Sulfobromophthalein sodium retention and morphological liver damage in dogs

TOXICOLOGY

AND

APPLIED

PHARMACOLOGY

5, 650-660

Sulfobromophthalein

Sodium

Morphological Departments Therapeutics

(1963)

Liver

Retention

Damage

and

in Dogs

D. HALLESY AND K.-F. BENITZ Pharmacology and Experimental

of Chemical Research, Lederle

Laboratories Pearl River, Received

Division, New York

January

14,

Pathology, Experimental American Cyanamid Company,

1963

The value of BSP retention as a test of liver function in man has been well established since it was first introduced by Rosenthal and White (1925). A significant correlation between elevated BSP retention values and diffuse liver cell damage in man was shown by Popper et al. (1949). The use of this liver function test to screen for the toxic effects of chemicals and drugs in man has been suggested by Popper and Schaffner (1957). Therefore, it seems desirable and logical to use the same liver function tests in laboratory animals as those used to detect harmful drug effects in man. Smith et al. (1940) could not establish a correlation between histologic changes in the liver and BSP retention in their experiments with cats and rabbits using selenium as a toxic agent. Only a few studies in dogs such as those published by Svirbely et al. (1946) and Gornall and Bardawill (1952) could document the usefulness of BSP determinations and establish a correlation between elevated BSP values and morphological liver changes. More recently, Poutsiaka et al. (1962) have shown that chemically induced regressive changes in the liver may be detected by rising BSP values as early as 2 to 5 days. The purpose of this report is to summarize observations which were made in our laboratories during the past six years in beagle dogs in order to establish the normal range of BSP retention for normal untreated beagles in our colony and to correlate pathologic liver changes with abnormal BSP retention values in drug-treated animals. MATERIALS AXD METHODS A total of 97 dogs, 50 males and 47 females, were used in this study. All these dogs were beagles, bred and reared in our colony; they ranged 650

BSP

RETENTION

AND

LIVER

MORPHOLOGY

651

in age from 12 to 40 months, the mean age being 21 months. All were immunized against distemper, rabies, and infectious hepatitis before they were 6 months of age. They were housed in windowless, air-conditioned quarters held at a temperature of 24.0” & l.O”C. Artificial illumination was automatically controlled 11 hours on and 13 hours off. The dogs were fed a 2.5: 1 mixture of dog food’ and Goff’s wet-packed, canned horsemeat. This diet was supplemented with 990 IU of vitamin A and 123 IU of vitamin D. Water was offered ad libitum. The drugs that were given to animals used for this study belonged to the following classes: antibacterial agents, tranquilizers, and glucocorticoids. Two dogs received carbon tetrachloride. All animals were fasted overnight prior to the test. An intravenous dose of 10 mg/kg of BSP was administered, and serum samples were taken exactly 30 minutes later. BSP retention values were determined using the method of Seligson et al. (1957). All values of the untreated control animals and all predose determinations of the drug-treated animals were used to calculate the mean and the standard deviation. Two animals, 1 male and 1 female, had to be excluded from this part of the study since they were experimental animals without pretest BSP determinations. Therefore, only 95 animals were used to establish the normal range of BSP retention values. All livers were examined grossly and weighed; representative samples were fixed in appropriate fixatives. Paraffin sections were stained with hematoxylin and eosin and, if necessary, with trichrome and periodic acidSchiff (PAS) methods. In some cases frozen sections stained with Sudan IV or Oil Red 0 and hematoxylin were also used for the histologic examination. RESULTS The means and ranges of BSP retention values of 95 normal untreated dogs are presented in Table 1. The inspection of these data shows that there was no difference between male and female beagles. The upper range of the 3 standard deviations was fairly close to the actual range. For further evaluation the assumption was made that any value above 7% would be suspicious or even indicative of morphologically detectable liver damage. The frequency distribution curve for these values is presented in Fig. I. As may be seen from this figure, these values approximate a normal diS1 Ken-L-Meal@,

Quaker

Oats

Company

652

MEANS

D. HALLESY

AND

RANGES

AND

K.-F.

