Biochemical effects of aflatoxin in pigs

TOXICOLOGYANDAPPLIEDPHARMACOLOGY

&393-404(1969)

Biochemical M.R.

Effects

of Aflatoxin

in Pigs

GUMBMANNANDS.N.WILLIAMS

Western Regional Research Laboratory, Agricuiturai Research Service, U.S. Department of Agriculture, Albany, California 94710 Received October 28,1968

Biochemical Effects of Aflatoxin in Pigs. GUMBMANN, M. R., and S. N. (1968).Toxicol. Appl. Pharmacol. 15,393-404.Biochemical changesaccompanyingthe developmentof aflatoxicosiswere observedin young pigs maintainedfor approximately 4 months on diets containing variousamountsof themetabolitesof Aspergillusflavus. Thesedietsprovided concentrationsof aflatoxin B1rangingfrom 2 to 810partsper billion (ppb). During the feeding period, serum glutamic-oxaloacetic transaminase, alkaline phosphatase,and isocitric dehydrogenasebecameelevated.Other factors in blood, namely, plasmaalbumin, albumin:globulin ratio, nonprotein nitrogen, urea nitrogen, and adeninenucleotides(I&, values)all decreased. At autopsythe concentrationin the liver of glutamic-oxaloacetic transaminase,isocitric dehydrogenase,lipid, vitamin A, glycogen, and total nitrogen decreasedasa function of increasingdietary aflatoxin. Thesechangesare discussedin relation to time of developmentand dietary level of aflatoxin. The lowestlevel of aflatoxin for which a biochemical responsewasdetectedwas51ppb. Reproduction studies with sows and boars maintained on 450 ppb aflatoxin for S-10 months failed to show any biochemicaleffects in the offspring which couldbeattributed to aflatoxin poisoning,eventhoughsuch alterationswereevident in the parents. WILLIAMS,

The aflatoxins, a group of closely related metabolites of Aspergillusflavus, are now well known to be potent hepatotoxins possessing carcinogenic activity and can occur as natural contaminants in animal feeds as well as in a wide variety of food material used for human consumption (Wogan, 1968). The hepatic lesionsand clinical signs associated with aflatoxicosis, including the broad range in species susceptibility, are similar to those caused by pyrolizidine alkaloid poisoning (Carnaghan and Crawford, 1964; Schoental, 1963). Biochemical changes studied during aflatoxin poisoning include short-term effects of either single or multiple doses of aflatoxin Bt on liver metabolism in the rat and duckling (Shank and Wogan, 1966; Clifford and Rees, 1967), serum proteins in the chick (Carnaghan et al., 1967), and liver function tests in the dog (Wilson et al., 1967). Enzymatic and other compositional changes in liver and serum following more extended periods of feeding aflatoxin-containing diets have been measured for chickens and ducklings (Platonow, 1965a, b; Camaghan et al., 1966; Brown and Abrams, 1965; Juszkiewicz et al., 1967), monkeys (Cuthbertson et al., 1967; Tupule et al., 1964), and

calves (Allcroft and Lewis, 1963). 393

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GUMBMANN

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WILLIAMS

Early work by Harding et al. (1963) showed that pigs fed a toxic groundnut meal, which presumably was contaminated with aflatoxin, responded with changes in serum enzymes and liver composition. Unfortunately, however, the aflatoxin content of this meal was unspecified. Bodnar et al. (1965) determined the activity of the serum enzymes alkaline phosphatase and glutamic-oxaloacetic and glutamic-pyruvic transaminases, in pigs fed aflatoxin-containing diets, but they found no biochemical response after feeding levels up to 800 ppb for 5.5 months. However, at levels of aflatoxin estimated to be 750 ppb or more, Annau et al. (1964) found alterations in the electrophoretic patterns of pig serum proteins. Acute intoxication by aflatoxin in pigs, leading to death between 24 and 72 hours, was accompanied by changes in serum enzyme levels and liver function indicative of marked hepatic damage (Cysewski et al., 1968). But at dosages great enough to cause death in l-32 days, Wilson et al. (1967) detected no change in serum transaminase activity. The purpose of the present investigation into the toxicity of aflatoxin in pigs was to characterize the chronic biochemical changes that occurred in blood and liver and to delineate the lowest dietary level of aflatoxin at which such responses could be elicited. Results are presented which extend this level considerably below those cited above. Reported elsewhere, but from this same toxicity study, are the effects on weight gain, feed conversion (Hintz et aE., 1967a), reproductive performance (Hintz et al., 1967b), and pathologic findings (Gag& et al., 1968). MATERIALS

