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Relative effects of various chlorinated hydrocarbons on liver and kidney function in dogs
rOXICOLOGY
AND
Relative
APPLIED
PHARMACOLOGY
Effects liver
10,
of Various and
Chlorinated
Kidney
CURTIS D. KLAASSEN~ Department
of Pharmacology, Iowa Received
(1967)
119-131
College City,
Function
Hydrocarbons in Dogs1 L. PLAA
AND GABRIEL of Medicine, Iowa 52240
October
on
The
University
of Iowa,
27, 1966
The altered morphology of the kidneys and liver produced by some of the chlorinated hydrocarbons has been studied quite extensively (Von Oettingen, 1964). However, comparisons between the various derivatives have been largely descriptive in nature. The reason for this is due in large part to the fact that the data obtained in these many studies were not readily translatable into the quanta1 dose-response data needed for quantitative comparisons. In the past few years, quantitative comparative studies have been made in mice after converting the various indices of hepatic function into dose-responsedata ( PIaa et al., 1958; Kutob and Plaa, 1962; Klaassen and Plaa, 1966). It has also been shown (Plaa and Larson, 1965) that the relative nephrotoxic properties of various chlorinated hydrocarbons could also be compared using phenolsulfonephthalein excretion and analyzing the data along similar lines. The dose-response data used in these comparisons are of the all-or-none type. These quanta1 data are usually handled by the probit method as described by Finney ( 1952) or the method of Litchfield and Wilcoxon ( 1949). Both of these methods suffer from the disadvantage that large numbers of animals are needed and, therefore, are not too practical for determining LDsO and organ dysfunction EDsO values in dogs. The “up-and-down” method of Brownlee et al. ( 1953) employs few animals. The purpose of the present study is to evaluate this method to estimate the median lethal (LD50) and organ dysfunction ( ED!x) values for a series of chlorinated hydrocarbons in dogs. Since it has been demonstrated that mice (Klaassen and Plaa, 1966) and rats ( Cornish and Adefuin, 1966) exposed both to ethanol and various chlorinated hydrocarbons are more susceptible to the liver-damaging properties of these substances, the effect of ethanol on the liver dysfunction induced by these hydrocarbons was also studied. METHODS
The following hydrocarbons were employed: dichloromethane, chloroform, carbon tetrachloride, l,l,l-trichloroethane, 1,1,2-trichloroethane, trichloroethylene, 1 Preliminary presentation of these data was made at the Fall Society for Pharmacology and Experimental Therapeutics, Mexico 1966. ’ U.S. Public Health Service Predoctoral Fellowship 6M-30996. 119
Meeting of the American City, Mexico, July 14-21,
120
CURTIS
D.
KLAASSEN
AND
GABRIEL
L.
PLAA
and tetrachloroethylene. All agents were made up in corn oil and administered intraperitoneally to mongrel male and female dogs weighing 7-14 kg. Lethality. The median lethal dose ( LDGo) was estimated by the method of Brownlee et al. (1953) 24 hours after the hydrocarbon was administered intraperitoneally. Lizjer fun&on. Liver function was assessed by determining the serum glutamic-pyruvic transaminase (SGPT) activity by the method of Reitman and Frankel ( 1957). The blood sample was obtained in a heparin-rinsed syringe from each dog 24 and 48 hours after the hydrocarbon was administered. A O.l-ml aliquot of plasma was added to 0.5 ml of alanine-ketoglutaric acid reagent. This mixture was incubated for 30 minutes at 40” on a module heater,” then 0.5 ml of the 2,4-dinitrophenylhydrazine reagent was added. Thirty minutes later, 5 ml of 0.4 iV NaOH was added; the absorbance of this solution was read at 505 rnp 30 minutes after the addition of NaOH. Kidney function. The dogs were anesthetized with intravenously administered pentobarbital sodium, and the ureters were cannulated. Saline (25 ml/kg) was given to the dogs intravenously over a 30-minute period. Phenolsulfonephthalein (PSP) was then administered intravenously at a dosage of 3 mg/kg (Poutsiaka et al., 1962) and the urine was collected for 30 minutes from the cannulated ureters. The urine was diluted with 0.1 N NaOH, and the concentration of PSP was determined spectrophotometrically, using the absorbance at 560 mp. Ethanol intoxication. Ethanol was diluted to 50% and administered by gavage. The dose of ethanol (4 g/kg) resulted in an observable central nervous system depression (primarily ataxia). The control dogs received an equicaloric amount (20 ml/kg) of 36% dextrose solution. Histopathology. Kidney and liver tissues were removed from the surviving dogs used in the LDso studies and fixed in 10% formalin. These tissues will be referred to as those from dogs administered hydrocarbons at the “near LDEo dose.” The tissues from 3 or 4 dogs were obtained for each compound. Tissues were also removed from the dogs used in the ED- 50 studies. These tissues, therefore, are from dogs that received doses both above and below the calculated EDao dose, and will be referred to as the tissues from dogs administered hydrocarbons at the “near ED,,, dose.” Liver samples from the ED,,, studies based on SGPT elevation were taken 48 hours after the administration of the hydrocarbon. In the PSP excretion EDso studies, the kidneys were removed 24 hours after the administration of the hydrocarbon. Livers were also removed from the dogs that were given threequarters of an LD 50 of the hydrocarbon after the SGPT values returned to normal values. Liver and kidney morphology was assessed after staining with hematoxylin and eosin. Statistics. In order to evaluate the method of Brownlee et al. (1953), the LD,,, and liver dysfunction ED ho values for chloroform and carbon tetrachloride in mice derived by this method were compared to those obtained using the method of Litchfield and Wilcoxon (1949). The ED,, values were calculated after the functional data were transformed to the all-or-none responses. ” “Temp-Blok,”
obtained
from
Lab-Line
Instruments,
Melrose
Park,
Illinois.
