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Metabolic fate of gossypol: The metabolism of gossypol-14C in laying hens
TOXICOLOGYANOAPPLIEDPHARMACOLOGY17,16@-173(1970)
Metabolic
Fate of Gossypol: The GossypoV4C in Laying
Metabolism Hens
of
M. B. ABOU-DONIA AND CARL M. LYMAN’ Department
of Biochemistry College
and Biophysics, Station, Texas
Received
August
Texas A & M University, 77843
7,1969
Metabolic Fate of Gossypol: The Metabolism of GossypoPC in Laying Hens.ABOU-DONIA, M. B., and LYMAN, CARL M. (1970). Toxicol. Appl. Pharmacol.17, 160-173.Orally administeredformyl-*4C-labeledgossypol wasrapidly excreted by laying hensinto the urine and feces.Only a smallportion wasdepositedin the tissues.A major part of the absorbedgossypol wasconcentratedin the eggs.The excretion of gossypolinto the intestine via the bile seemedto be a major pathway by which absorbedgossypol wasexcreted.The decarbonylationof gossypolto carbon dioxide and apogossypolwasnot a major route for gossypolmetabolismin the laying hen. The time necessaryto eliminateone-halfof the radioactive gossypol(l0 mg, 0.35&i) from the bird body (1 kg) under the conditionsusedwasfound to be 30 hr. Gossypol is a yellow coloring matter occurring in cottonseed. Its toxicity to chickens haslong beenrecognized. Couch et al. (1955), reported that gossypol in the diet affected the growth and development of hens. Hill and Tatsuka (1964) showed that high levels of gossypol causeda significant growth retardation in chicks, and a marked reduction in feed and metabolizable energy. They also found that gossypol causeda significant increasein the weight of the pancreasrelative to body size. Couch et al. (1955) reported the lowering of the blood hemoglobin of the chicks due to the feeding of free gossypol and postulated that free gossypol in the diet leads to interference with the synthesis of the proteins of the blood in thesechicks. Narain et al. (1961) found that feeding of free gossypol to chicks caused a lowering of total serum protein. Schaible et al. (1933) showed that gossypol was responsible for the discoloration of egg yolks laid by hens fed a diet containing cottonseed meal. Woronick and Grau (1955) found that gossypol fed to laying hens wasdeposited in the yolks asgossypol-cephalin and gossypol-protein complexes. Lyman et al. (1969)recovered most of the gossypol activity from the fecesof chicks fed r4C-labeled gossypol. They also found that most of the labeled compound retained wasin the liver, muscle,and kidneys, with the highestspecific activity in theliver. The purpose of the present work was to investigate the metabolic fate of gossypol in laying hens.This included study of the elimination of gossypol from the bird body and of its distribution in different tissues. METHODS The radioactive gossypol (2,2’-binaphthalene-8,8’-dicarboxaldehyde-l,1’,6,6’,7,7’hexahydroxy-5,5’-diisopropyl-3,3’-dimethyl) usedwaslabeled in the formyl group only. ’ Deceased March9, 1969. 160
METABOLISM
OF GOSSYPOL
IN LAYING
HENS
161
The specific activity was 19.75 $i/mmole. It was synthesized according to Lyman et al. (1969). Infrared analysis and thin-layer chromatography showed that the material had a radiochemical purity of at least 99 %. Care and treatment of birds. Midget laying hens (also called Mini, Dwarf), selected for uniformity of weight, were placed in individual metabolic cages. Each animal weighed approximately 1 kg. The metabolic cages were designed for studies of expired i4C02 ; they were made of glass and contained coarse- and fine-mesh screens to isolate the excrements. The expired air was continuously drawn out through the rear end of the cage to eliminate the possibility of high specific activity air being trapped in this volume and slowly leaking back into the cages to distort expiration rate records. Water and solid food containers were provided. The birds were allowed to adjust to their environment for 1 week; after this each hen received a single IO-mg dose of gossypol in a gelatin capsule to ensure that the complete sample entered the stomach. The birds were returned to their cages and given free access to food and water. Only hens that laid eggs regularly were used. The hens were sacrificed after varying intervals of time (1,2,4, or 8 days). For each time period 3 birds were used. The excrements and the eggs were collected daily. The eggs were stored in a refrigerator. At the end of the experimental periods, the birds were anesthetized with chloroform and decapitated. The blood was collected, and the individual organs were removed and weighed. Determination of 14C activity. i4C activity was determined by the use of a Beckman Model LS250 liquid scintillation counter after preparation of the samples as indicated below. The scintillation medium was toluene-ethylene glycol monomethyl ether solution (2: 1 v/v) containing 5 g of 2,5-diphenyloxazole (PPO) (obtained from Beckman, Houston, Texas) per liter. A solution of ethanolamine-ethylene glycol monomethyl ether (1:2 v/v) (both obtained as purified reagents from Fisher Scientific Company, Houston, Texas) was used in two traps (100 and 75 ml) to trap i4C02. Samples from tissues, feces and eggs were prepared for liquid scintillation counting by oxygen combustion. The combined feces and urine were lyophilized, weighed, and ground; samples of 50-100 mg were placed in “combustion envelopes” made of cellophane (obtained from Ivers Lee Company, Newark, New Jersey). Two-tenths milliliter of a 10 % sucrose solution and enough water to wet the entire sample were added. The envelopes were then air-dried under the hood. The contents of the gastrointestinal tract parts were separated and the different parts were washed with ethanol-ether (5 : 2 v/v). The wash of each part was added to its corresponding content, dried and analyzed as described for the feces. Fresh tissues (200-300 mg) were placed directly into combustion envelopes. Two-tenths milliliter of a 10 % sucrose solution was added, and the envelopes were dried as above. Eggs were collected daily and stored in the refrigerator until the end of the experiment. The yolk was carefully separated from the white, and fresh samples were weighed and prepared for oxygen combustion as described for the tissues. A bag containing the dried sample was placed in the platinum basket, where it was carried by a glass rod through the glass stopper which was firmly positioned in a 2-liter heavy-walled Erlenmeyer flask that had previously been purged with oxygen for 10 sec.
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The commercially available Thomas-Ogg infrared igniter (A. H. Thomas Company, Philadelphia, Pennsylvania) was used for ignition of samples. Since the infrared ignition depends upon absorption of the light, it was necessary to wrap the cellophane bag in a black filter paper to assure prompt ignition. Combustion of a sample is normally complete within less than 1 min. The next step was the addition of the solvent. The composition of the solvent was ethanolamine-ethylene glycol monomethyl ether (1: 2 v/v). The flask was first cooled for 5 min, then 10 ml of solvent was added through the sidearm. The flask was swirled gently to distribute the solvent over the entire bottom and an inch or two up on the sidewalls. The flask was then placed in an ice water bath with the cooling confined to the bottom inch of the fask and maintained in this manner for 15-20 min. At the end of this period, an additional 5 ml of the solvent was delivered again through the sidearm into the flask to rinse any activity that may have sequestered in the solvent that wetted the sidearm initially. The flask was swirled to mix the solvents, and 5 ml was added to 10 ml of the scintillation solvent in a sample counting vial. In this investigation free gossypol was determined by extracting the tissues and feces with ethanol-ethyl ether (5:2 v/v). Bound gossypol was estimated by the difference between total and free gossypol. It is recognized that a definition of free gossypol based on solubility relationship is quite empirical. In this study, a standard quenching curve was prepared for quench corrections utilizing a certified “C-benzoic acid standard (New England Nuclear, Boston, Massachusetts). RESULTS
14C Activity in Expired Air Figure 1 shows the accumulated total and the daily rate of 14C activity in the expired air from hens (average values for 3 birds) given radioactive gossypol. During the
Days
after
dosing
1. Daily rate and accumulated total ‘T activity in the expired air from hens following a single lO-mg oral dosing of formyl-labeled gossypol. FIG.