AND

METHODS

Aflatoxin was supplied in the experimental diets by the addition of 15 % peanut meal made from naturally infected peanuts and spiked where necessary with mold cultures to achieve the desired concentrations. In the first trial 6 diets were fed; 5 contained peanut meal with final aflatoxin Br concentrations of approximately 2,851, 105, and 233 parts per billion (ppb). The sixth which contained 15 % soybean meal in place of peanut meal had no detectable aflatoxin. In the second trial there were 5 diets, 4 with peanut meal furnishing final aflatoxin Bi concentrations of 5, 450, 615, and 810 ppb, and 1 containing soybean meal as described above. The low level peanut meal diets of both trials, which contained negligible aflatoxin (2 and 5 ppb), and the soy diets were considered negative controls. Details on mixing and basal composition of the diets, including analyses for aflatoxin Bi, B2, and Gi have been described elsewhere (Hintz et al., 1967a). Each dietary group consisted of 10 Duroc-Jersey pigs (6 barrows and 4 gilts) except the one at 105 ppb in the first trial which had 8 (6 barrows and 2 gilts). Experimental feeding began when the pigs were 12-14 weeks old and continued until autopsy 112-134 days later. Additional pigs (5 boars and 5 gilts) were maintained on the diet of trial 2 containing 450 ppb aflatoxin and used for reproduction studies when they were approximately 8 months old. The boars were autopsied 248 days and the sows 325 days after the start of the trial. Three piglets from each litter of the 5 sows were selected randomly for autopsy and clinical investigation at an age that varied from 56 to 73 days. For a control reference, 6 piglets 74 to 84 days old were obtained from sows fed a diet containing soybean meal with no detectable aflatoxin. Blood samples were taken 4 times during the course of trial 1 (at 24, 55, 88, and 102 days) from the group on the highest level of aflatoxin (233 ppb) and from the control

BIOCHEMICAL

EFFECTS

OF AFLATOXIN

395

group fed either soy or peanut meal. In the second trial, a control group and one of the 3 groups receiving aflatoxin were selected each week for blood analyses starting at 20 days. For both trials only one-half of a group was sampled at any given time ; blood from the other half was obtained on alternate occasions. Bleeding was from the anterior vena cava, and clotting was prevented with potassium oxalate. The plasma was proportioned for various tests and kept at -20”. Blood obtained at autopsy was pooled for each group in trial 1, but not in trial 2, where individual samples were collected. No anticoagulant was used, thus autopsy analyses were performed on serum. Liver samples were also taken at this time and placed in dry ice. Enzyme activities determined in serum (or plasma) and liver were: glutamicoxaloacetic transaminase (GOT) (Umbreit et al., 1957), alkaline phosphatase (AP) (King and Wotton, 1956), isocitric dehydrogenase @CD), and malic dehydrogenase (MDH) (Ochoa, 1955a, b). Glutamic-pyruvic transaminase (GPT) (Umbreit et al., 1957) was measured only in serum (or plasma). The Ez6,, values, a measure of adenine nucleotides (Siekevitz and Potter, 1955), were determined from the light absorption at 260 rnp of acid-soluble material extracted from liver and serum (or plasma) by the method of Gallagher (1961). Other nonenzymatic constituents measured were: blood nonprotein nitrogen (NPN), blood urea nitrogen (BUN), serum (or plasma) total protein, albumin and globulin (Annino, 1964), total serum (or plasma) cholesterol (Babson et al., 1962), serum (or plasma) and liver copper (Gubler et al., 1952), and liver glycogen (van der Vies, 1954). Liver vitamin A and lipids were extracted with petroleum ether from 25 % aqueous homogenates containing an equal volume of ethanol; vitamin A was determined by the antimony trichloride method according to Embree et al. (1957), and lipids were determined gravimetrically. Liver nitrogen was measured by the Kjeldahl technique. Analysis of variance and Duncan’s multiple range test were used for comparison of group means (Duncan, 1955). Relationships among biochemical factors and trends related to time and dose level were estimated by calculation of correlation coefficients and slopes of linear regression. At autopsy, liver vitamin A was found to be significantly lower in both trials for the control animals fed soybean meal compared to the control animals fed peanut meal. Since this reflected a dietary or nutritional difference unrelated to aflatoxin, the peanut meal controls were preferred for a basis of comparison whenever possible, and results with the soy diets have not been specifically reported. RESULTS Trial 1 Blood Analyses