CHLORINATED
HYDROCARBONS
AND
ORGAN
FUNCTION
121
After finding the method quite suitable for the purposes of this study, the “upand-down” method of Brownlee et al. (1953) was used to estimate the 24-hour LDb0 values and the organ dysfunction ED 5o values as measured by PSP and SGPT. This method consists of giving a dose of a compound to one animal and noting the presence or absence of an effect. If it has an effect, one decreases the dose and repeats the trial on a second animal. If it does not have an effect, one increases the dose and repeats the trial. This is repeated for 3 times after one has obtained one positive and one negative response in two successive doses. The increment in dosage used was a 40% increase of the first negative response, as suggested by Deichmann and Le Blanc ( 1943). A formula is used to determine the EDs0 or LDsO. Thus, one can obtain an estimate of the potency ratio (PR) by making a ratio of the LD 50 to the EDs0 but cannot test the significance of this PR statistically. The ethanol-pretreated dogs were compared to the dextrose dogs by the MannWhitney U test as described by Goldstein ( 1964). RESULTS
Evaluation of the “Up-and-doum” Method Mice were used to compare the LDjo and EDso values obtained by the method of Brownlee et al. (1953) to those obtained by the method of Litchfield and Wilcoxon. The 24-hour LDso and the liver dysfunction EDs0 as measured by SGPT were both measured after intraperitoneal injection of the test compounds. The results are shown in Table 1. The LDso values by the method of Brownlee et al. for chloroform and carbon tetrachloride were I.2 and 2.5 ml/kg, respectively, and 1.1 (0.9-1.3) and 2.7 (2.4-3.0) ml/kg, respectively by the method of Litchfield and Wilcoxon. The liver dysfunction as measured by SGPT is shown in Table 1. The EDEOvalues for liver dysfunction for chloroform and carbon tetrachloride by the method of Brownlee et al. were 0.18 and 0.008 ml/kg, respectively, and those obtained by the method of Litchfield and Wilcoxon were 0.17 (0.14-0.21) and 0.0092 (0.0058-0.015) ml/kg, respectively. Since the LDso and organ damage EDzO as estimated by the method of Brownlee et al. did not fall outside the confidence limits determined by the method of Litchfield and Wilcoxon, the former method was used for estimating the LDB, and organ dysfunction ED,,, values in the dog studies. Lethality Table 2 shows the LD,, values, based on the incidence of mortality within 24 hours, for the halogenated hydrocarbons tested. In dogs as in mice (Klaassen and Plaa, 1966), the least potent hydrocarbon tested as far as death is concerned was 1,&l-trichloroethane, and its isomer, 1,1,2-trichloroethane, was the most potent. Liver Function In control dogs it was found that the SGPT activity was 36 =I=(SD) 7 units. Thus, a value of 50 units (36 + 2 SD) was chosen as the upper limit of the nor-
tetrachloride
tetrachloride
Dose (ml/kg) Response” Dose (ml/kg) Response”
Dose (ml/kg) Response” Dose (ml/kg) Responseb
0.14 N 0.011 E
determination 0.20 E 0.008 N
0.14 N 0.011 E
0.95 S 3.3 D
LDso determination 0.95 1.35 S D a.3 3.2 S D ED,
Mouse 3
at 44 hours
0.20 E 0.008 E
1.35 D 1.4 S
Mouse 4
after
IN MICE
the dose.
0.14 N 0.005 N
0.95 S 12.3 S
Mouse 5
TABLE 1 OF SERUM GLUTAMIC-PYRUVIC TRANSAMINASE ACTIVITY DOSE OF CHLOROFORM OR CARBON TETRACHLORIDE~ Mouse a
Mouse 1
OF THE LDSo AND ED,, FOR ELEVATION AFTER A SINGLE INTRAPERITONEAL
0 The determinations were performed by the method of Brownlee et al. (1953). b Response is indicated by a “D” for death or an “S” for survival at 94 hours after the dose. c Response is indicated by an “N” for a normal SGPT activity, and an “E” for an elevated activity
Carbon
Chloroform
Carbon
Chloroform
Compound
DETERMINATION
0.008
0.18
a.5
I.2
LDb,, or EDso (ml/kg)
m
? g
F r
2 E
i
$ * B 2
P
a Response
Tetrachloroethylene
Trichloroethylene
l,l,%Trichloroethane
is indicated
tetrachloride
l,l,l-Trichloroethane
Carbon
Chloroform
Dichloromethane
Compound
an “S”
by a “D”
for death
for survival
S 0.35 S 2.0 D 3.0 D
Response Dose (ml/kg) Response Dose (ml/kg) Response
Dose (ml/kg) Response
D 1.4 S a.5
e
at $24 hours
1.46 S
S a.55 D
D 2.0 S after
a.0
S 1.45
S
D 1.45 S
the dose.