TABLE

1

OF SULFOBROMOPHTHALEIN

SODIUM

OF 95 NORMAL

Number of animals

Sex Males Females Males and females combined

Number of BSP determinations

BENITZ

UNTREATED

Mean

(BSP)

RETENTION

VALUES

DOGS

(%)

SD (%,

49

84

2.9

-c 0.92

46 95

82 166

3.1 3.0

-c 1.14 * 1.03

Range of 3 SD (%) 0.1-5.7 CM.5 1X6.1

Actual range (%) 1.0-5.7 0.7-7.0 0.7-7.0

tribution with a tendency to be skewed in the direction of increasing BSP retention. There were only 3 animals (D60-M, G35-M, and E81-M) in which the liver was grossly and microscopically normal whereas the BSP retention values showed a slight increase over the presumptive upper normal

0

2 BSP

FIG.

1.

Frequency

distribution

4 RETENTION

of BSP retention

6

8

&I

values

in normal

male

and female

beagle dogs. range. These animals had been subjected to drug treatment and their BSP values prior to autopsy were 8.3%) 8.6%, and 7.8%. These observations indicated that the statistical range of the 3 standard deviations did not present a hard and fast borderline since values between 770 and 9% are occasionally associated with normal hepatic structures. Twelve of 97 animals had normal BSP values but showed pathologic

BSP

RETENTION

AND

LIVER

MORPHOLOGY

653

conditions in the liver as listed in Table 2. Only 4 of the 12 animals showed abnormal increasesof the absolute organ weights (normal ranges: males 213-398 g, females 212432 g). The relative weights were found to be abnormally high in 4 dogs. This was partly caused by the increase in organ massand partly by loss of body weight. Seven of the 12 beagles had morphologic changes which did not affect the liver cord cells as such. These animals showed focal changes or storage phenomena in Kupffer cells which are known to be of no consequenceto the excretory and metabolic functions of the liver. It is not surprising that single liver cell necroseswhich were found in dog K86-F did not lead to an elevated BSP retention since the vast majority of the liver cord cells was still intact. Although the slight vacuolization caused by the increased storage of glycogen which was found in K74-F represents a deviation from the statistical norm, one cannot regard these changes as a serious, harmful drug effect since the storage of glycogen is a normal function of the liver cord cell. Occasionally a slightly increased storage of glycogen has been observed in well-nourished beagles of our colony, and one could therefore consider an increased glycogen content of hepatic cells as normal variation. However, since the animal K74-F received a glucocorticoid, this glycogen storage phenomenon was considered a drug-induced change. The slight fatty infiltration of the lobular centers in the animals D47-M and K72-F was not associatedwith any other regressivechangesof the hepatic cord cells, a morphological indication that these cells apparently maintained their functional ability. The hydropic vacuolization of hepatic cells in dog K84-M was regarded as a drug-induced change since qualitatively similar but quantitatively more pronounced changes were seen in other dogs receiving higher dose levels of this particular compound. The vacuolization, however, was not severe enough to alter the BSP retention value prior to sacrifice. Animals with elevated BSP retention values and extensive pathologic changesof the liver cord cells are listed in Table 3. All these animals had normal BSP retention values prior to drug treatment with the exception of K60-F and D14-M on which no pretreatment determinations were done. All animals showed elevated BSP retention values except for K43-F ( 10%) and G69-M (8%)) which one has to consider as borderline values. All livers of these animals showed morphologic abnormalities: 4 of the absolute liver weights were increased and 9 relative weights were also abnormally high (see normal ranges mentioned previously). Only 3

6.5 3.0 3.5 2.2 4.7 5.3

4.9

K74-F E23-M” D4S-Mb D47-M K72-F E86-F

E83-M

63.1”

28.9 31.3 27.4 32.2 32.6 60.5

385 354 323 373 222 375

410a

44.1’”

58P

50.P 28.3 27.8 35.4

Rel. k/W

446a 289 320 44Za

weights

Liver

Abs. cd

increased weights. control animal.