After 88 days experimental feeding, significant alterations were evident in the blood from pigs receiving 233 ppb aflatoxin. Plasma AP became elevated and blood NPN’and BUN decreased. Plasma GOT was found to be elevated at 55 and 88 days, but this did not persist until 102 days. At autopsy these changes in AP, NPN, and BUN were found in all groups at aflatoxin levels of 51 ppb or more (Table 1). Determinations on blood from the 2 ppb group are not included because of extensive hemolysis that occurred in this sample. Although serum GOT was progressively elevated by diets containing up to

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GUMBMANNANDWILLIAMS

105 ppb aflatoxin, the 233 ppb group did not show this effect. Total cholesterol concentration also tended to increase with increasing dietary aflatoxin. Serum EzeOvalue and copper concentration were unaffected. TABLE 1 SERUM

CONSTITUENTS

OF PEELED BLOOD SAMPLES OBTAINED OF PIGS FED AFLATOXIN IN TRIAL 1

AT AUTOPSY

Aflatoxin level (ppb) Constituent

8

Glutamic-oxaloacetic transaminase’ Alkaline phosphataseb Nonprotein nitrogenC Urea nitrogenC Total cholesterol’

3.8 9.8 28.8

21.3 164

51

10.5

233

4.5 13.3 25.6 18.2

7.4 13.2 25.4 19.4

3.8 14.8 21.6

172

174

15.0

190

g Micromoles of oxaloacetate per 100 ml/min. b King-Armstrong

c Milligrams

units

per 100 ml.

per 100 ml.

Trial 2 Blood Analyses

Mean plasma GOT activity in trial 2 (Fig. 1) became elevated in all test groups over the 5 ppb control after about 60 days. Comparison of means showed that the elevation was significant at the 95% level or higher for each group determined after this time through 98 days. After 98 days GOT activity of two groups declined. As in trial 1, aflatoxin again brought about increased levels of plasma AP (Fig. 1). By 49 days the pigs receiving 615 ppb had significantly elevated AP activity (P < 0.05) as did all test groups sampled thereafter. The sharp peak shown for the highest level of aflatoxin was the result of extreme elevation in 3 animals and was exaggerated by the death of one before the conclusion of the feeding trial. A lesser maximum, however, occurring near 96 days could be seen in the individual data of 5 out of the 9 surviving animals of this group. The remaining 4, in which AP activity continued to increase, were those that had the lowest AP levels of the group at 96 days. Plasma AP in control animals distinctly decreased for approximately the first 80 days. Plasma ICD activity was consistently less than that of the controls throughout most of the feeding period, especially at 810 ppb aflatoxin, and shortly before autopsy, it became elevated in all test groups. The remaining 2 enzyme activities followed during the second feeding trial-plasma GPT and MDHwere unaffected. Table 2 presents the rate of change of NPN and plasma proteins during feeding trial 2, as influenced by dietary aflatoxin. For animals fed aflatoxin, NPN, albumin and A/G ratio showed significant linear rates of decrease, while remaining essentially constant in the controls. Total plasma protein, on the other hand, remained constant in the test animals while significantly increasing in the controls. Comparison of only the control and test group sampled at any given time revealed that the depressing effects of aflatoxin on NPN had taken place as early as the first sampling date, at 20 days in the 810 ppb

