lucier et al.la. The activity of the latter enzyme required higher mercury levels before inhibition was obsec’red. The purpose of this investigation WPSto determine the effects of exposure of chicks to mercuric chloride administered in the drinking water or by injection on the hepatic activity of fatty acid synthetase, and on the metabolism of ff%]acetate in intact chicks. MATERIALS
AND METHODS
Male chicks were maintained in electrically heated batteries from day-old to 3 weeks of age. A commercially prepared chick starter diet was available a& ~~~~~u~ throughout each ex~riment. In Ex~rimen~ 1 through 4, mercury was administered ia deionized drinking water as mercuric chloride. A stock solution of mercuric chloride (1.84 - lo-sM) was prepared weekly and stored in a polyethylene container. This solution was diluted daily to provide mercury levels of 50, 100, 150, 200 or 300 pg of mercury/ml of drinking water as specified for each experiment. in each experiment, a control group was maintained in which the drinking water contained no mercury. In all treatments, the drinking water was available ad ~j~~f~~and was provided in Pyrex fountains. Subsequent to the ~ornpl~tiol~ of these experiments, we became aware of the report of Greenwood and Clarkson’s concerning losses of mercury from submolar solutions. Based upon that report, our best estimate of possible mercury loss in the present experiments is from < 5 % to 15 %, with the lower value most plausible. Individual body weights were recorded at weekly intervals beginning when the
237 chicks were day-old. Feed and water consumption were measured at weekly intervals. Mortality was recorded daily. In all experiments, additional parameters were measured with 6 chicks selected at random from each treatment. In Experiments i and 2, the incorporation of [rJC]acetate into respiratory COz and into long-chain fatty acids in the liver and carcass was measured. In these experiments, 2 ,&i of sodium [I-i%]acetate were injected into the abdominal cavity. The chicks were placed immediately in a glass respiratory chamber and expired COZ was collected in NaOH during a 3O-min post-injection period. One-half ml samples of the NaOH solution were taken and radioassays were made using a liquid scintillation spectrometer. Total 14COz activity of samples collected from each chick was assayed by the method of HarlanIs. The chicks were then killed by cervical dislocation, the livers were removed immediately, weighed, and placed in cold 0.25 M sucrose. Each liver was minced, rinsed, and homogenized in a Potter-Elvehjem tissue grinder with a Teflon pestle. The homogenates were centrifuged for 10 min at 800 x gav. The pellet was discarded and the supernatant fractions were used to assay for fatty acid synthetase by a previously described method17. All steps were carried out at O-5”. All assays were with freshly prepared fractions except where noted. Prior to centrifugation, 10 % ofthe liver homogenate was set aside for extraction and radioassay of fatty acids. After liver removal, the skin, head, shanks and feet were removed from each carcass. The remaining carcass was then homogenized for 5 min in a Waring Blender with an equal weight of distilled water. Samples equal to 4% of this homogenate were taken for extraction and radioassay of fatty acids. The extraction and assay procedures have been described previouslyl*. TABLE I EFFECTS OF MERCURY EN THE DRINKMG
3
WATER ON MORTALITY,
Es-perhncnt No.
Number of chicks
Percent nrortaiity
-
1 2
Treat
fmnt
f&in drinking water ylglml)
Body weighta (g)
Liver weight” (g)
351 & 6
11.0 f 0.3
--
16
0
16
37.5
12 12
0 0 0
12
17
12 12 12 12
0 0 0 0
12
3
BODY WEIGHT AND LIVER WEIGHT AT
WEEKS OF AGE
* Mean i SE. b Significantly different from control, P < 0.01.