Dog 4 1.25 D 1.00 S 1.8 D 3.5 D 0.45
CRLORINATED
S 0.35
1.00 S 0.75 s 1.4 S a.5
3
Erg
OF VARIOUS
D 0.46
D 3.5
1.8
0.75 S 0.50 S
4
Dog
1
Erg
TABLE LDso’s
INTRAPER~TIONAL
Response Dose (ml/kg) Response Dose (ml/kg)
and
ACUTE
1.00 D 0.75
OF THE
Dose (ml/kg) Responsea Dose (ml/kg)
DETERMINATION
S
St.0
S
S 0.55 D 2.0
a.5
1.00 D 1.25 S 1.4 D
5
Dog
HYDROCARBONS
=ho
1.5
1.0
0.95
(ml/W
a.1
1.9
0.45
3.1
FOR DOGS
L&o
a1
%l
4.9
91
16
13
15
bmoWkd
6” 2 =1 2
2 0 :: G
ti 2: v)
3 8
X
8
2
i c!
9
124
CURTIS
D.
KLAASSEN
AND
GABRIEL
L.
PLAA
ma1 range. Any SGPT value greater than 50 units was considered significant and indicative of liver dysfunction. Blood was obtained from the dogs 24 and 48 hours after administration of the hydrocarbon. The results are shown in Table 3. Here, as in mice (Klaassen and Plaa, 1966), there is a great difference in the ability of these hydrocarbons to produce liver dysfunction. Carbon tetrachloride causes liver dysfunction at much lower doses than the other hydrocarbons. Liver
Histopathology
The hydrocarbons could be divided into two classes according to the changes seen with the light ,microscope at near-lethal doses. In the first class one would group carbon tetrachloride, chloroform, and 1,1,2-trichloroethane from which the most severe damage was seen, consisting of centrolobular necrosis. Carbon tetrachloride caused severe centrolobular necrosis, chloroform caused moderate centrolobular necrosis, and 1,1,2-trichloroethane caused mild centrolobular necrosis. All the hydrocarbons caused slight subcapsular necrosis. The second class would consist of dichloromethane, I,I,l-trichloroethane, trichloroethylene, and tetrachloroethylene which, at near-lethal doses, produced moderate neutrophilic infiltrations in the sinusoids and portal areas, but no necrosis. Near the EDs0 all the hydrocarbons, except l,l,l-trichloroethane, caused vacuolization of centrolobular hepatocytes in about half of the animals. l,l,l-Trichloroethane, at the near-ED,, doses caused subcapsular necrosis but no centrolobular lesion. 1,1,2-Trichloroethane caused centrolobular necrosis in this dose range. With chloroform, 2 of the 7 livers examined had necrosis, but with 1,1,2-trichloroethane all livers exhibited necrosis. Severity
and Length
of Duration
of Liver
Dysfunction.
Figure 1 shows the results obtained when single doses of the hydrocarbons (0.75 X LD6,,) were given to the dogs. Each point is the mean obtained from 3 dogs. Carbon tetrachloride caused the greatest elevation of SGPT activity. Although chloroform did not cause as great an elevation in SGPT, the time needed for the values to return to the normal range was about the same time as that needed for carbon tetrachloride. The rest of the hydrocarbons caused less elevation of SGPT than chloroform and carbon tetrachloride. These SGPT values returned to the normal range within 7 to 10 days compared to 17-18 days with chloroform and carbon tetrachloride. After the SGPT values had returned to the normal range, the livers were removed from the dogs and examined histologically. In most cases the livers exhibited moderate vacuolation of the centrolobular and midzonal hepatocytes as well as traces of brown material in the cytoplasm of centrolobular Kupff er cells. Kidney
Function
In control dogs, 56* following administration - 2 SD) was considered was determined 24 hours
(SD) 8.3% of the PSP was excreted within 30 minutes to dogs. Thus, a PSP excretion of less than 39% (56 to be indicative of significant kidney dysfunction. This after the administration of the hydrocarbon. Only dogs
Dog
(1 Response
Tetrachloroethylene
Trichloroethylene
I,l,%Trichloroethane
is indicated
tetrachloride
I,l,l-Trichloroethane
Carbon
Chloroform
by an “E”
for elevation
N
activity
and
E
N of SGPT
0.84
N
0.45
0.60
E
0.6
0.37
N 0.50 E
E
E 0.75
N 1.05
0.019
0.50 N 0.20 E
2
Dog
CHLORINATED
0.014
E 0.15 N
0.75
Dose (ml/kg) Response” Dose (ml/kg) Response Dose (ml/kg) Response Dose (ml/kg) Response Dose (ml/kg) Response Dose (ml/kg) Response Dose (ml/kg) Response
TABLE
3
an “N”
N
for normal
N
0.60
E
0.6
E
0.37
E
1.05
activity
E
0.84
N
0.45
N
0.24
N
0.75
N
0.019
N
0.014
0.20
N
0.50 N
4
Dog
34 hours
after
N
0.60
N
0.6
E
0.37
E
1.05
E
0.024
E
0.25
E
0.75
Dog 5
the dose.