0.96 2.26 3.06 3.58 4.09 4.36 4.52 4.57
xkO.31 AZ0.42 f 0.56 f 0.63 f 0.65 4 0.68 zk0.70
z!z0.08
Egg white
a Each value is an average of six determinations
3.13 kO.35 3.21 k 0.43 3.26 zk0.47
1.15* 0.10 1.84 +z0.17 2.48 f 0.21 2.82 f 0.27 3.00 f 0.30
1
2 3 4 5 6 7 8
Expired Air
Days
f 1.05
+I 0.18 zt 0.70 f 0.81 f 0.93 f 0.95 f 0.98 f 1.01
lo-MG ORAL
41.94 f 4.17 54.78 rk 5.01 64.64 f 6.22 70.29L!=6.40 74.18 f 7.01 76.40k7.31 77.85 iz7.33 78.55 f 7.45
Feces
1
from 3 hens % the standard error.
1.97 4.69 6.27 7.40 8.04 8.52 8.87 9.10
Egg yolk
PERCENTAGE RECOVERY OF 14CACTIVITYFROM HENS GIVENA SINGLE
TABLE
1.09+ 0.09
0.92 f 0.10
97.93zk9.86
-
97.06 zt 9.88
7.74 * 0.95
97.53 zt 9.74 97.26 k9.50
-
43.37 5 4.11 23.81 f 2.10
Total
GOSSYPOL”
5.23 *0.77
8.14 h 1.10 9.88 zt 1.21
Tissuecontents
(0.35 &i) OF Y-LABELED
Tissues
DOSE
g Q x % Cl 0 B s 0 r z $ 3 0 X B
L E: $
164
ABOU-DONIAANDLYMAN
period of the experiments there was a steady increase in the total 14C activity in the expired air. The daily rate of r4C activity in the expired air reached a peak on the first day after the administration, then sharply decreased till the fifth day and leveled off thereafter. The total 14C activity in the expired air throughout the experiment (8 days) was 3.26 % of the administered dose (Table 1). Combined Urinary-Fecal Excretion of 14CActivity
Figure 2 shows daily rate and accumulated total combined urinary-fecal excretion of r4C activity from the hens. A sharp increase in the accumulated total urinary-fecal
-Accumulated Daily
----
---c, 0
1
2
3
-c-w-
4 Days
5 after
6
7
8
dosing
FIG. 2. Daily rate and accumulated total urinary-fecal excretion of Y activity from hens following a single IO-mg oral dosing of formyl-labeled gossypol.
excretion through the first day after the administration and a slow increase thereafter is indicated. Table 2 lists the ratio of free to bound gossypol in the urinary-fecal excretion TABLE 2 RATIO OF FREE TO BOUND GOSSWOL IN THE URINARY-FECAL EXCRETION AND EGGS FROM HENS GIVEN A SINGLE lo-MG ORAL DOSE (0.35 &i) OF 14C-L~~~~~ GOSSYPOL’ Days
Urinary-fecal excretion 0.02 0.05 0.04 0.05 0.06 0.11 0.28 0.36
Egg albumen 0.22 0.21 0.35 0.51 0.77 0.83 0.91 1.18
a Each value is an average of six determinations
Egg yolk 0.10 0.18 0.37 0.41 0.53 0.67 0.85 0.91
from 3 hens.
165
METABOLISM OF GOSSYPOL IN LAYING HENS
of hens fed a single oral dose of “C-labeled gossypol. It is interesting to note that this ratio had a very small value in the beginning of the experiment, i.e., 0.02 in the first days, which successively increased with time to 0.36 in the last day. Table 1 shows that 8 days after administration of 10 mg of gossypol, 78.55 ‘A of the i4C activity was recovered in the feces of the hens. 14CActivity in the Egg
The daily rate and accumulated total 14C activity in the egg white are presented in Fig. 3. It shows a rapid increase in accumulated total i4C activity in the egg white from hens given a lo-mg single dose of gossypol. The daily rate of 14C activity reached a peak 5
0
1
2
3
4 Days
5 after
6
7
8
dosing
FIG. 3. Daily rate and accumulated total % activity in the egg white from hens following a single IO-mg oral dosing of formyl-labeled gossypol.
2 days after the administration of the gossypol. Figure 4 shows the daily rate and accumulated total i4C activity in the egg yolk. It also shows a rapid increase in the accumulated total activity. The daily rate of i4C activity reached a peak 2 days after the administration, sharply decreased till the fifth day, then leveled off thereafter. Table 1 shows the total i4C activity in the egg white was 4.57 ‘A of the administered dose as compared to 9.10 % in the egg yolk. Table 2 also shows the ratios of free to bound gossypol in the egg. This ratio, while generally higher in the egg white than in the egg yolk, progressively increased by time in both parts of the egg. Histologic and General Observations
At the end of each experiment the animals were sacrificed and the tissues were excised. When a IO-mg single dose of gossypol was given, there were severe lesions in the lungs
166
ABOU-DONIA
AND
Days
after
LYMAN
dosing
FIG. 4. Daily rate and accumulated total ‘T activity in the egg yolk from hens following a single oral IO-mg dosing of formyl-labeled gossypol.
and livers. The hens lost some weight (13 %) the first 2 days, then gained some weight and had comparable weights to that of the control birds at the end of the experiments. At that level of gossypol there was normal egg production, and the feed consumption was similar to that of the control. ‘TissueDeposit of 14CActivity
The data obtained by radiochemical analysis of the tissues from hens fed formyl-i4Clabeled gossypol are summarized in Tables 3 and 4 and Fig. 5. In both tables the data consisted of radioactivity from all tissues and from the contents of different parts of the alimentary canal. Table 3 shows the distribution of the recovered 14C activity in different tissues and tissue contents from the birds. One day after the administration all tissues contained radioactivity, the spleen and the brain having the lowest activity. The liver had the highest activity followed by the bile. The alimentary canal tissues contained high activity, but less than that in the liver and the bile. The activity in all tissues was 8.14 % of the total dose. Two days after the administration, most of the tissues contained higher i4C activity than after 1 day, with the liver, ovary, and bile having the highest concentrations. The intestinal tract tissues had less activity after 2 days. At the fourth day there was a general decrease in the radioactivity in all tissues, with no radioactivity detected in the brain and the lungs. The liver and bile still had the highest activity. At 8 days after the oral dosing most of the tissues had no detectable activity. Only in the blood, spleen, kidney, liver, ovary, and bile was radioactivity detected. In the alimentary canal, the
METABOLISM
OF GOSSYPOL
IN LAYING
167
HENS
small intestine, ceca and large intestine contained radioactivity. The activity recovered from all tissues after 8 days was 0.92 % of the administered dose. The contents of different parts of the alimentary canal contained a much larger amount of radioactivity than the tissues at all times. After one day the gastrointestinal tract contents had 43.37 % of the total dose. The ventriculus had the highest amount followed by the ceca, small intestine, proventriculus, crop, and large intestine.‘The activity decreased after 2 days, but the total amount of the activity in the contents of the alimentary canal was 23.81% of the applied dose. After 4 days the activity dropped sharply to 7.74 ‘A, and after 8 days it was only 1.09 ‘A of the administered dose. TABLE DISTRIBUTION
OF RADIOACTIVITY IN V~mous SINGLE lo-MG ORAL DOSE (0.35
3 TISSUES OF HENS 1, 2, 4, AND 8 DAYS &i) OF 14C-L~~~~~~ GOSSWOL”
Total Sample
1 Day
dpm
2 Days
AFTER
per Tissue 4 Days
8 Days
Tissue Brain Lungs Heart Blood Spleen Kidney Liver Bile Pancreas Ovary Oviduct Muscles Fatty tissues Crop Proventriculus Ventriculus Small intestine Ckca Large intestine Total dpm % of Total
245 743 576 4749 224 1403 14902 8634 252 3939 1723 7500 450 3409 1076 5424 3970 2619 906
dose
* 7 f 22 f 17 f 147 f 6 zt 42 * 510 f 270 f 8 =k 127 =!r 51 f. 329 f 14 + 107 f 29 f 155 f 119 f 61 zt 20
62644 8.14
424 f 4OOkll 457 f 3015 f 249 f 1298 5 22765 zk 12810 + 254 f 14527 zt 1437 f 10500 * 1632 zk 1101 f 874 f 2135 f 1178 f 540 f 557 *
0 0
12 14 91 7 35 690 370 8 430 41 298 47 29 20 65 35 17 15
76053 9.88
156f5 1047 f 157*5 629 f 17512 f 9063 f 332 f 1822 zk 449+14 4782 rt 696 f 123 *4 487 f 1099 * 940 * 757 f 299 +
0 0 0 332 50 237 4784 696 0 530 0 0 0 0 0 50 135 56 82
30 18 51 29 10 46 143 21 15 31 28 25 30
40350 5.23
zt f zk f f
10 2 7 141 21
f
15
f I f 4 f 2 f 3
7103 0.92
Gastrointestinal tract contents Crop Proventriculus Ventriculus Small intestine Ceca Large intestine Total dpm % of Total
dose
a Each value
6012 29360 191939 47895 53771 4714 333548 43.37
is an average
f f f f f f
185 854 854 1451 1630 141
5431 3365 106390 24703 38157 4874
f 149 z!z 121 h 3250 * 790 f 1065 f 132
182821 23.81 of six determinations
1398f40 1454 + 24423 f 19829 f 10137 f 2524 f
0 104Oi31 3808 f 114 2182 f 66 1044*30 382 f 12
41 785 601 313 94
59766 7.74 from
3 hens f the standard
8446 1.09 error.