None 300
90 f
14b
3.5 f 0.6b
None
331 f
9
11.9 f 0.8
50 100
327 f 323 f
12
10.6 rt 0.4
2aO
12 202 + Ilb
10.7 j, 0.s 6.4 & 0.4b
None 50 100 200
343 rt 336 f 308 f 164 f
11 14 12 14b
12.6 f 12.0 & 10.7 & 6.3 f
1.1 0.6 0.6 O.gb
TABLE iI EFFECTS OF MERCURY
IN THE DRINKING WATER ON FEED INTAKE, WATER INTAKE AND EST&IATED MERCURY
INTAKE DURING 3-WEEK EXPERIMENTS*
Experiment Number of chicks1 No. treatment
1
16
Feed intake (g]chickldayl
None
1 2 3 1 2 3
13 29 41 6 6 8
38 64 96 8 13 18
2.4 3.9 5.4
! 2 3 1 2 3 1 2 3 1 2 3
12 26 42 11 25 42 10 25 41 8 14 24
24 57 99 22 53 88 18 56 76 16 39 42
-
1 2 3 1 2 3 1 2 3
11 30 43 11 29 41 11 29 38 8 15 19
26 64 118 26 58 105 27 55 92 23 40 45
300
2
12
None
50
100
200
3
12
Water intake Estimated” (mllchickjaiay) Hg intake (t4birdlday.J
Wee& Treatment (Hg in drinking water, &ml)
None
50
100
200 : 3
1.1 2.7 4.4 1.8 5.6 7.6 3.2 7.8 8.4 1.3 2.9 5.3 2.7 5.5 9.2 4.6 8.0 9.0
U Each value in the table is the mean for that treatment group. b The estimation assumes no losses of Hg during storage (see text and ref. 15).
In Experiments 1, 3 and 5, fatty acid synthetase activity was determined in the 800 x gav supernatant fraction of livers. In Experiment 5, 12 untreated 3-weekold chicks were starved for 1 h, and then permitted access to feed for 2 h. Each chick was palpated to be sure that its digestive tract (crop) was full. Six chicks were injected with mercury in the form of mercuric chloride at a level of 6 mg of mercury/l00 g of body weight. The injection was made into the abdominal cavity and the chicks were killed 1 h post-injection. Six noninjected chicks served as controls. Fatty acid synthetase activity is expressed in Units where 1 Unit = 1 pmole malonyl-CoA incorporated into long-chain fatty acids/min at 25”. Protein content
239 TABLE III EFFECTS OF 0 AND INTAKE,
MERCURY
PERIMENT
300 ,ug MERCURY/d
OF DRINKING WATER ON BODY WEIGHT, FEED INTAKE,
INTAKE AND LIVER WElGHT WITH TREATMENTS REVERSED AT
2
WATER
WEEKS OF AGE (EX-
4)”
wg in ~~~k~~ water f pgjml) O-2 wk
2-3 wk
None None 300 300
None 300 300 None
2wk 2-3 wk &dy weight goin (gl (g)
3rd wk feed intake (gfchick)
3& wk ~r~in~red water intake 3rd wk (ml/chick) mercury intake img/chick)
236 225 75 74
310 104 81 138
612 133 140 356
-I- 147b 9 + 40 + 96
0 40 42 0
Li%P
weight Igt
12.@ 7.5 4.2 6.2
B Each value in the table is the mean for that treatment group. b Analysis of variance (2 x 2 factorial) indicated: significant mercury effect P < 0.01. significant reversal effect P c 0.01 non-significant interaction between the 0-2 week treatment and no treatment groups. c Analysis of variance (2 x 2 factorial) indicated: significant mercury effect P < 0.01 significant reversa1effect P < 0.05 significant interaction P < 0.01 between the 0-2 week treatment and no treatment groups.