0.74
0.57
0.35
0.87
0.02
0.20
0.60
(ml/W
EDm
IX DOGSBY A SINGLE INTRAPERITONEAL
0.15
E
0.75
Dog 3
HYDROCARBONS
OFSERUM GLUTAMIC-PYRUVICTRANSAMINASE
Dichloromethane
FOR ELEVATION
1
OFTHE ED,,
Compound
DETERMINATION
DOSE
7.2
6.3
3.8
8.6
0.2
2.5
9.4
(mmole/kg)
EDso
OF VARIOUS
8
2
2 z =I
8 5
5
E !z cn
!4
8
$
U
z
B
a Response
Tetrachlorethylene
l,l,%Trichloroethane
Chloroform
Compound
DETERMINATION
is indicated
OF THE
by a “D”
for diminished
Dose (ml/kg) Responsea Dose (ml/kg) Response Dose (ml/kg) Response
EDSo FOR DIMINUTION
excretion
and an “N”
E
1.05
0.56 D 0.40 D
2
0.4 N 0.28 N 1.45 N
Dog
1
4
for normal
excretion.
0.4 D 0.28 N 1.45 N
3
Dog
EXCRETION HYDROC.~RBONS
TABLE
Dog
OF PHENOLSULFONEPHTHALEIN CHLORINATED
0.84 N 0.40 N 1.05 E
4
0.4 N 0.52 D 1.45 N
5
Dog
BY A SINGLE
Dog
IN DOGS
IXTIL~PERITONEAL
1.4
0.40
0.43
DOSE
14
4.3
5.4
EDSO (mmole/kg)
OF VARIOUS
CHLORINATED
HYDROCARBONS
AND
TABLE
ORGAN
127
FUNCTION
5
COMPARISON OF VARIOUS CHLORINATED HYDROCARBONS FOR ACUTE LETHALITY AND THEIR EFFECTS ON PHENOLBULFONEPHTHALEIN EXCRETION AND SERUM GLUTAMIC-PYRUVIC TRANSAMINASE ACTIVITY AFTER A SINGLE INTRAPERITONEAL DOSE T O MICE AND DOGS~ PSP excretion
Species
LDSO (ml/kg)
Dichloromethane
Mouse
1.50
Chloroform
Dog Mouse
0.95 1.9 1.0
Compound
Dog
Carbon
tetrachloride
Mouse Dog
l,l,l-Trichloroethane
Mouse Dog
l,l,‘&Trichloroethane Trichloroethylene Tetrachloroethylene
3.1 0.35
Dog
0.45
Mouse Dog Mouse Dog
a.a 1.9 a.9 9.1
0 The data on mice are reproduced * The “potency ratio” is the ratio
given kidney Kidney
chloroform, disfunction
Potency ratio”
0.078 0.45 -
Potency ratio”
0.60
1.6
15
0.18
6.4 5.0
0.17 0.40
a.3
O.&O
-
0.0098 0.019
-
a.5 0.87
2.1
0.10 0.35
1.1 -
1.4
or
-
-
-
from Klaassen and Plaa of the LDso to the EDw.
1,1,2-trichloroethane, (Tables 4 and 5).
ED@ (dkd
elevation
-
a.7 1.5 3.8
Mouse
SGPT
1.5
280 79 1.5 3.5 3.4 1.3
1.6
1.4
0.57
3.8
2.9
0.98
0.74
2.8
(1966).
tetrachloroethylene
demonstrated
Histopathology
At near-lethal doses all the hydrocarbons caused some alteration of the kidneys. The changes do not appear to be as severe in the dog as those seen in mice (Klaassen and Plaa, 1966). Only with l,l,&-trichloroethane was necrosis observed. With carbon tetrachloride, Bowman’s capsules appeared dilated with some glomerular tufts contracted; calcification of a small number of tubules in the medulla was also seen. The rest of the compounds, chloroform, l,l,l-trichloroethane, dichloromethane, trichloroethylene, and tetrachloroethylene, showed slight calcification of the tubules. At the near-PSP excretion-EDbO doses, only slight changes such as mild dilatation of the collecting ducts were seen in some of the kidneys; the rest exhibited no lesions. Efect
of Ethanol on Hydrocarbon-Induced
Liver Dysfunction
Table 6 shows the results when the dogs were pretreated once with ethanol or dextrose 24 hours prior to the administration of an approximate ED50 dose of the hydrocarbon. The SGPT levels were measured 48 hours later. Ethanol potentiated the SGPT activity produced by carbon tetrachloride, chloroform, and trichloroethylene. However, no significant difference could be demonstrated between the SGPT activity of dextrose and alcohol-treated dogs prior to l,l,ltrichloroethane or corn oil. These results were confirmed by histopathologic
oil vehicle
a Difference
Corn
Trichloroethylene
between
tetrachloride
l,l,l-Trichloroethane
Carbon
Chloroform
Compound
TABLE
6
alcohol-pretreated
Yes No Yes No Yes No Yes NO Yes No
Ethanol
Pretreatment
0.20 0.20 0.024 0.024 0.87 0.87 0.57 0.57 -
and dextrose-pretreated
No Yes No Yes No Yes No Yes No Yes
Dextrose
Hydrocarbon dose (ml/kg)
groups,
P < 0.05
400 28 270 as 33 26 63 23 24 20
Dog 1
(one-tail,
Mann-Whitney
5600 57 8200 61 43 28 107 50 32 29
Dog 4
(units)
U test).