3.2

K84-M

a Abnormally b Untreated

4.8 2.9 1.6 2.6

K103-M DS6-M D54-M K86-Fb

Animal no. and sex

BSP value prior to autopsy (%I

MORPHOLOGICAL

Normal

Distinct yellow cast Normal Normal Normal Yellow Yellow Normal

Normal Normal Normal Normal

Gross findings

CHANCES OF

findings

BEAGLES ASSOCIATED VALUES WITH

N~RM.~L

Marked siderosis of Kupffer cells Small scattered foci of lymphocytes, macrophages, and granulocytes Small scattered foci of lymphocytes, macrophages, and granulocytes Several focal accumulations of lymphocytes and mesenchymal elements sometimes associated with single liver cell necroses 1. Scattered focal collections of lymphocytes and fibroblasts occasionally surrounding small fragments of worm cuticle 2. Hydropic vacuolization of liver cord cells Slight vacuolization of liver cord cells; vacuoles contain glycogen Focal lymphocytic infiltration around bile ducts Chronic inflammatory focus in a portal tract Slight centrilobular fatty infiltration Slight centrilobular fatty infiltration Slight siderosis of Kupffer cells; perivascular infiltrations consisting of mesenchymal cells, granulocytes, plasma cells, and multinucleated giant cells Slight siderosis of Kupffer cells

Microscopic

RETENTION

TABLE 2 12 MALE AND FEMALE

SULFOBROMOPHTHALEIN

IN LIVERS

Drug

induced

2. Drug induced Drug induced Spontaneous Spontaneous Drug induced Drug induced Drug induced

1. Spontaneous

Drug induced Spontaneous Spontaneous Spontaneous

Classification of lesions

@ 3 N

5 2: $

F E CE *

BSP

RETENTION

AND

LIVER

MORPHOLOGY

655

animals had livers that appeared normal on gross examination, Microscopic studies revealed that the livers of all animals had widespread regressive changes up to the point of necroses, occasionally associated with some unspecific inflammatory reactions. On the basis of our past experience all these regressive liver cell changes had to be regarded as drug induced since they were never observed in untreated control animals, Since the final BSP determination of dog D40-F was done when the animal was already moribund, one could speculate that the hepatic circulation was already insufficient, thus affecting the BSP retention. There is also some discrepancy between the negative gross findings and the severe centrilobular fatty infiltration that was found microscopically. It is reasonable to assume, however, that postmortem autolysis hampered the finer morphological evaluation of these changes. DISCUSSION

The usefulness of the BSP liver function test in dogs has been reviewed recently by Hoe (1960). Drill and Ivy (1944) used the BSP test in dogs that were poisoned with carbon tetrachloride, but these authors used 5 mg BSP per kilogram body weight. They concluded from their studies that this liver function test was very sensitive, but the livers of these animals were not examined morphologically. Svirbely et al. (1946) have used the BSP retention to study xylidene poisoning of dogs. Retention values up to 15% were considered normal by these authors whereas 1 S207~ were regarded as suggestive of liver damage and values above 20% were considered definitely abnormal. These authors found a good correlation between elevated BSP values and the hepatotoxic effects of xylidene inhalation, which consisted of fatty metamorphosis, precirrhotic changes, and cirrhosis. Gornall and Bardawill (1952) used 10 mg BSP/kg and collected blood samples after 15 minutes. Using carbon tetrachloride as a hepatotoxic agent, these investigators established a relationship between BSP retention and anatomical liver damage. Cornelius (1958) suggested using a dose of 10 mg BSP per kilogram body weight for dogs. However, this author was not able to correlate the retention of the dye with a specific type of lesion. On the basis of the results presented in Table 3 one can state, however, that any diffuse and severe hepatic cell damage is associated with an increase of dye retention, This is true for cases with widespread liver cell necroses, but one can also consider regressive changes, such as severe fatty infiltration and hydropic vacuolization, as necrobiotic processes that may ultimately result in hepatic necroses.