of the liver fractions was determined by the biuret method of Cleland and SlaterlQ. Statistical significance was determined by analysis of variance or t test. The ex~rimental desigqwas considered to be completely randomized, and within treatment variance was used as the error term. RESULTSAND DISCUSSION
Efict ~~~?ercu~y on g~~~~~ ~erf~r~ff~ce. Data on body weight, liver weight, feed consumption, water intake, mercury intake and mortality for Experiments 1, 2 and 3 are summarized in Tables I and II. A mercury level of 300 pg/ml of drinking water depressed body and liver weights as compared with controls (P c 0.01) in Experiment 1. Feed and water intakes were also depressed by mercury. In experiment 2 and 3, growth was depressed by 200 pg mercury~ml, but body weight was not markedly affected by 50 and 100 pg mercury/ml. Liver weight and feed and wuter intakes paralleled changes in body weight. Mortality was observed with 300 pg mercury/ml in Experiment 1 and 200 ,ug of mercury/ml in Experiment 2. There was no mortality in Experiment 3. An evaluation of the mortality and body weight data in Experiment 1 showed an interesting pattern. Approximately one-third of the chicks which received 300 pg of mercury/ml of drinking water seemed to be unable to make the necessary physiological adjustment and died during the first week. Another one-third grew little, if any, during the 3week feeding period. The remaining one-third grew at a rate about 33% as fast as
240 TABLE IV EFFECT OF MERCURY in vivo [%]ACETATE
IN THE DRINKING WATER ON HEPATlC FATTY ACID SYNTHETASE ACTIVITY AND INCORPORATION INTO RESPIRATORY COa, LIVER FATTY ACIDS, AND CARCASS
3 WJZKSOFAGE"
FAlTYACIDSAT
~~~~~~ Experiment No.
Number Treatment (Hg in chicks drinking water, PglmU
Fatty acid synthetase activity (mUnits/mg protein)
[14ClAcetate incorporafion Respirutory COa ( % of dose)
Long-chain fatty acids ( ohof available dose)e Liver
Carcass
1
6 6
None 300
0.54 f 0.07” 0.20 f 0.051
15.32 f 1.09 26.56 + 1.67’
3.79 f 0.43 1.81 f 0.31’
9.62 f 1.17 2.27 f 0.68’
2
6 6 6 6
None 50 100 200
-
17.34 rt 2.35 15.03 f 1.30 16.60 rt: 1.58 16.74 + 2.50
3.13 3.86 3.65 3.40
3
6 6 6 6
None 50 100 200
4.22 3.90 2.86 2.37
-
-
-
-
-
a b c d e * g
of
zk 0.51~~’ + 0.31 i 0.24s f 0.11’
f f C f
0.32 0.53 0.74 0.51
6.44 3.27 4.61 2.81
f f It f
0.76 0.57’ 1.26 0.57’
Values in the tables are means f standard errors. Liver fraction frozen prior to assay. Enzyme activity determined with freshly prepared liver fraction. Significant linear effect, P < 0.01 Available dose = total dpm in dose - dpm in respiratory COs. Significantly different from control P < 0.01. Significantly different from control P < 0.05.
the chicks not receiving mercury. The basis for this differential response is unknown. In Experiment 4 chicks were administered either 0 or 300 yg of mercury/ml of drinking water for 2 weeks. During the third week, one-half of the chicks in each group were continued on the same treatment while the treatment was reversed for the remaining chicks. The results are shown in Table III. Chicks maintained on 0 pg of mercury/ml of drinking water grew normally while those shifted from 300 pg to 0 pg of mercury/ml gained considerably less weight. Conversely, chicks maintained on 300 yg of mercury/ml grew while those shifted from 0 to 300 yg of mercury/ml lost weight. Feed intake and liver weights paralleled body weight. The data clearly indicate that chicks shifted to 300 ,ug were not as tolerant of mercury as those maintained on 300 ,ug throughout. This effect may be related to the differential response mentioned above (Experiment 1). Efic? oSmercury op1Zipid metabolism. The effects of mercury in the drinking water on hepatic fatty acid synthetase activity of randomly selected chicks from Experiments 1 and 3 are shown in Table IV. In both experiments, the addition of mercury to the drinking water depressed enzyme activity. The mercury effect was dose-related. It should be mentioned here that freezing of liver fractions prior to assay (Experiment 1) reduces fatty acid synthetase activity as compared with freshly
241 TABLE V HEPATIC FATTY ACID SYN’KHFXASf3 ACTIVITY WITH INTRA-ABDOMINAL JNJECTJON OF MERCURY AT
3 WEEKS
OF AGE (EXP~ZRIMENT5p
None Injected
338 =t 25 315 & 24
13.11 f 1.24 13.96 4 1.38
7.66 f 0.85 3.42 IJ; 0.530
a V&m in the table are means f stantierrors. b Activity detemrined on unfro?sn liver frrrction. C SingScantly different from non-injected, P < 0.01.