48 82 500 78 43 34
61 -
Dog 5
120 210 580 310 92 43
-
Dog 6
GLUTAMIC-PYRUVIC TRANSAMINASE CHLORINATED HYDROC LRBONS activity
610 42 7300 48 43 27 82 31 31 2s
Dog 3
SGPT
OF SERUM OF VARIOUS
420 39 400 37 34 26 66 24 31 20
Dog 2
EFFECT OF 24-HOUR PRETRE.
No
Yes
No
Yes
Yes
Significanta
CHLORINATED
HYDROCARBONS
AND
ORGAN
FUNCTION
129
*--a Dichloromethane )r.......~Chloroform Carbon Tetrachloride .+..A I, I, I - Tr ichloroet hone I, I ,2 -Trichloroethone G---O Trichloroethylene o-)-a Tetrochloroethylene
2
4
6
8 IO 12 14 I6 DAYS
I8
FIG. 1. Duration of elevation of serum glutamic-pyruvic transaminase (SGPT) after a single intraperitoneal dose of various chlorinated hydrocarbons to dogs. The dosage of each compound was three-quarters of the single intraperitoneal LD,,. Each point on the curves is the mean of the results from three dogs.
examination, in that alcohol pretreatment caused more severe morphologic changes in the dogs treated with carbon tetrachloride, chloroform, and trichloroethylene, but not in those treated with l,l,l-trichloroethane. DISCUSSION
The method of Brownlee et al. ( 1953) was used in this study in order to conserve animals. This method appears to be quite satisfactory for estimating median effective doses. It has the advantage in that it is possible to determine median doses with as few as 5 animals. The values obtained are also quite reproducible. The liver dysfunction ED 5Ovalues as measured by serum glutamic-pyruvic transaminase (SGPT) demonstrate that all the halogenated hydrocarbons tested are capable of producing some degree of liver dysfunction. Table 5 compares the EDso values obtained in mice and dogs and also contains the SGPT-potency ratio (PR). This ratio compares the dose required to produce death to that required
130
CUFtTIS
D. KLAASSEN
AND
GABRIEL
L. PLAA
to produce organ dysfunction. It is felt (Klaassen and Plaa, 1966) that the PR is more informative about the relative capacities of these agents to produce organ dysfunction than is a comparison of ED 50 values alone. The 24-hour LD50 is largely a measure of the deep narcosis caused by these hydrocarbons. Therefore, a large PR indicates a high capacity for producing organ dysfunction, since the dose required to produce this injury is considerably lower than that required to produce deep narcosis. When one looks at the PR values in Table 5, it can be seen that carbon tetrachloride is vastly different from the other hydrocarbons. This table also shows that when the effects of these halogenated hydrocarbons in dogs are compared to those in mice, there is not much of a species difference in the relative capacity of these agents to produce liver dysfunction. However, it should be noted that dichloromethane produces some liver dysfunction in dogs but not in mice. Chloroform and carbon tetrachloride caused the highest elevation following a dose of three-quarters of the LDsO (Fig. 1). Even though carbon tetrachloride caused a peak SGPT elevation 5 times that of chloroform, both values returned to the normal range at about the same time. The rest of the hydrocarbons produced only moderate SGPT elevation which returned to normal levels in 7-10 days, thus demonstrating a difference in the amount of dysfunction produced. The kidney dysfunction phenolsulfonephthalein (PSP) PR values (Table 5) demonstrate that chloroform, 1,1,2-trichloroethane, and tetrachloroethylene can produce kidney dysfunction in dogs only at doses in the near-lethal range. In the mouse (Plaa and Larson, 1965; Klaassen and Plaa, 1966) only chloroform and 1,1,2-trichloroethane produce kidney damage as measured by PSP excretion. However, chloroform seems to have a greater nephrotoxic capacity in mice. It should be noted that carbon tetrachloride did not produce altered PSP excretion; however, some histologic changes were seen in the kidneys of dogs treated with nearlethal doses of all the hydrocarbons. With chloroform and carbon tetrachloride, which produce liver dysfunction at relatively low doses, a marked potentiation occurs when the dogs are pretreated with ethanol. However, with trichloroethylene and l,l,l-trichloroethane, which produce liver dysfunction at relatively high doses, it is difficult to demonstrate a potentiation of the hydrocarbon-induced liver dysfunction by alcohol. This has also been demonstrated in mice (Klaassen and Plaa, 1966) and in rats (Cornish and Adefuin, 1966). Therefore, from a practical standpoint, it would appear that alcohol intoxication would have its most important effect only on subsequent exposures to carbon tetrachloride or chloroform, agents which cause severe alterations in organ function at relatively low doses. SUMMARY The “up-and-down” statistical method was used to estimate the L&o and liver and kidney dysfunction EDEo values in dogs, and serum glutamic-pyruvic transaminase was used to determine hepatic function following intraperitoneal injection of dichloromethane, chloroform, carbon tetrachloride, l,l,l-trichloroethane, 1,1,2-trichloroethane, trichloroethylene, and tetrachloroethylene. Median effective doses for inducing dysfunction were calculated and compared to the lethal dose. Carbon tetrachloride caused elevated transaminase values at much lower doses than the rest of the hydrocarbons. Renal function was tested by urinary phenol-