397

BIOCHEMICALEFFECTSOFAFLATOXIN 8.0

\ E a

_

L

24

GLUTAMIC-OXALACETIC TRANSAMINASE

-

ALKALINE

PHOSPHATASE

-0-I5

20-

8

5 PPB AFLATOXIN

-

c

450

PPB AFLATOXIN

6

615 PPB AFLATOXIN

-

810

PPB AFLATOXIN

v) g. 3

16-

d si 12 -

8

1 20

I

I

I

40

I 60

I

80

DAYS

FIG. 1. Mean plasma glutamic-oxaloacetic fed aflatoxin in trial 2.

I

1

ON

I 100

I

I 120

I 1 0

DIET

transaminase and alkaline phosphatase activities in pigs

TABLE 2 RATEOF CHANGEOFNONPROTEINN~TRCGENAND PLASMAPROTEINSIN INTRIAL 2, FORTHEPERIODFROM 20 TO~~DAYS

PIGS FEDAFLATOXIN

Percentchangeper week” Dietary group Control (5 ppb aflatoxin) Test”

Nonprotein nitrogen

- 0.1 (NS) - 1.5(< 0.005)

Albumin

+ 1.0 (NS) - 3.1 (< 0.01)

A/G ratio

- 0.1 (NS) - 3.1 (< 0.01)

Total plasma protein

+ 1.o (< 0.001) + 0.2 (NS)

’ Calculated from theregressioncoefficient, with significance given in parentheses. Degrees of freedom : control, 25; test 42. NS = not significant. * The test group includes all animals receiving 450,615, and 810 ppb aflatoxin.

398

GUMBMANN

3.

0.2

AND

WILLIAMS

0

I 450

’ 5

AFLATOXIN

IN DIET,

I 615

810

(PPB)

FIG. 2. Serum enzymes at autopsy in pigs fed aflatoxin in trial 2. Means are plotted with lines of linear regression calculated from 35 degrees of freedom. Significance of slope: AP, P -e0.001; GOT, ICD, P < 0.025. Solid points indicate a significant difference of means from those of the control (P -x0.05).

TOTAL

PROTEIN

12

d

0 0

10

GLOBULIN

1 0

0

8 f

I

11

ALBUMIN

o.3, 5

o-

450

AFLATOXIN

IN DIET,

o

1

615

810

(PPB)

FIG. 3. Serum proteins at autopsy in pigs fed aflatoxin in trial 2. Presentation of data is the same as in Fig. 2. Significance of slope: total protein, P < 0.1; globulin, P = 0.05; albumin, P i 0.025; A/G ratio, P < 0.01.

BIOCHEMICAL

EFFECTS

399

OF AFLATOXIN

group. Plasma albumin and A/G ratio became significantly depressed at 42 days in the 810 ppb group, as did BUN. Serum Ez6e values became significantly lowered in the 450 and 615 ppbgroups at 27 and 34 days, respectively, and were always less than those of the control in the 810 ppb group, but never significantly so. Plasma cholesterol was unaffected. Marked visible icterus developed midway in the second feeding trial in 2 pigs from the 810 ppb group, one of which died on day 110. Icterus was accompanied by considerably elevated plasma AP, GOT, and GPT and depressed BUN and NPN. Upon _ TOTAL

NITROGEN 0 .