prepared fractions ~Ex~riment 3). However, the relative ~s~nse of frozen and fresh fractions to prior mercury treatment is unaifected~~. The inh~bjtory effect of mercury on fatty acid synthetase could possibly be an artifact induced by translocation of mercury during tissue preparation from some inert protein to the e&zyme molecule. To rule out this possibility, the in viva incorporation of [l-f4C@e~ate into liver and carcass fatty acids was measured in Experiments I and 2. These results are shown in Table IV also. In Exp~iment 1, 300 pug of mercury/ml of drinking water increased the production of respiratory 14C02 from [1-i*C]acotate and decreased incorporation of acetate into liver and carcass fatty acids. In Experiment 2, up to 200 ,ug of mercury had no effect on production of respiratory 14CQ? from acetate as compared with contruls nor was incorporation of [l-l”C]acetate jnta liver fatty acids affected by mercury. In~rporation of [I-14C]acetate into carcass fatty acids was reduced, in comparison with controls, by 50 and 200 lug, but not by 100 pg of mercury/ml of drinking water. The latter datum was the result of uncharacteristically high incorporations by 2 of the 6 chicks. These results suggest that thee dj~eren~es in fatty acid synthetase activities observed are reflections of in v&o mercury ef&ts and not of translocation of mercury to the enzyme during tissue preparation, O’I-Iea and LeveilW showed that 90-95% of lipogenesis in chicks occurs in liver. Hence, [Wjacetate incorporation into both liver and carcass fatty acids is presumably a fun~ion of the rate of hepatic li~ogenesis. In Experiment 2, rner~~ry reduced acetate incorporation into carcass but not liver fatty acids (Table III). Such a result could be a reflection of reduced hepatic lipogenesis coupled with an impairment by rner~u~ of lipid transport from liver to other body sites. There is no immediately apparent explanation for the differences in respiratory l*C02 production betw~n ~x~riment 1 and 2 except for the differences in levels of mercury admin~stran tion. The data presented above show a definite reduction of fatty acid synthetase activity in chicks by the ingestion of mercury. Starvation also is known to depress the hepatic activity of fatty acid synthetase 2~~23,In view of the reduced growth and
242
feed intake observed with high (100 pg or more of mercury/ml of drinking water) levels of mercury ingestion, it is possible that the results obtained with fatty acid synthetase are a reflection of inanition rather than a direct effect of mercury on the enzyme. To test this possibility, normal chicks were starved for 1 h and then realimented for 2 h. The purpose of the l-h starvation was to insure that the chicks would consume feed when it was made available, and thus would be in a fed state during subsequent treatment. Then one-half of the chicks received an intra-abdominal injection of 6 mg of mercury/100 g of body weight while the remaining one-half received no further treatment. All chicks had access to feed during the post-injection period. The chicks were killed 1 h post-injection and hepatic fatty acid synthetase activity was measured. The results are shown in Table V. Mercury injection caused a marked reduction of fatty acid synthetase activity despite the fact that the mercurytreated chicks were in a fed state (palpable feed in the crop) and had body weights similar to controls. This result is consistent with the hypothesis that mercury directly inhibits fatty acid synthetase, and that the inhibition is not dependent entirely upon prior inanition. We have observed24 that 6-phosphogluconate dehydrogenase activity of chick liver was unaffected by a 3-week period of administration of 150 pg or 300 lug of mercury in the drinking water although fatty acid synthetase activity in the same livers was depressed. This observation is also consistent with the hypothesis of direct inhibition of fatty acid synthetase by mercury rather than inanition effects since dphosphogluconate dehydrogenase activity is correlated to fatty acid synthesis in liver in a variety of physiological states25326. ACKNOWLEDGEMENT
The authors wish to recognize the skillful technical assistance of Mrs. F. D. Suggs and Mr. C. Strickland. The assistance of Mr. Hong-i Yang in making the statistical analyses is gratefully acknowledged. This research was supported in part by the U.S. Public Health Service, grant No. HD-02887, National institutes of Health.
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