CHLORINATJZD
HYDROCARBONS
AND
ORGAN
FUNCTION
131
sulfonephthalein excretion, and this test demonstrated that chloroform, 1,1,2-trichloroethane, and tetrachloroethylene produced altered renal function. Enhancement of the hepatic dysfunction produced by the hydrocarbons was assessed using ethanol in intoxicating doses. ACKNOWLEDGMENTS This work was supported by funds from the U.S. Public Health Service (Research Grant AM-05802) and by funds from the Dow Chemical Co., Midland, Michigan. Histologic evaluation of the tissues was performed by Dr. Robert G. Geil, International Research and Development Corporation, Mattawan, Michigan. REFERENCES BROWNLEE, K. A., HODGES, J. L., and ROSENBLAIT, M. ( 1953). The up-and-down method with small samples. Am. Statist. Assoc. J. 48, 262-277. CORNISH, H., and ADEFUIN, J. ( 1966). Ethanol potentiation of halogenated aliphatic solvent toxicity. Am. lnd. Hyg. Assoc. J. 27, 57-61. DEICHMANN, W. B., and LEBLANC, T. J. ( 1943). Determination of the approximate lethal dose with about six animals. J. Ind. Hyg. ToxicoZ. 25, 415417. FINNEY, D. J. ( 1952). Statistical Method in Biological Assay. Charles Griffin, London. GOLDSTEIN, A. ( 1964). Biostatistics: An Introductory Text. ‘Macmillan, New York. KLAASSEN, C. D., and PLAA, G. L. (1966). The relative effects of various chlorinated hydrocarbons on liver and kidney function in mice. Toxicol. Appl. Pharmucot. 9, 139-151. KUTOB, S. D., and PLAA, G. L. ( 1962). A procedure for estimating the hepatotoxic potential of certain industrial solvents. Toxicol. Appl. Phurmucol. 4, 345-361. LITCHFIELD, J. T., JR. and WILCOXON, F. ( 1949). A simplified method for evaluating doseeffect experiments. J. Pharmacol. Exptl. Therup. 96, 99-113. PLAA, G. L., and LARSON, R. E. (1965). Relative nephrotoxic properties of chlorinated methane, ethane, and ethylene derivatives in mice. Toxicol. Appl. Pharmacol. 7, 37-44. PLAA, G. L., EVANS, E. A., and HINE, C. H. ( 1958). Relative hepatotoxicity of seven halogenated hydrocarbons. J. Phurm~.~ol. Exptl. Therap. 123, 224-229. POUTSIAKA, J. W., KEYSSER, C. H., THOMAS, B. G. H., and LINEGAR, C. H. (1962). Simultaneous determination in dogs of liver and kidney functions with bromosulfalein and phenolsulfonephthalein. Toxicol. Appl. Phmmacol. 4, 55-69. REITMAN, S., and FRANK=, S. ( 1957). A calorimetric method for the determination of serum glutamic oxalacetic and glutamic pyruvic transaminases. Am. J. Clin. Puthol. 28, 56-63. VON OETITNGEN, W. F. (1964). The Halogenuted Hydrocarbons of Industrial and Toxicological Importance. Elsevier, Amsterdam.
AND
Relative
APPLIED
PHARMACOLOGY
Effects liver
10,
of Various and
Chlorinated
Kidney
CURTIS D. KLAASSEN~ Department
of Pharmacology, Iowa Received
(1967)
119-131
College City,
Function
Hydrocarbons in Dogs1 L. PLAA
AND GABRIEL of Medicine, Iowa 52240
October
on
The
University
of Iowa,
27, 1966
The altered morphology of the kidneys and liver produced by some of the chlorinated hydrocarbons has been studied quite extensively (Von Oettingen, 1964). However, comparisons between the various derivatives have been largely descriptive in nature. The reason for this is due in large part to the fact that the data obtained in these many studies were not readily translatable into the quanta1 dose-response data needed for quantitative comparisons. In the past few years, quantitative comparative studies have been made in mice after converting the various indices of hepatic function into dose-responsedata ( PIaa et al., 1958; Kutob and Plaa, 1962; Klaassen and Plaa, 1966). It has also been shown (Plaa and Larson, 1965) that the relative nephrotoxic properties of various chlorinated hydrocarbons could also be compared using phenolsulfonephthalein excretion and analyzing the data along similar lines. The dose-response data used in these comparisons are of the all-or-none type. These quanta1 data are usually handled by the probit method as described by Finney ( 1952) or the method of Litchfield and Wilcoxon ( 1949). Both of these methods suffer from the disadvantage that large numbers of animals are needed and, therefore, are not too practical for determining LDsO and organ dysfunction EDsO values in dogs. The “up-and-down” method of Brownlee et al. ( 1953) employs few animals. The purpose of the present study is to evaluate this method to estimate the median lethal (LD50) and organ dysfunction ( ED!x) values for a series of chlorinated hydrocarbons in dogs. Since it has been demonstrated that mice (Klaassen and Plaa, 1966) and rats ( Cornish and Adefuin, 1966) exposed both to ethanol and various chlorinated hydrocarbons are more susceptible to the liver-damaging properties of these substances, the effect of ethanol on the liver dysfunction induced by these hydrocarbons was also studied. METHODS
The following hydrocarbons were employed: dichloromethane, chloroform, carbon tetrachloride, l,l,l-trichloroethane, 1,1,2-trichloroethane, trichloroethylene, 1 Preliminary presentation of these data was made at the Fall Society for Pharmacology and Experimental Therapeutics, Mexico 1966. ’ U.S. Public Health Service Predoctoral Fellowship 6M-30996. 119
Meeting of the American City, Mexico, July 14-21,
120
CURTIS
D.