A
ACTIVITY”
4
0.01 0.03 0.13 0.11 0.002 0.01
6113 48934 35545 14087 56012 9428
0.01
0.33 0.47 0.41 0.70 0.77 0.03 0.29 0.17 0.07 0.18 0.09
0.25 0.80 0.16 0.05 0.10 0.43 0.33
Free/ bound
88 99 91 239 109 123 556 3084 90 129 55 7 12 338 186 190 131 494 100
Specific activity
1 Day
tract.
4314 5616 19685 7266 39748 9748
116 53 73 152 116 114 849 4577 91 478 46 10 31 109 151 75 51 222 59
Specific activity
0.01 0.06 0.74 0.94 0.08 0.34
0.37 0.89 0.54 0.12 0.12 0.72 0.49 0.09 0.57 0.47 0.41 0.80 0.92 0.06 0.31 0.41 0.29 0.53 0.21
Free/ bound
2 Days
976 3756 41554 5831 10561 5044
0 0 25 53 112 55 653 3236 119 60 14 5 17 12 84 38 31 143 33
Specific activity
4 Days
0.03 1.25 1.03 1.82 0.21 0.84
0.87 0.66 0.38 0.88 0.65 0.20 0.92 0.71 0.67 1.40 1.54 0.50 0.47 0.64 0.64 0.72 0.04
Free/ bound
0 1734 706 642 1264 776
0 0 0 17 36 21 165 248 0 18 0 0 0 0 0 0 5 11 9
Specific activity
7.73 8.54 6.30 11.89 1.50
0.92 1.31 0.85
$ 2
3 s
?
0.83 0.36 1.19
$
0.75 0.62 0.99
Free/ bound
8 Days
IN VARIOUS TISSUES OF HENS 1,2,4, and 8 DAYS AFTER A SINGLE lo-MG ORAL DOSE (0.35 PCi) OF 14C-L~~~~~~ GOSSYPOL~
a DPM per gram fresh tissue or per gram dry contents gastrointestinal b Each value is an average of six determinations from 3 hens. - %Tq.z--..-z”-._ _-/. ..,, _..^.._. ..._._*..__ ~_ _- -I ___ ,_
Brain Lungs Heart Blood Spleen Kidney Liver Bile Pancreas Ovary Oviduct Muscles Fatty tissues Crop Proventriculus Ventriculus Smallintestine Ceca Largeintestine Gastrointestinaltract contents Crop Proventriculus Ventriculus Smallintestine Ceca Large intestine
Tissue
Sample
SPECIFIC
TABLE
METABOLISM
OF GOSSYPOL
IN LAYING
169
HENS
Table 4 shows the same data as specific activity in various tissues and tissue contents of the bird. In the tissues, after 1 day the highest specific activity was in the bile, followed by the liver. Muscle and fatty tissues had the lowest specific activity. The different tissues of the alimentary canal had less specific activity than the liver. That general pattern was noticed at the other periods. The contents of the alimentary canal had very high specific activity after 1 day, the ceca having the highest, followed by the crop, proventriculus, ventriculus, small intestine and large intestine. The ceca had the highest specific activity after 2 days, 4 days, and 8 days.
0
1
2
3
4 Days
5 after
6
7
a
dosing
FIG. 5. Change in level of radioactivity in hen tissues following a single oral dosing of IO-mg formyllabeled gossypol.
Table 4 also lists the ratios of the free to bound gossypol recovered from different tissues and tissue contents. Bound gossypol is that portion remaining in the sample after the extraction of the free gossypol and is probably in combination with the free epsilon amino groups of lysine (Lyman ef al., 1959). This ratio was generally less than 1 and higher in the tissues than in the contents of the alimentary canal after 1 day. However, the value of these ratios had progressively increased after 2,4, and 8 days in all tissues and tissue contents. After 8 days the free to bound gossypol ratio was much higher in the contents of the intestinal tract than in the tissues.
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ABOtJ-DONIA
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By plotting the percentage of 14C activity deposited in the tissues on semilogarithmic paper, it was possible to calculate the half-life (ti ,2) value of gossypol in the hens. The t, ,* value is the biological half-life of the radioactivity in the bird and is defined as the time necessary to eliminate one-half of the radioactive compounds from the bird. That value was 30 hr in the hens when a IO-mg single dose of gossypol was administered. DISCUSSION In the preparation of samples, no heat was used for drying. In our experience with metabolic fate studies of labeled materials in rats and chicks, we have found that the use of heat in the process of sample drying results in loss of activity. Thus, in this study, there is very small chance for error from evaporation loss of the label, as indicated by the good recoveries of radioactivity. Excretion via urine andfeces. Since the urine and feces of the birds are voided into the cloaca, the combined urinary-fecal excretion of i4C activity will be discussed. There is abundant evidence that the secretory mechanism for aromatic acids in the kidney is in most respects very similar in all vertebrate groups (Shannon, 1939), and the same seems to be true with regard to the secretion of the bile components (Sperber, 1959). The kidneys of birds are relatively larger than those of mammals, ranging from 1 to 2.6 % of the body weight (Benoit, 1950). The results show that gossypol is rapidly excreted from the bird through the combined feces and urine. One day after the oral administration of gossypol, 41.94 % of the dose was recovered in the excrement. After 8 days the total activity recovered in the combined urine and feces was 78.55% of the dose. The i4C activity in the urine could not be separately determined; however, Abou-Donia and Lyman (unpublished observation) found that gossypol was least excreted (0.77-3.14 %) via urine in rats fed formyl-‘4Clabeled gossypol. They suggested that gossypol, which is a weak acid, might be filtered and secreted by the tubular mechanism reported for organic acids in mammals (Weiner, 1967). Such compounds are reabsorbed from the tubules by nonionic diffusion. The tubular epithelium of the distal convoluted tubule is selectively permeable or more permeable to the nonionized lipid-soluble molecules than the poorly lipid-soluble anion or cation (Milne et al., 1958). In hens reabsorption of gossypol, with a pK, of 7(Tanksley, 1968), would be favored by the low urinary pH of the chicken urine (ranges from 6.22 to 6.7; Hester et al., 1940), which increases the portion of nonionized molecules. Also the high solubility of gossypol in lipid solvents should result in a high rate of reabsorption. Decarbonylation of gossypol. The results indicate that decarbonylation of gossypol to carbon dioxide and presumably the unstable apogossypol is not a major route for gossypol metabolism in the laying hen. This interpretation is based on the fact that only a total of 3.26% of the activity of the administered dose was recovered in the expired air from the time at which the dose was administered until 8 days later, when only very small amounts of 14C activity remained in the tissues. Gossypol in the egg. The data indicate that a major portion of the gossypol that was absorbed into the bloodstream was concentrated in the egg. Thus at the end of 8 days 13.67 % of the activity of the administered dose was recovered in the eggs. The ovary and oviduct had considerable activity 1 day after the ingestion of gossypol. The total activity accumulated in the egg albumen and the egg yolk was 4.57 and 9.10 %,
METABOLISM
OF GOSSYPOL
IN LAYING
HENS
171