UREA

NITROGEN

EzbO VALUE

(AS ADENINE

5

450

AFLATOXIN

IN DIET,

NITROGEN)

615

810

(PPB)

FIG. 4. Nonprotein nitrogenous constituents of serum at autopsy in pigs fed aflatoxin in trial 2. Presentation of data is the same as in Fig. 2. Significance of slope: Total and urea nitrogen, P < 0.05; Ezaovalue, P 4 0.025.

microscopic examination, the livers of both pigs were found to have suffered the greatest histopathologic damage observed in these feeding experiments (W. E. Gag&, personal communication). The relationships of the biochemical alterations found in blood at the end of trial 2 to dietary concentration of aflatoxin are presented in Figs. 2,3, and 4. With increasing aflatoxin, the serum enzyme activities AP, GOT, and ICD increased, while albumin and nonprotein nitrogenous substances decreased. Significant slopes of regression, except for total serum protein, indicated that the observed responses tended to be proportional to aflatoxin level. Still unaltered at this time were GPT, MDH, and cholesterol. Trial 1 Liver Analyses

In feeding trial 1, only liver AP activity and lipid content responded consistently to the level of dietary aflatoxin. Both were positively correlated to aflatoxin concentration (P < 0.01) and significantly elevated above the control at the highest level fed, 233 ppb (P < 0.05). The maximum mean increase was about 40 % for AP and 13 % for lipid. Liver GOT activity was significantly lower in the same 2 groups for which it was elevated in plasma, namely, 51 and 105 ppb. However, no difference from the control was noted

400

GUMBMANN

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WILLIAMS

at 233 ppb. The remaining substances determined in liver, vitamin A, glycogen, and copper, and the Ezeo value, were not affected by the dietary levels of the first trial. Trial 2 Liver Analyses

The results of liver analyses performed at autopsy in feeding trial 2 are summarized in Fig. 5. Mean GOT activity was found to be significantly elevated for the two intermediate

I”

4

100

5;s

1.4

2 f 5 I -0

1.2 0 1.0

- ISOCITRIC

DEHYDROGENASE 0

-

0 0

I LIPID h’ ,.o”c 4.0

c

0 0

AFLATOXIN

Q

IN DIET, (PPB)

FIG. 5. Liver constituents at autopsy in pigs fed aflatoxin in trial 2. Solid lines represent slopes of linear regression on aflatoxin concentration. Significance of slope: lipid, glycogen, P -C 0.05; ICD, vitamin A, P-C 0.01; E2ho value, Pi 0.005; GOT, total nitrogen, P < 0.001. Means signticantly different from those of the control : l , P < 0.05 ; 8, P c 0.01.

BIOCHEMICAL

EFFECTS

OF AFLATOXIN

401

levels and depressed below all other means in the 810 ppb group. The individual GOT activities of these 3 test groups formed a highly significant inverse relationship to aflatoxin concentration. Of the two liver dehydrogenases measured, MDH and ICD, only the latter was influenced by aflatoxin. Activity of ICD was negatively correlated to increasing aflatoxin concentration with the means of the 450 and 810 ppb groups being significantly lower than that of the control. Similarly, lipid, vitamin A, glycogen, and total nitrogen were negatively correlated to aflatoxin concentration with significant depression of mean values occurring in the 450 and 810 ppb groups for lipid and total nitrogen and in the 8 10 ppb for vitamin A. This response of lipid content is opposite to that observed in trial 1, in which a gradual but small increase was measured up to 233 ppb aflatoxin. There were no significant differences between mean glycogen values. Mean liver Ezao values were similar to those of GOT in that significant elevation occurred at 450 and 615 ppb accompanied by significant depression at 810 ppb. Liver AP activity, unlike that of trial 1, showed no trends to aflatoxin or differences between groups. Reproduction

Studies

Some of the same biochemical alterations resulting from aflatoxin in feeding trial 2 were apparent in the animals used for reproduction studies when autopsied approximately 4-6 months later. In the sows, serum albumin, A/G ratio, BUN, liver nitrogen, and liver ICD were lowered, and plasma ICD was considerably increased, even beyond levels found for the comparable group (450 ppb) at the end of trial 2. These substances, with the exception of liver nitrogen, were similarly altered in the boars, but to a lesser degree. Serum AP and GOT were not elevated in either sex but were close to the control values of trial 2 at autopsy. Analysis of blood and liver from the piglets produced in this reproduction study failed to reveal any changes that could be attributed to aflatoxin poisoning. DISCUSSION