29.5

23.0

15.5

10.0

12.2

K60-F

D14-M

DS2-F

K43-F

KlOO-M

Animal no. and sex

BSP value prior to autopsy (%)

606a

727”

292

323

61 .P

54.3n

26.1

37.1a

40.0a

Rel. k/W

265

weights

Liver

CHANGES

Ahs. k)

MORPHOLOGICAL

findings

Distinct

Distinct

Normal

yellow

yellow

cast

cast

Very soft and brittle, diffuse yellow discoloration Diffuse yellow discoloration

Gross

3 WITH

focal liver cell necroses with and histiocytic infiltration

findings

ASSOCIATED

Microscopic

BEAGLES VALUES

granulo-

ELEVATED

Hemorrhagic liver cell necroses with granulocytic and histiocytic infiltration; fatty infiltration of liver cells Marked glycogen depletion with areas of fatty and hydropic vacuolization; scattered foci of lymphocytes, granulocytes, and macrophages Scattered focal collections of lymphocytes and fibroblasts occasionally surrounding small fragments of worm cuticle; hydropic vacuolization of liver cord cells Pronounced vacuolization (glycogen storage?) of liver cells resulting in considerable cellular distortion and enlargement; no fat in vacuoles

Multiple cytic

AND FFXALE RETENTION

TABLE IN LIVERS OF 11 MALE SULFOBROMOPHTHALEIN

Drug

Drug

induced

induced

induced

induced

Drug

Drug

induced

Drug

Classification of lesions

2

$ n 8

E

$

k 2

E

P

12.6

11.4

29.0 2 1 .o

D57-M

D34-F

D40-F E44-M

46.8”

318

485” 323

weights.

48.0a 52.1a

46.3n

30.6

331

264

98.0”

(g/k)

Rel.

weights

735a

Abs. (!a

Liver

increased

8.0

G69-M

IJ Abnormally

16.6

K31-F

Animal no. and sex

BSP value prior to autopsy (%) yellow

findings cast

Normal Moderate yellowbrown surface with hemorrhages on ventral surface, 36 mm in diameter

Yellow

Yellow

Normal

Distinct

Gross

TABLE

Microscopic

findings

Pronounced vacuolization (glycogen storage?) of liver cells resulting in considerable cellular distortion and enlargement; no fat in vacuoles Numerous focal liver cell necroses; maximal diameter approximately 400 p Severe centrilobular fatty infiltration ; moderate amount of bile plugs; slight chronic inflammatory cell infiltrates Severe centrilobular fatty infiltration; moderate amount of bile plugs; slight chronic inflammatory cell infiltrates Severe centrilobular fatty infiltration; autolytic Numerous bile plugs; complete glycogen loss; increased nuclear variability with discharge of nucleoli; one necrotic area

3 (Continued)

induced

Drug

Drug Drug

induced induced

induced

induced

Drug

Drug

induced

Drug

Classification of lesions

658

D. HALLESY

AND

K.-F.

BENITZ

Popper and Schaffner (1957) have stated that human cases of acute hepatitis, cirrhosis, extrahepatic biliary obstruction, hepatic tumor metastases, and chronic passive congestion of the liver result in 79-930/O of the cases in abnormal BSP retention. Table 4 shows the correlation of BSP retention values with normal and pathologic hepatic structures. The BSP values were divided into three groups: normal, borderline, and definitely elevated values. The TABLE CORRELATION

OF

SULFOBROMOPHTHALEIN MORPHOL~CY

Normal livers

BSP Values Normal values (< 7%) Borderline values (F-10%) Elevated values (>lO%) a Represents b Represents C Represents

correct incorrect doubtful

71a

(73.2%) 3~ (3.1%) 0

4 (BSP)

RETENTION

IN 97 MALE

AND FEMALE

Regressive

hepatocellular

Slight 3c (3.1%) 0 0

VALUES

WITH

LIVER

Does changes Severe 2b

(2.1%) (2.:;) 95

Other changes 7a (7.2%) 0 0

(9.3%) predictions, predictions, predictions,

total = 89.7%. total = 2.1%. total = 8.3%.

regressive hepatocellular changes were divided into two categories: minor degrees of hydropic vacuolization, glycogen storage, and fatty infiltration were classified as slight whereas necrobioses and necroses of the liver cord cells were regarded as severe. Marked degrees of fatty infiltration, vacuolization together with bile plugs, and cellular reactions were also put in this group. Pathologic structures which did not affect the liver cord cells proper, for instance, siderosis of Kupffer cells or scattered cellular infiltrations, were summarized under other changes. Twenty-three out of 97 animals showed pathologic conditions in the liver. As shown in Table 4 correct predictions from normal or definitely elevated BSP retention values could be made in 89.7%. The incorrect predictions totaling 2.1% were found in animals with normal BSP retention values, yet the hepatic structures of these dogs showed severe morphological damage. Finally, there was a small group of animals showing borderline BSP retention values associated either with normal livers or slight or severe regressive hepatocellular changes. BSP retention values above 10% were always associated with severe degrees of liver damage.