respectively. The high activity in the egg might be explained by the fact that eggs are very rich in protein, i.e., 16.6 and 10.6 % for yolk and albumen, respectively (Sturkie, 1965). The higher contents of the yolk over the albumen is due to the higher lipid content of the former (32.6 %). Evidence has been presented which indicated that gossypol fed to laying hens is deposited in the yolk as gossypol-cephalin and gossypol-protein complexes (Woronick and Grau, 1955). Gossypol could have reached the egg from the ovary through the blood, before the formation of the egg shell. The high concentration of gossypol in the egg yolk is in harmony with the finding that gossypol is responsible for a discoloration of egg yolk in eggs laid by hens fed a diet containing cottonseed meal (Schaible et al., 1933). The signijicance of the bile in gossypol metabolism. The data indicate the excretion into the intestine via the bile is a major pathway by which absorbed gossypol is removed from the body. At 1, 2,4, and 8 days after the administration of the dose, the specific activity of the bile was much greater than in any tissue of the body, including the liver. These data suggest that gossypol is concentratively transferred from blood into bile. Gossypol in circulation enters the liver, from which it emerges (as such or in a degraded state) with the bile, and passes into the gallbladder. At intervals, bile leaves the gallbladder by the bile duct, which discharges into the small intestine. Gossypol, a weak acid, with a pK, value of 7, should be highly ionized in the chicken blood which has a pH value of 7.54 (Johnson and Bell, 1936). Thus the transfer process resembles in some respects that which transports organic anions across the kidney tubule. Gossypol seems to fall in Class B of Brauer’s classification. Brauer (1959) originally divided the substances that are excreted into bile into three classes according to their bile:plasma concentration ratios. Substances of Class A are those with a ratio of nearly 1. Class B includes substances with bile: blood ratios usually ranging from 10 to 1000. Class C compounds consist of those with a ratio of less than 1. In this investigation, the bile: plasma ratios of gossypol in the laying hens were found to be 12.04, 20.12, 28.89, and 14.59 after 1, 2, 4, and 8 days of the administration. The high concentration of total gossypol in the bile might suggest that it is “actively” secreted into bile. This is in agreement with the concept of Stowe and Plaa (1968), who reported that the transport of compounds in Class B across the biliary epithelium into bile appears to require some kind of active secretory process. They also reported that characteristically these substances compete for transport and the transport mechanism can be saturated by an excess of compound. The high biliary excretion of gossypol is in harmony with the tentative conclusion of Williams et al. (1965) that compounds of high molecular weight (more than 300) and containing two or more aromatic rings tended to be excreted into bile. It is also in agreement with Millburn et al. (1967), who concluded that for appreciable biliary excretion in the rat, a compound should have polar anionic groups and a molecular weight of about 350 or should be able to be converted metabolically into such compound. Gossypol possesses these properties with six polar hydroxyl groups and two less polar carbonyl groups and a molecular weight of 5 18. All reports show that the bile of chickens is acid with a pH value of 5.88 (Winget et al., 1962). The low bile pH increases the proportion of nonionized molecules to about 97%. The net neutral charge and the high solubility of gossypol in lipid solvents would tend to cause gossypol to be excreted more
172
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AND
LYMAN
extensively in the bile. It also seems that these two factors should favor the biliary excretion over the urinary excretion of gossypol. The small values for the ratio of free to bound gossypol in the bile indicate that most of the gossypol in the bile is in some conjugated form. Since gossypol combined with small peptides analyzed as free gossypol (Cater, 1968), it is more likely that this is not the form of gossypol in the bile. It might be possible to speculate as to why gossypol with its anion polar groups and high molecular weight is mainly excreted via the feces. Gossypol might be excreted through the transport mechanism that is normally used for biliary excretion of endogeneous compounds, such as glycocholic acid (molecular weight 466; pK, 4.54), taurocholic acid (molecular weight 516; pK, 1.56) (Sobotka, 1938), and bilirubin monoand diglucuronides (molecular weight 761 and 739; most glucuronides have pK, 3-4) (Williams, 1959), as has been suggested for foreign compounds by Millburn et al. (1967). It is to be noted that these compounds possess highly polar anionic groups and a relatively high molecular weight. It is therefore possible that gossypol with its anionic polar groups and high molecular weight will be excreted in the bile in relatively large amounts. Gossypol in the tissues.The data obtained from radiochemical analysis of different tissues show that 1 day after the oral administration of radioactive gossypol only 8.14 % was deposited in the tissues, while 43.47 % was recovered from the contents of the intestinal tract. The radioactivity recovered from the tissues increased after 2 days, but dropped thereafter. Generally the accumulation of dietary gossypol in the tissues of the hens was similar to that observed in rats (Abou-Donia and Lyman, unpublished observation), for swine by Smith and Clawson (1965) and Sharma et al. (1966), and for the rainbow trout by Roehm et al. (1967). Among the various tissues, the liver contained the largest total activity. However, bile had a higher specific activity. Blood had a relatively high specific activity 1 day after administration. Gossypol may penetrate the erythrocyte due to its lipophilic character, probably through a lipid-like membrane similar to that reported for mammalian erythrocytes (Schanker, 1962). These results are in harmony with the finding that feeding free gossypol to chicks caused lowering of the blood hemoglobin (Chang, 1955). The brain, which is protected by the blood-brain barrier, had a significant amount of activity 1 and 2 days after administration. This penetration is attributed to the high lipophilic character of gossypol. The high bound gossypol in the brain may be related to the level of phospholipids in this organ as suggested by Smith and Clawson (1965). ACKNOWLEDGMENT
Acknowledgment is made to Mrs. Susan G. Els for her technical assistance in the metabolic and radioactive studies reported here. This study was supported by USDA-ARS Contract No: 12-14-100-9497
(72).
REFERENCES J. (1950). In: Trait& de Zoologie (P.-P. Gras&, ed.), Vol. XV, “Oiseaux”, p. 341. Masson, Paris. BRAUER,R. W-,, JR. (1959). Mechanisms of bile secretion. J. Am. Med. Assoc.1@,1462-1466.
BENOIT,
METABOLISMOF GOSSYPOLIN LAYING HENS
173
CATER, C. M. (1968). Studies on the reaction products of gossypol with amino acids, peptides and proteins. Ph.D. Dissertation, Texas A & M University, College Station, Texas. CHANG, W. Y. (1955). The chemical characteristics of cottonseed meal as related to its biological utilization. M.S. thesis, Texas A & M University, College Station, Texas. COUCH, J. R., CHANG, W. Y., and LYMAN, C. M. (1955).The effect of free gossypolon chick growth. Poultry Sci. 34, 178-183. HESTER, H. R., ESSEX, H. E., and MANN, F. C. (1940).Secretionof urine in the chicken.Am. J. Physiol. 128, 592-602. HILL, F. W., and TATSUKA, K. (1964).Studiesof the metabolizableenergyof cottonseedmeals for chicks,with particular referenceto the effect of gossypol.Poultry Sci. 43,362-370. JOHNSON, E. P., and BELL, W. B. (1936).The blood pH of leukotic fowls and the filterability of leukosisagent.J. Infect. Diseases 58,342-348. LYMAN, C. M., BALIGA, B. P., and SLAY,M. W. (1959).Arch. Biochem.Biophys.84,486497. LYMAN, C. M., CRONIN, J. T., TRANT, M. T., and ODELL, G. V. (1969).Metabolism of gossypol in the chick. J. Am. Oil ChemistsSot. 46, 100-104. MILLBURN, P., SMITH,R. L., and WILLIAMS, R. T. (1967).Biliary excretion of foreign compounds; biphenyl, stilbestrol,and phenolpthaleinin the rat; molecularweight, polarity, and metabolismasfactors in biliary excretion. Biochem.J. 104, 1275-1281. MILNE, M. D., SCHRIBNER, B. N., and CRAWFORD, M. A. (1958).Non-ionic diffusion and excretion of weak acidsand bases.Am. J. Med. 24,709-729. NARAIN, R., LYMAN, C. M., DEYOE, C. W., and COUCH, J. R. (1961).Paperelectrophoresisand albumin/globulin ratios of the serumof normal chickensand chickensfed free gossypolin the diet. Poultry Sci. 40,21-23. ROEHM, M. P., LEE,D. T., and SINNHUBER, R. 0. (1967).Accumulation and elimination of dietary gossypolin the organsof Rainbow trout. J. Nutr. 92,425428. SCHAIBLE, P. J., MOORE,L. A., and MOORE, J. M. (1933).Gossypol,a causeof discolorationin eggyolks from hensfed cottonseedmeal.Poultry Sci. 12,344. SCHANKER, L. S. (1962).Passageof drugsacrossbody membranes.Pharmacol.Rev. 14, 501530. SHANNON, J. A. (1939).Renal tubular excretion. Physiol. Rev. 19,63-93.