Some of the biochemical changes that characterize developing aflatoxicosis are not unlike those generally associated with other hepatotoxins. Marked increasesin serum AP, GOT, and ICD (as observed in the present study) and in other enzymes are known to result from poisoning by carbon tetrachloride, thioacetamide, and dimethyl nitrosamine (Rees and Sinha, 1960; Rees et al., 1962) and have been related to progressive liver necrosis (Cornelius et al., 1959, 1963; Rees and Sinha, 1960; Musser et al., 1966). Alterations in liver function as represented by changes in these enzymes occurred in pigs after feeding 450 ppb aflatoxin or more, with the exception of AP activity which became elevated at aflatoxin levels as low as 51 ppb. The individual pigs of the 810 ppb group, in which elevated serum AP declined after 98 days, were consistently found to have greater severity of liver involvement as determined microscopically (Gagne, personal communication). This decline in serum AP activity after initial elevation was also observed for calves fed 2400 ppb aflatoxin, a level that was eventually fatal (Allcroft and Lewis, 1963). Other evidence of developing liver damage was given by decreased serum albumin concentration and A/G ratio in the 8 10 ppb group after 42 days and was later apparent

402

GUMBMANN

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WILLIAMS

at 450 ppb or more. Alterations in pig serum protein, including decreased albumin, have also been reported to occur after 75 days of feeding toxic peanut meal containing 7501500 ppb aflatoxin Bi (Annau et al., 1964). Consistent with the ability of aflatoxin to inhibit or otherwise alter protein synthesis (Clifford and Rees, 1967; Shank and Wogan, 1966) was the decrease in concentration of urea in blood, the chief end product of protein metabolism, and the marked decrease in liver nitrogen content at autopsy. These changes in blood took place early in the feeding trial at 8 10 ppb aflatoxin and were later evident at 51 ppb. Acute poisoning in the rat by the pyrrolizidine alkaloid, lasiocarpine, and by carbon tetrachloride has been shown by Gallagher (1961) to bring about a sharp rise of &so value in serum accompanied by a fall of EZ6,, value in liver after only a few hours. In contrast, the subacute poisoning of pigs by aflatoxin as observed in the present study resulted in consistently lowered serum EZ6,, values, which first occurred early in the feeding trial and at 450 ppb aflatoxin or more. The same effect has been obtained with chickens fed 3060 ppb aflatoxin for several weeks (Platonow, 1965b). Liver Ez6,, values in pigs became significantly elevated at the intermediate aflatoxin levels, 450 and 615 ppb, and fell only at 8 10 ppb. Although the small increase in liver lipid observed at the end of trial 1 appeared to be related to the ingestion of aflatoxin, it was not observed histologically and did not represent the pathologic condition of fatty degeneration commonly associated with more severe poisoning by aflatoxin or other hepatotoxins (GagnC et al., 1968). At the end of trial 2, significant decreases in liver lipid were clearly indicated in 2 of the 3 groups receiving aflatoxin. Whether temporary elevation occurred earlier in the experiment as observed in the case of chickens fed 1500 ppb aflatoxin (Carnaghan et al., 1966) is not known. Pigs fed toxic peanut meal of unspecified aflatoxin content but of general greater toxicity than the diets of the present feeding trials consistently accumulated more liver lipid thandidcontrols(Harding et al., 1963). Inspection of these data revealed, however, that the greatest liver lipid concentrations were measured in pigs autopsied between 29 and 61 days, lower levels being found before and after this period. Aflatoxin at 450 ppb or more brought about decreased storage of liver glycogen and vitamin A. Such an effect on vitamin A has been reported for chickens (Carnaghan et al., 1966) calves (Allcroft and Lewis, 1963), and also pigs (Harding et al., 1963) and is characteristic of chronic liver damage (Haig and Post, 1941). Decreased glycogen, also observed in ducklings and to a lesser extent in rats after multiple sublethal doses of aflatoxin Bi (Shank and Wogan, 1966), may be related in part to feed consumption, which showed small but progressive decrease with increasing aflatoxin levels beginning at 450 ppb (Hintz et al., 1967a). In general, the biochemical changes occurring at 450 ppb aflatoxin and above were found to be closely related to the degree of liver pathology, as graded by GagnC et al., (1968). Those changes detected at lower levels may be regarded as adaptive responses to continued aflatoxin ingestion which may or may not have developed into observable liver lesions. ACKNOWLEDGMENTS Thanks are expressedto Mrs. Paulette Hendersonfor her valuable technical assistance during theseinvestigationsand to Miss Linda C. Whitehand, who generouslyaided in the statisticalcalculations.