BSP

RETENTION

AND

LIVER

MORPHOLOGY

659

In summary, there was a positive correlation between BSP retention values and liver morphology in 87/97 cases (89.7%). We therefore conclude that the BSP retention test is a useful tool for the early detection of severe hepatotoxic effects of chemicals and drugs, and elevated values should always direct the attention of the pathologist toward the liver especially in respect to sampling, application of multiple fixatives, special stains, and histochemical analysis. SUMMARY The normal range of sulfobromophthalein (BSP) retention values was determined in 49 male and 46 female beagle dogs 30 minutes after a single injection of 10mg BSP per kilogram body weight. All livers of these animals were examined grossly and microscopically and were found to be normal. The mean value of 166 determinations was 3.0% (SD & 1.03%). There was no difference between males and females. Hepatotoxic effects of drugs (antibacterial agents, tranquilizers, and glucocorticoids) resulted in elevated BSP retention values which were aIways associated with diffuse, regressive changes of the hepatic parenchyma. Correct predictions (normal BSP retention values below 7% associated with normal hepatic structures and elevated BSP retention values above 10% associated with severe, regressive hepatocellular changes) were found in 89.7% of the animals examined. Incorrect predictions (normal BSP retention values associated with liver damage) were found in 2.1%. One can therefore recommend the use of the BSP liver function test as a useful aid for the detection of drug-induced liver damage in dogs. REFERENCES C. E. (1958). The diagnosis of liver disease in the dog and the horse. Iowa State Coil. Vet. 20, 155-162. DRILL, V. A., and IVY, A. C. (1944). Comparative value of bromsulphalein, serum phosphatase, prothrombin time, and intravenous galactose tolerance test in detecting hepatic damage produced by carbon tetrachloride. J. Clin. Invest. 23, 209-216. GORNALL, A. G., and BARDAWILL, C. J. (1952). The study of liver function in dogs. Can. J. Med. Sci. 30, 256-271. HOE, C. M. (1960). Tests for liver function in domestic animals. Vet. Rev. Annotations 6, (Pt. I), 1-26. POPPER, H., and SCHAFFNER, F. (19.57). Liver: Strzccture and Function, p. 371. McGraw-Hill, New York. POPPER, H., STEIGMANN, F., MEYER, K. A., KOZOLL, D., and FRANKLIN, M. (1949). Correlation of liver function and liver structure: clinical applications. Am. J. Med. 6, 278-291. POUTSIAKA, J. W., KEYSSER, C. H., THOMAS, B. G. H., and LINEGAR, C. R. (1962). Simultaneous determination in dogs of liver and kidney functions with bromosulfalein and phenolsulfonephthalein. Toxicol. AppZ. Pharmacol. 4, 55-69. ROSENTHAL, S. M., and WEIITE, E. C. (1925). Clinical application of the bromsulphalein test for hepatic function. J. Am. Med. Assoc. 64, 1,112-1,114. CORNELIUS,

660

D. HALLESY

AND

K.-F.

BENITZ

D., MARINO, J., and DODSON, E. (1957). Determination of sulfobromophthalein in serum. Clin. Chew. 3, 638-645. SMITH, M. I., LILLIE, R. D., STOHLMAN, E. F., and WESTPALL, B. B. (1940). Studies in chronic selenosis. Natl. Inst. Health Bull. 174, 21-44. SVIRBELY, J. L., MONACO, A. R., and ALFORD, W. C. (1946). The comparative efficiency of various liver function tests in detecting hepatic damage produced in dogs by xylidene. J. Lab. Clin. Med. 31, 1133-1143. SELIGSON,