SHARMA,M. P., S~rrrr, F. H., andCLAWSON, A. J. (1966).Effectsof levelsof protein and gossypol, and lengthof feedingperiod on the accumulationof gossypolin tissuesof swine.J. Nutr. tQ(434-438. SMITH,F. H., and CLAWSON,A. J. (1965).Effect of diet on accumulationof gossypolin the organsof swine.J. Nutr. 87, 317-321. SOBOTKA, H. (1938).Chemistryof Steroids,p. 140.Williams &Wilkins, Baltimore, Maryland. SPERBER, I. (1959).Secretionof organic anionsin the formation of urine and bile. Pharmacol. Rev. 11, 109-155. STOWE,C. M., and PLAA, G. L. (1968).External excretion of drugsand chemicals.Ann. Rev. Pharmacol.8,337-356. STURKIE,P. D. (1965).Avian Physiology,2nd ed., p. 474. ComstockPubl., Ithaca, New York. TANKSLEY, T. D. (1968).The effect of gossypolon someenzyme systems.Ph.D. Dissertation, TexasA & M University, CollegeStation, Texas. WEINER, I. M. (1967). Mechanismof drug absorption and excretion: the renal excretion of drugsand related compounds.Ann. Rev.Pharmacol.7,39-56. WILLIAMS, R. T. (1959).Detoxification Mechanisms, 2nded., p. 729.Chapman&Hall, London. WILLIAMS, R. T., MILLBURN, P., and SMITH,R. L. (1965).The influenceof enterohepaticcirculation on toxicity of drugs.Ann. N. Y. Acad. Sci. 123,110-l 24. WINGET, C. M., ASHTON, G. C., and CAWLEY,A. J. (1962).Changesin gastrointestinalpH associatedwith fasting in the laying hen.Poultry Sci. 41, 1115-l120. WORONICK, C. L., and GRAU, C. R. (1955).Gossypol-cephalincompoundfrom fresheggsof hensfed cottonseedmeal.J. Agr. Food Chem.3,706707.
Metabolic
Fate of Gossypol: The GossypoV4C in Laying
Metabolism Hens
of
M. B. ABOU-DONIA AND CARL M. LYMAN’ Department
of Biochemistry College
and Biophysics, Station, Texas
Received
August
Texas A & M University, 77843
7,1969
Metabolic Fate of Gossypol: The Metabolism of GossypoPC in Laying Hens.ABOU-DONIA, M. B., and LYMAN, CARL M. (1970). Toxicol. Appl. Pharmacol.17, 160-173.Orally administeredformyl-*4C-labeledgossypol wasrapidly excreted by laying hensinto the urine and feces.Only a smallportion wasdepositedin the tissues.A major part of the absorbedgossypol wasconcentratedin the eggs.The excretion of gossypolinto the intestine via the bile seemedto be a major pathway by which absorbedgossypol wasexcreted.The decarbonylationof gossypolto carbon dioxide and apogossypolwasnot a major route for gossypolmetabolismin the laying hen. The time necessaryto eliminateone-halfof the radioactive gossypol(l0 mg, 0.35&i) from the bird body (1 kg) under the conditionsusedwasfound to be 30 hr. Gossypol is a yellow coloring matter occurring in cottonseed. Its toxicity to chickens haslong beenrecognized. Couch et al. (1955), reported that gossypol in the diet affected the growth and development of hens. Hill and Tatsuka (1964) showed that high levels of gossypol causeda significant growth retardation in chicks, and a marked reduction in feed and metabolizable energy. They also found that gossypol causeda significant increasein the weight of the pancreasrelative to body size. Couch et al. (1955) reported the lowering of the blood hemoglobin of the chicks due to the feeding of free gossypol and postulated that free gossypol in the diet leads to interference with the synthesis of the proteins of the blood in thesechicks. Narain et al. (1961) found that feeding of free gossypol to chicks caused a lowering of total serum protein. Schaible et al. (1933) showed that gossypol was responsible for the discoloration of egg yolks laid by hens fed a diet containing cottonseed meal. Woronick and Grau (1955) found that gossypol fed to laying hens wasdeposited in the yolks asgossypol-cephalin and gossypol-protein complexes. Lyman et al. (1969)recovered most of the gossypol activity from the fecesof chicks fed r4C-labeled gossypol. They also found that most of the labeled compound retained wasin the liver, muscle,and kidneys, with the highestspecific activity in theliver. The purpose of the present work was to investigate the metabolic fate of gossypol in laying hens.This included study of the elimination of gossypol from the bird body and of its distribution in different tissues. METHODS The radioactive gossypol (2,2’-binaphthalene-8,8’-dicarboxaldehyde-l,1’,6,6’,7,7’hexahydroxy-5,5’-diisopropyl-3,3’-dimethyl) usedwaslabeled in the formyl group only. ’ Deceased March9, 1969. 160
METABOLISM
OF GOSSYPOL
IN LAYING
HENS
161
The specific activity was 19.75 $i/mmole. It was synthesized according to Lyman et al. (1969). Infrared analysis and thin-layer chromatography showed that the material had a radiochemical purity of at least 99 %. Care and treatment of birds. Midget laying hens (also called Mini, Dwarf), selected for uniformity of weight, were placed in individual metabolic cages. Each animal weighed approximately 1 kg. The metabolic cages were designed for studies of expired i4C02 ; they were made of glass and contained coarse- and fine-mesh screens to isolate the excrements. The expired air was continuously drawn out through the rear end of the cage to eliminate the possibility of high specific activity air being trapped in this volume and slowly leaking back into the cages to distort expiration rate records. Water and solid food containers were provided. The birds were allowed to adjust to their environment for 1 week; after this each hen received a single IO-mg dose of gossypol in a gelatin capsule to ensure that the complete sample entered the stomach. The birds were returned to their cages and given free access to food and water. Only hens that laid eggs regularly were used. The hens were sacrificed after varying intervals of time (1,2,4, or 8 days). For each time period 3 birds were used. The excrements and the eggs were collected daily. The eggs were stored in a refrigerator. At the end of the experimental periods, the birds were anesthetized with chloroform and decapitated. The blood was collected, and the individual organs were removed and weighed. Determination of 14C activity. i4C activity was determined by the use of a Beckman Model LS250 liquid scintillation counter after preparation of the samples as indicated below. The scintillation medium was toluene-ethylene glycol monomethyl ether solution (2: 1 v/v) containing 5 g of 2,5-diphenyloxazole (PPO) (obtained from Beckman, Houston, Texas) per liter. A solution of ethanolamine-ethylene glycol monomethyl ether (1:2 v/v) (both obtained as purified reagents from Fisher Scientific Company, Houston, Texas) was used in two traps (100 and 75 ml) to trap i4C02. Samples from tissues, feces and eggs were prepared for liquid scintillation counting by oxygen combustion. The combined feces and urine were lyophilized, weighed, and ground; samples of 50-100 mg were placed in “combustion envelopes” made of cellophane (obtained from Ivers Lee Company, Newark, New Jersey). Two-tenths milliliter of a 10 % sucrose solution and enough water to wet the entire sample were added. The envelopes were then air-dried under the hood. The contents of the gastrointestinal tract parts were separated and the different parts were washed with ethanol-ether (5 : 2 v/v). The wash of each part was added to its corresponding content, dried and analyzed as described for the feces. Fresh tissues (200-300 mg) were placed directly into combustion envelopes. Two-tenths milliliter of a 10 % sucrose solution was added, and the envelopes were dried as above. Eggs were collected daily and stored in the refrigerator until the end of the experiment. The yolk was carefully separated from the white, and fresh samples were weighed and prepared for oxygen combustion as described for the tissues. A bag containing the dried sample was placed in the platinum basket, where it was carried by a glass rod through the glass stopper which was firmly positioned in a 2-liter heavy-walled Erlenmeyer flask that had previously been purged with oxygen for 10 sec.