KLAASSEN
AND
GABRIEL
L.
PLAA
and tetrachloroethylene. All agents were made up in corn oil and administered intraperitoneally to mongrel male and female dogs weighing 7-14 kg. Lethality. The median lethal dose ( LDGo) was estimated by the method of Brownlee et al. (1953) 24 hours after the hydrocarbon was administered intraperitoneally. Lizjer fun&on. Liver function was assessed by determining the serum glutamic-pyruvic transaminase (SGPT) activity by the method of Reitman and Frankel ( 1957). The blood sample was obtained in a heparin-rinsed syringe from each dog 24 and 48 hours after the hydrocarbon was administered. A O.l-ml aliquot of plasma was added to 0.5 ml of alanine-ketoglutaric acid reagent. This mixture was incubated for 30 minutes at 40” on a module heater,” then 0.5 ml of the 2,4-dinitrophenylhydrazine reagent was added. Thirty minutes later, 5 ml of 0.4 iV NaOH was added; the absorbance of this solution was read at 505 rnp 30 minutes after the addition of NaOH. Kidney function. The dogs were anesthetized with intravenously administered pentobarbital sodium, and the ureters were cannulated. Saline (25 ml/kg) was given to the dogs intravenously over a 30-minute period. Phenolsulfonephthalein (PSP) was then administered intravenously at a dosage of 3 mg/kg (Poutsiaka et al., 1962) and the urine was collected for 30 minutes from the cannulated ureters. The urine was diluted with 0.1 N NaOH, and the concentration of PSP was determined spectrophotometrically, using the absorbance at 560 mp. Ethanol intoxication. Ethanol was diluted to 50% and administered by gavage. The dose of ethanol (4 g/kg) resulted in an observable central nervous system depression (primarily ataxia). The control dogs received an equicaloric amount (20 ml/kg) of 36% dextrose solution. Histopathology. Kidney and liver tissues were removed from the surviving dogs used in the LDso studies and fixed in 10% formalin. These tissues will be referred to as those from dogs administered hydrocarbons at the “near LDEo dose.” The tissues from 3 or 4 dogs were obtained for each compound. Tissues were also removed from the dogs used in the ED- 50 studies. These tissues, therefore, are from dogs that received doses both above and below the calculated EDao dose, and will be referred to as the tissues from dogs administered hydrocarbons at the “near ED,,, dose.” Liver samples from the ED,,, studies based on SGPT elevation were taken 48 hours after the administration of the hydrocarbon. In the PSP excretion EDso studies, the kidneys were removed 24 hours after the administration of the hydrocarbon. Livers were also removed from the dogs that were given threequarters of an LD 50 of the hydrocarbon after the SGPT values returned to normal values. Liver and kidney morphology was assessed after staining with hematoxylin and eosin. Statistics. In order to evaluate the method of Brownlee et al. (1953), the LD,,, and liver dysfunction ED ho values for chloroform and carbon tetrachloride in mice derived by this method were compared to those obtained using the method of Litchfield and Wilcoxon (1949). The ED,, values were calculated after the functional data were transformed to the all-or-none responses. ” “Temp-Blok,”
obtained
from
Lab-Line
Instruments,
Melrose
Park,
Illinois.