BIOCHEMICAL

EFFECTS

OF AFLATOXIN

403

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CORNELIUS, C. E., BISHOP,J., SWITZER,J., and RHODE,E, A. (1959).Serumand tissuetransaminaseactivities in domesticanimals.Cornell Vet. 49, 116-126. CORNELIUS, C. E., DOUGLAS,G. M., GRONWALL,R. R., and FREEDLAND, R. A. (1963).Comparative studieson plasmaarginaseand transaminasein hepatic necrosis.Cornell Vet. 53, 181-191. CUTHBERTSON, W. F. J., LAURSEN, A. C., andPRATT,D. A. H. (1967).Effect ofgroundnut meal containingaflatoxin on cynomolgusmonkeys.Brit. J. Nutr. 21,893-908. CYSEWSKI, S. J., PIER,A. C., ENGSTROM, G. W., RICHARD,J. L., DOUGHERTY, R. W., and THURSTON, J. R. (1968).Clinical pathologic featuresof acute aflatoxicosisof swine.Am. J. Vet. Res.29, 1577-1590. DUNCAN,D. B. (1955).Multiple rangeandmultiple F tests.Biometrics11,142. EMBREE,N. D., Ams, S. R., LEHMAN,R. W., and HARRIS,P. L. (1957). Determination of vitamin A. In: Methods of BiochemicalAnalysis (D. Glick, ed.), Vol. 4, pp. 89-90. Wiley (Interscience),New York. GAGN~,W. E., DUNGWORTH, D. L., and MOULTON,J. E. (1968).Pathologiceffectsof aflatoxin in pigs.Pathol. Vet. 5,370-384. GALLAGHER, C. H. (1961).The effect of hepatotoxins on E260valuesof liver and of serum. AustralianJ. Exptl. Biot. Med. Sci. 39, 323-332. GUBLER, C. J., LAHEY, M. E., ASHENBRUCKER, H., CARTWRIGHT, G. E., and WINTROBE, M. M. (1952).Studieson coppermetabolism.I. A methodfor the determinationof copperin whole blood, red blood cells,and plasma.J. Biol. Chem.196,209-220. HAIG, C., and POST, J. (1941).Vitamin A concentrationin rat liver during recovery from CCL, cirrhosis.Proc. Sot. Exptl. Biol. Med. 48, 710-714. HARDING,J. D. J., DONE,J. T., LEWIS,G., and ALLCROFT, R. (1963).Experimentalgroundnut poisoningin pigs.Res.Vet. Sci. 4,217-229. HINTZ,H. F., BOOTH,A. N., CUCULLU, A. F., GARDNER, H. K., and HEITMAN,H., JR. (1967a). Aflatoxin toxicity in swine.Proc. Sot. Exptl. Biol. Med. 124,266-268. HINTZ,H. F., HEITMAN,H. JR., BOOTH,A. N., and GAGN&W. E. (1967b).Effectsof aflatoxin on reproduction in swine.Proc. Sot. Exptl. Biol. Med. 126, 146-148. JUSKIEWICZ, T., STEC,J., STEFANIAK, B., RAKALSKA,Z., and MADEJSKI,Z. (1967).Biochemical and pathologicaleffectsof aflatoxin poisoningin ducklings. Vet. Record.81,297-298.

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