162
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The commercially available Thomas-Ogg infrared igniter (A. H. Thomas Company, Philadelphia, Pennsylvania) was used for ignition of samples. Since the infrared ignition depends upon absorption of the light, it was necessary to wrap the cellophane bag in a black filter paper to assure prompt ignition. Combustion of a sample is normally complete within less than 1 min. The next step was the addition of the solvent. The composition of the solvent was ethanolamine-ethylene glycol monomethyl ether (1: 2 v/v). The flask was first cooled for 5 min, then 10 ml of solvent was added through the sidearm. The flask was swirled gently to distribute the solvent over the entire bottom and an inch or two up on the sidewalls. The flask was then placed in an ice water bath with the cooling confined to the bottom inch of the fask and maintained in this manner for 15-20 min. At the end of this period, an additional 5 ml of the solvent was delivered again through the sidearm into the flask to rinse any activity that may have sequestered in the solvent that wetted the sidearm initially. The flask was swirled to mix the solvents, and 5 ml was added to 10 ml of the scintillation solvent in a sample counting vial. In this investigation free gossypol was determined by extracting the tissues and feces with ethanol-ethyl ether (5:2 v/v). Bound gossypol was estimated by the difference between total and free gossypol. It is recognized that a definition of free gossypol based on solubility relationship is quite empirical. In this study, a standard quenching curve was prepared for quench corrections utilizing a certified “C-benzoic acid standard (New England Nuclear, Boston, Massachusetts). RESULTS
14C Activity in Expired Air Figure 1 shows the accumulated total and the daily rate of 14C activity in the expired air from hens (average values for 3 birds) given radioactive gossypol. During the
Days
after
dosing
1. Daily rate and accumulated total ‘T activity in the expired air from hens following a single lO-mg oral dosing of formyl-labeled gossypol. FIG.
0.96 2.26 3.06 3.58 4.09 4.36 4.52 4.57
xkO.31 AZ0.42 f 0.56 f 0.63 f 0.65 4 0.68 zk0.70
z!z0.08
Egg white
a Each value is an average of six determinations
3.13 kO.35 3.21 k 0.43 3.26 zk0.47
1.15* 0.10 1.84 +z0.17 2.48 f 0.21 2.82 f 0.27 3.00 f 0.30
1
2 3 4 5 6 7 8
Expired Air
Days
f 1.05
+I 0.18 zt 0.70 f 0.81 f 0.93 f 0.95 f 0.98 f 1.01
lo-MG ORAL
41.94 f 4.17 54.78 rk 5.01 64.64 f 6.22 70.29L!=6.40 74.18 f 7.01 76.40k7.31 77.85 iz7.33 78.55 f 7.45
Feces
1
from 3 hens % the standard error.
1.97 4.69 6.27 7.40 8.04 8.52 8.87 9.10
Egg yolk
PERCENTAGE RECOVERY OF 14CACTIVITYFROM HENS GIVENA SINGLE
TABLE
1.09+ 0.09
0.92 f 0.10
97.93zk9.86
-
97.06 zt 9.88
7.74 * 0.95
97.53 zt 9.74 97.26 k9.50
-
43.37 5 4.11 23.81 f 2.10
Total
GOSSYPOL”
5.23 *0.77
8.14 h 1.10 9.88 zt 1.21
Tissuecontents
(0.35 &i) OF Y-LABELED
Tissues
DOSE
g Q x % Cl 0 B s 0 r z $ 3 0 X B
L E: $
164
ABOU-DONIAANDLYMAN
period of the experiments there was a steady increase in the total 14C activity in the expired air. The daily rate of r4C activity in the expired air reached a peak on the first day after the administration, then sharply decreased till the fifth day and leveled off thereafter. The total 14C activity in the expired air throughout the experiment (8 days) was 3.26 % of the administered dose (Table 1). Combined Urinary-Fecal Excretion of 14CActivity
Figure 2 shows daily rate and accumulated total combined urinary-fecal excretion of r4C activity from the hens. A sharp increase in the accumulated total urinary-fecal
-Accumulated Daily
----
---c, 0
1
2
3
-c-w-
4 Days
5 after
6
7
8
dosing
FIG. 2. Daily rate and accumulated total urinary-fecal excretion of Y activity from hens following a single IO-mg oral dosing of formyl-labeled gossypol.
excretion through the first day after the administration and a slow increase thereafter is indicated. Table 2 lists the ratio of free to bound gossypol in the urinary-fecal excretion TABLE 2 RATIO OF FREE TO BOUND GOSSWOL IN THE URINARY-FECAL EXCRETION AND EGGS FROM HENS GIVEN A SINGLE lo-MG ORAL DOSE (0.35 &i) OF 14C-L~~~~~ GOSSYPOL’ Days
Urinary-fecal excretion 0.02 0.05 0.04 0.05 0.06 0.11 0.28 0.36
Egg albumen 0.22 0.21 0.35 0.51 0.77 0.83 0.91 1.18
a Each value is an average of six determinations
Egg yolk 0.10 0.18 0.37 0.41 0.53 0.67 0.85 0.91
from 3 hens.
165
METABOLISM OF GOSSYPOL IN LAYING HENS
of hens fed a single oral dose of “C-labeled gossypol. It is interesting to note that this ratio had a very small value in the beginning of the experiment, i.e., 0.02 in the first days, which successively increased with time to 0.36 in the last day. Table 1 shows that 8 days after administration of 10 mg of gossypol, 78.55 ‘A of the i4C activity was recovered in the feces of the hens. 14CActivity in the Egg
The daily rate and accumulated total 14C activity in the egg white are presented in Fig. 3. It shows a rapid increase in accumulated total i4C activity in the egg white from hens given a lo-mg single dose of gossypol. The daily rate of 14C activity reached a peak 5
0
1
2
3
4 Days
5 after
6
7
8
dosing
FIG. 3. Daily rate and accumulated total % activity in the egg white from hens following a single IO-mg oral dosing of formyl-labeled gossypol.
2 days after the administration of the gossypol. Figure 4 shows the daily rate and accumulated total i4C activity in the egg yolk. It also shows a rapid increase in the accumulated total activity. The daily rate of i4C activity reached a peak 2 days after the administration, sharply decreased till the fifth day, then leveled off thereafter. Table 1 shows the total i4C activity in the egg white was 4.57 ‘A of the administered dose as compared to 9.10 % in the egg yolk. Table 2 also shows the ratios of free to bound gossypol in the egg. This ratio, while generally higher in the egg white than in the egg yolk, progressively increased by time in both parts of the egg. Histologic and General Observations
At the end of each experiment the animals were sacrificed and the tissues were excised. When a IO-mg single dose of gossypol was given, there were severe lesions in the lungs
166
ABOU-DONIA
AND
Days
after
LYMAN
dosing
FIG. 4. Daily rate and accumulated total ‘T activity in the egg yolk from hens following a single oral IO-mg dosing of formyl-labeled gossypol.
and livers. The hens lost some weight (13 %) the first 2 days, then gained some weight and had comparable weights to that of the control birds at the end of the experiments. At that level of gossypol there was normal egg production, and the feed consumption was similar to that of the control. ‘TissueDeposit of 14CActivity
The data obtained by radiochemical analysis of the tissues from hens fed formyl-i4Clabeled gossypol are summarized in Tables 3 and 4 and Fig. 5. In both tables the data consisted of radioactivity from all tissues and from the contents of different parts of the alimentary canal. Table 3 shows the distribution of the recovered 14C activity in different tissues and tissue contents from the birds. One day after the administration all tissues contained radioactivity, the spleen and the brain having the lowest activity. The liver had the highest activity followed by the bile. The alimentary canal tissues contained high activity, but less than that in the liver and the bile. The activity in all tissues was 8.14 % of the total dose. Two days after the administration, most of the tissues contained higher i4C activity than after 1 day, with the liver, ovary, and bile having the highest concentrations. The intestinal tract tissues had less activity after 2 days. At the fourth day there was a general decrease in the radioactivity in all tissues, with no radioactivity detected in the brain and the lungs. The liver and bile still had the highest activity. At 8 days after the oral dosing most of the tissues had no detectable activity. Only in the blood, spleen, kidney, liver, ovary, and bile was radioactivity detected. In the alimentary canal, the
METABOLISM
OF GOSSYPOL
IN LAYING
167
HENS
small intestine, ceca and large intestine contained radioactivity. The activity recovered from all tissues after 8 days was 0.92 % of the administered dose. The contents of different parts of the alimentary canal contained a much larger amount of radioactivity than the tissues at all times. After one day the gastrointestinal tract contents had 43.37 % of the total dose. The ventriculus had the highest amount followed by the ceca, small intestine, proventriculus, crop, and large intestine.‘The activity decreased after 2 days, but the total amount of the activity in the contents of the alimentary canal was 23.81% of the applied dose. After 4 days the activity dropped sharply to 7.74 ‘A, and after 8 days it was only 1.09 ‘A of the administered dose. TABLE DISTRIBUTION
OF RADIOACTIVITY IN V~mous SINGLE lo-MG ORAL DOSE (0.35
3 TISSUES OF HENS 1, 2, 4, AND 8 DAYS &i) OF 14C-L~~~~~~ GOSSWOL”
Total Sample
1 Day
dpm
2 Days
AFTER
per Tissue 4 Days
8 Days
Tissue Brain Lungs Heart Blood Spleen Kidney Liver Bile Pancreas Ovary Oviduct Muscles Fatty tissues Crop Proventriculus Ventriculus Small intestine Ckca Large intestine Total dpm % of Total
245 743 576 4749 224 1403 14902 8634 252 3939 1723 7500 450 3409 1076 5424 3970 2619 906
dose
* 7 f 22 f 17 f 147 f 6 zt 42 * 510 f 270 f 8 =k 127 =!r 51 f. 329 f 14 + 107 f 29 f 155 f 119 f 61 zt 20
62644 8.14
424 f 4OOkll 457 f 3015 f 249 f 1298 5 22765 zk 12810 + 254 f 14527 zt 1437 f 10500 * 1632 zk 1101 f 874 f 2135 f 1178 f 540 f 557 *
0 0
12 14 91 7 35 690 370 8 430 41 298 47 29 20 65 35 17 15
76053 9.88
156f5 1047 f 157*5 629 f 17512 f 9063 f 332 f 1822 zk 449+14 4782 rt 696 f 123 *4 487 f 1099 * 940 * 757 f 299 +
0 0 0 332 50 237 4784 696 0 530 0 0 0 0 0 50 135 56 82
30 18 51 29 10 46 143 21 15 31 28 25 30
40350 5.23
zt f zk f f
10 2 7 141 21
f
15
f I f 4 f 2 f 3
7103 0.92
Gastrointestinal tract contents Crop Proventriculus Ventriculus Small intestine Ceca Large intestine Total dpm % of Total
dose
a Each value
6012 29360 191939 47895 53771 4714 333548 43.37
is an average
f f f f f f
185 854 854 1451 1630 141
5431 3365 106390 24703 38157 4874
f 149 z!z 121 h 3250 * 790 f 1065 f 132
182821 23.81 of six determinations
1398f40 1454 + 24423 f 19829 f 10137 f 2524 f
0 104Oi31 3808 f 114 2182 f 66 1044*30 382 f 12
41 785 601 313 94
59766 7.74 from
3 hens f the standard
8446 1.09 error.
A
ACTIVITY”
4
0.01 0.03 0.13 0.11 0.002 0.01
6113 48934 35545 14087 56012 9428
0.01
0.33 0.47 0.41 0.70 0.77 0.03 0.29 0.17 0.07 0.18 0.09
0.25 0.80 0.16 0.05 0.10 0.43 0.33
Free/ bound
88 99 91 239 109 123 556 3084 90 129 55 7 12 338 186 190 131 494 100
Specific activity
1 Day
tract.
4314 5616 19685 7266 39748 9748
116 53 73 152 116 114 849 4577 91 478 46 10 31 109 151 75 51 222 59
Specific activity
0.01 0.06 0.74 0.94 0.08 0.34
0.37 0.89 0.54 0.12 0.12 0.72 0.49 0.09 0.57 0.47 0.41 0.80 0.92 0.06 0.31 0.41 0.29 0.53 0.21
Free/ bound
2 Days
976 3756 41554 5831 10561 5044
0 0 25 53 112 55 653 3236 119 60 14 5 17 12 84 38 31 143 33
Specific activity
4 Days
0.03 1.25 1.03 1.82 0.21 0.84
0.87 0.66 0.38 0.88 0.65 0.20 0.92 0.71 0.67 1.40 1.54 0.50 0.47 0.64 0.64 0.72 0.04
Free/ bound
0 1734 706 642 1264 776
0 0 0 17 36 21 165 248 0 18 0 0 0 0 0 0 5 11 9
Specific activity
7.73 8.54 6.30 11.89 1.50
0.92 1.31 0.85
$ 2
3 s
?
0.83 0.36 1.19
$
0.75 0.62 0.99
Free/ bound
8 Days
IN VARIOUS TISSUES OF HENS 1,2,4, and 8 DAYS AFTER A SINGLE lo-MG ORAL DOSE (0.35 PCi) OF 14C-L~~~~~~ GOSSYPOL~
a DPM per gram fresh tissue or per gram dry contents gastrointestinal b Each value is an average of six determinations from 3 hens. - %Tq.z--..-z”-._ _-/. ..,, _..^.._. ..._._*..__ ~_ _- -I ___ ,_
Brain Lungs Heart Blood Spleen Kidney Liver Bile Pancreas Ovary Oviduct Muscles Fatty tissues Crop Proventriculus Ventriculus Smallintestine Ceca Largeintestine Gastrointestinaltract contents Crop Proventriculus Ventriculus Smallintestine Ceca Large intestine
Tissue
Sample
SPECIFIC
TABLE
METABOLISM
OF GOSSYPOL
IN LAYING
169
HENS
Table 4 shows the same data as specific activity in various tissues and tissue contents of the bird. In the tissues, after 1 day the highest specific activity was in the bile, followed by the liver. Muscle and fatty tissues had the lowest specific activity. The different tissues of the alimentary canal had less specific activity than the liver. That general pattern was noticed at the other periods. The contents of the alimentary canal had very high specific activity after 1 day, the ceca having the highest, followed by the crop, proventriculus, ventriculus, small intestine and large intestine. The ceca had the highest specific activity after 2 days, 4 days, and 8 days.
0
1
2
3
4 Days
5 after
6
7
a
dosing
FIG. 5. Change in level of radioactivity in hen tissues following a single oral dosing of IO-mg formyllabeled gossypol.
Table 4 also lists the ratios of the free to bound gossypol recovered from different tissues and tissue contents. Bound gossypol is that portion remaining in the sample after the extraction of the free gossypol and is probably in combination with the free epsilon amino groups of lysine (Lyman ef al., 1959). This ratio was generally less than 1 and higher in the tissues than in the contents of the alimentary canal after 1 day. However, the value of these ratios had progressively increased after 2,4, and 8 days in all tissues and tissue contents. After 8 days the free to bound gossypol ratio was much higher in the contents of the intestinal tract than in the tissues.
170
ABOtJ-DONIA
AND
LYMAN
By plotting the percentage of 14C activity deposited in the tissues on semilogarithmic paper, it was possible to calculate the half-life (ti ,2) value of gossypol in the hens. The t, ,* value is the biological half-life of the radioactivity in the bird and is defined as the time necessary to eliminate one-half of the radioactive compounds from the bird. That value was 30 hr in the hens when a IO-mg single dose of gossypol was administered. DISCUSSION In the preparation of samples, no heat was used for drying. In our experience with metabolic fate studies of labeled materials in rats and chicks, we have found that the use of heat in the process of sample drying results in loss of activity. Thus, in this study, there is very small chance for error from evaporation loss of the label, as indicated by the good recoveries of radioactivity. Excretion via urine andfeces. Since the urine and feces of the birds are voided into the cloaca, the combined urinary-fecal excretion of i4C activity will be discussed. There is abundant evidence that the secretory mechanism for aromatic acids in the kidney is in most respects very similar in all vertebrate groups (Shannon, 1939), and the same seems to be true with regard to the secretion of the bile components (Sperber, 1959). The kidneys of birds are relatively larger than those of mammals, ranging from 1 to 2.6 % of the body weight (Benoit, 1950). The results show that gossypol is rapidly excreted from the bird through the combined feces and urine. One day after the oral administration of gossypol, 41.94 % of the dose was recovered in the excrement. After 8 days the total activity recovered in the combined urine and feces was 78.55% of the dose. The i4C activity in the urine could not be separately determined; however, Abou-Donia and Lyman (unpublished observation) found that gossypol was least excreted (0.77-3.14 %) via urine in rats fed formyl-‘4Clabeled gossypol. They suggested that gossypol, which is a weak acid, might be filtered and secreted by the tubular mechanism reported for organic acids in mammals (Weiner, 1967). Such compounds are reabsorbed from the tubules by nonionic diffusion. The tubular epithelium of the distal convoluted tubule is selectively permeable or more permeable to the nonionized lipid-soluble molecules than the poorly lipid-soluble anion or cation (Milne et al., 1958). In hens reabsorption of gossypol, with a pK, of 7(Tanksley, 1968), would be favored by the low urinary pH of the chicken urine (ranges from 6.22 to 6.7; Hester et al., 1940), which increases the portion of nonionized molecules. Also the high solubility of gossypol in lipid solvents should result in a high rate of reabsorption. Decarbonylation of gossypol. The results indicate that decarbonylation of gossypol to carbon dioxide and presumably the unstable apogossypol is not a major route for gossypol metabolism in the laying hen. This interpretation is based on the fact that only a total of 3.26% of the activity of the administered dose was recovered in the expired air from the time at which the dose was administered until 8 days later, when only very small amounts of 14C activity remained in the tissues. Gossypol in the egg. The data indicate that a major portion of the gossypol that was absorbed into the bloodstream was concentrated in the egg. Thus at the end of 8 days 13.67 % of the activity of the administered dose was recovered in the eggs. The ovary and oviduct had considerable activity 1 day after the ingestion of gossypol. The total activity accumulated in the egg albumen and the egg yolk was 4.57 and 9.10 %,
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respectively. The high activity in the egg might be explained by the fact that eggs are very rich in protein, i.e., 16.6 and 10.6 % for yolk and albumen, respectively (Sturkie, 1965). The higher contents of the yolk over the albumen is due to the higher lipid content of the former (32.6 %). Evidence has been presented which indicated that gossypol fed to laying hens is deposited in the yolk as gossypol-cephalin and gossypol-protein complexes (Woronick and Grau, 1955). Gossypol could have reached the egg from the ovary through the blood, before the formation of the egg shell. The high concentration of gossypol in the egg yolk is in harmony with the finding that gossypol is responsible for a discoloration of egg yolk in eggs laid by hens fed a diet containing cottonseed meal (Schaible et al., 1933). The signijicance of the bile in gossypol metabolism. The data indicate the excretion into the intestine via the bile is a major pathway by which absorbed gossypol is removed from the body. At 1, 2,4, and 8 days after the administration of the dose, the specific activity of the bile was much greater than in any tissue of the body, including the liver. These data suggest that gossypol is concentratively transferred from blood into bile. Gossypol in circulation enters the liver, from which it emerges (as such or in a degraded state) with the bile, and passes into the gallbladder. At intervals, bile leaves the gallbladder by the bile duct, which discharges into the small intestine. Gossypol, a weak acid, with a pK, value of 7, should be highly ionized in the chicken blood which has a pH value of 7.54 (Johnson and Bell, 1936). Thus the transfer process resembles in some respects that which transports organic anions across the kidney tubule. Gossypol seems to fall in Class B of Brauer’s classification. Brauer (1959) originally divided the substances that are excreted into bile into three classes according to their bile:plasma concentration ratios. Substances of Class A are those with a ratio of nearly 1. Class B includes substances with bile: blood ratios usually ranging from 10 to 1000. Class C compounds consist of those with a ratio of less than 1. In this investigation, the bile: plasma ratios of gossypol in the laying hens were found to be 12.04, 20.12, 28.89, and 14.59 after 1, 2, 4, and 8 days of the administration. The high concentration of total gossypol in the bile might suggest that it is “actively” secreted into bile. This is in agreement with the concept of Stowe and Plaa (1968), who reported that the transport of compounds in Class B across the biliary epithelium into bile appears to require some kind of active secretory process. They also reported that characteristically these substances compete for transport and the transport mechanism can be saturated by an excess of compound. The high biliary excretion of gossypol is in harmony with the tentative conclusion of Williams et al. (1965) that compounds of high molecular weight (more than 300) and containing two or more aromatic rings tended to be excreted into bile. It is also in agreement with Millburn et al. (1967), who concluded that for appreciable biliary excretion in the rat, a compound should have polar anionic groups and a molecular weight of about 350 or should be able to be converted metabolically into such compound. Gossypol possesses these properties with six polar hydroxyl groups and two less polar carbonyl groups and a molecular weight of 5 18. All reports show that the bile of chickens is acid with a pH value of 5.88 (Winget et al., 1962). The low bile pH increases the proportion of nonionized molecules to about 97%. The net neutral charge and the high solubility of gossypol in lipid solvents would tend to cause gossypol to be excreted more
172
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extensively in the bile. It also seems that these two factors should favor the biliary excretion over the urinary excretion of gossypol. The small values for the ratio of free to bound gossypol in the bile indicate that most of the gossypol in the bile is in some conjugated form. Since gossypol combined with small peptides analyzed as free gossypol (Cater, 1968), it is more likely that this is not the form of gossypol in the bile. It might be possible to speculate as to why gossypol with its anion polar groups and high molecular weight is mainly excreted via the feces. Gossypol might be excreted through the transport mechanism that is normally used for biliary excretion of endogeneous compounds, such as glycocholic acid (molecular weight 466; pK, 4.54), taurocholic acid (molecular weight 516; pK, 1.56) (Sobotka, 1938), and bilirubin monoand diglucuronides (molecular weight 761 and 739; most glucuronides have pK, 3-4) (Williams, 1959), as has been suggested for foreign compounds by Millburn et al. (1967). It is to be noted that these compounds possess highly polar anionic groups and a relatively high molecular weight. It is therefore possible that gossypol with its anionic polar groups and high molecular weight will be excreted in the bile in relatively large amounts. Gossypol in the tissues.The data obtained from radiochemical analysis of different tissues show that 1 day after the oral administration of radioactive gossypol only 8.14 % was deposited in the tissues, while 43.47 % was recovered from the contents of the intestinal tract. The radioactivity recovered from the tissues increased after 2 days, but dropped thereafter. Generally the accumulation of dietary gossypol in the tissues of the hens was similar to that observed in rats (Abou-Donia and Lyman, unpublished observation), for swine by Smith and Clawson (1965) and Sharma et al. (1966), and for the rainbow trout by Roehm et al. (1967). Among the various tissues, the liver contained the largest total activity. However, bile had a higher specific activity. Blood had a relatively high specific activity 1 day after administration. Gossypol may penetrate the erythrocyte due to its lipophilic character, probably through a lipid-like membrane similar to that reported for mammalian erythrocytes (Schanker, 1962). These results are in harmony with the finding that feeding free gossypol to chicks caused lowering of the blood hemoglobin (Chang, 1955). The brain, which is protected by the blood-brain barrier, had a significant amount of activity 1 and 2 days after administration. This penetration is attributed to the high lipophilic character of gossypol. The high bound gossypol in the brain may be related to the level of phospholipids in this organ as suggested by Smith and Clawson (1965). ACKNOWLEDGMENT
Acknowledgment is made to Mrs. Susan G. Els for her technical assistance in the metabolic and radioactive studies reported here. This study was supported by USDA-ARS Contract No: 12-14-100-9497
(72).
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