CHLORINATED
HYDROCARBONS
AND
ORGAN
FUNCTION
121
After finding the method quite suitable for the purposes of this study, the “upand-down” method of Brownlee et al. (1953) was used to estimate the 24-hour LDb0 values and the organ dysfunction ED 5o values as measured by PSP and SGPT. This method consists of giving a dose of a compound to one animal and noting the presence or absence of an effect. If it has an effect, one decreases the dose and repeats the trial on a second animal. If it does not have an effect, one increases the dose and repeats the trial. This is repeated for 3 times after one has obtained one positive and one negative response in two successive doses. The increment in dosage used was a 40% increase of the first negative response, as suggested by Deichmann and Le Blanc ( 1943). A formula is used to determine the EDs0 or LDsO. Thus, one can obtain an estimate of the potency ratio (PR) by making a ratio of the LD 50 to the EDs0 but cannot test the significance of this PR statistically. The ethanol-pretreated dogs were compared to the dextrose dogs by the MannWhitney U test as described by Goldstein ( 1964). RESULTS
Evaluation of the “Up-and-doum” Method Mice were used to compare the LDjo and EDso values obtained by the method of Brownlee et al. (1953) to those obtained by the method of Litchfield and Wilcoxon. The 24-hour LDso and the liver dysfunction EDs0 as measured by SGPT were both measured after intraperitoneal injection of the test compounds. The results are shown in Table 1. The LDso values by the method of Brownlee et al. for chloroform and carbon tetrachloride were I.2 and 2.5 ml/kg, respectively, and 1.1 (0.9-1.3) and 2.7 (2.4-3.0) ml/kg, respectively by the method of Litchfield and Wilcoxon. The liver dysfunction as measured by SGPT is shown in Table 1. The EDEOvalues for liver dysfunction for chloroform and carbon tetrachloride by the method of Brownlee et al. were 0.18 and 0.008 ml/kg, respectively, and those obtained by the method of Litchfield and Wilcoxon were 0.17 (0.14-0.21) and 0.0092 (0.0058-0.015) ml/kg, respectively. Since the LDso and organ damage EDzO as estimated by the method of Brownlee et al. did not fall outside the confidence limits determined by the method of Litchfield and Wilcoxon, the former method was used for estimating the LDB, and organ dysfunction ED,,, values in the dog studies. Lethality Table 2 shows the LD,, values, based on the incidence of mortality within 24 hours, for the halogenated hydrocarbons tested. In dogs as in mice (Klaassen and Plaa, 1966), the least potent hydrocarbon tested as far as death is concerned was 1,&l-trichloroethane, and its isomer, 1,1,2-trichloroethane, was the most potent. Liver Function In control dogs it was found that the SGPT activity was 36 =I=(SD) 7 units. Thus, a value of 50 units (36 + 2 SD) was chosen as the upper limit of the nor-
tetrachloride
tetrachloride
Dose (ml/kg) Response” Dose (ml/kg) Response”
Dose (ml/kg) Response” Dose (ml/kg) Responseb
0.14 N 0.011 E
determination 0.20 E 0.008 N
0.14 N 0.011 E
0.95 S 3.3 D
LDso determination 0.95 1.35 S D a.3 3.2 S D ED,
Mouse 3
at 44 hours
0.20 E 0.008 E
1.35 D 1.4 S
Mouse 4
after
IN MICE
the dose.
0.14 N 0.005 N
0.95 S 12.3 S
Mouse 5
TABLE 1 OF SERUM GLUTAMIC-PYRUVIC TRANSAMINASE ACTIVITY DOSE OF CHLOROFORM OR CARBON TETRACHLORIDE~ Mouse a
Mouse 1
OF THE LDSo AND ED,, FOR ELEVATION AFTER A SINGLE INTRAPERITONEAL
0 The determinations were performed by the method of Brownlee et al. (1953). b Response is indicated by a “D” for death or an “S” for survival at 94 hours after the dose. c Response is indicated by an “N” for a normal SGPT activity, and an “E” for an elevated activity
Carbon
Chloroform
Carbon
Chloroform
Compound
DETERMINATION
0.008
0.18
a.5
I.2
LDb,, or EDso (ml/kg)
m
? g
F r
2 E
i
$ * B 2
P
a Response
Tetrachloroethylene
Trichloroethylene
l,l,%Trichloroethane
is indicated
tetrachloride
l,l,l-Trichloroethane
Carbon
Chloroform
Dichloromethane
Compound
an “S”
by a “D”
for death
for survival
S 0.35 S 2.0 D 3.0 D
Response Dose (ml/kg) Response Dose (ml/kg) Response
Dose (ml/kg) Response
D 1.4 S a.5
e
at $24 hours
1.46 S
S a.55 D
D 2.0 S after
a.0
S 1.45
S
D 1.45 S
the dose.
Dog 4 1.25 D 1.00 S 1.8 D 3.5 D 0.45
CRLORINATED
S 0.35
1.00 S 0.75 s 1.4 S a.5
3
Erg
OF VARIOUS
D 0.46
D 3.5
1.8
0.75 S 0.50 S
4
Dog
1
Erg
TABLE LDso’s
INTRAPER~TIONAL
Response Dose (ml/kg) Response Dose (ml/kg)
and
ACUTE
1.00 D 0.75
OF THE
Dose (ml/kg) Responsea Dose (ml/kg)
DETERMINATION
S
St.0
S
S 0.55 D 2.0
a.5
1.00 D 1.25 S 1.4 D
5
Dog
HYDROCARBONS
=ho
1.5
1.0
0.95
(ml/W
a.1
1.9
0.45
3.1
FOR DOGS
L&o
a1
%l
4.9
91
16
13
15
bmoWkd
6” 2 =1 2
2 0 :: G
ti 2: v)
3 8
X
8
2
i c!
9
124
CURTIS
D.
KLAASSEN
AND
GABRIEL
L.
PLAA
ma1 range. Any SGPT value greater than 50 units was considered significant and indicative of liver dysfunction. Blood was obtained from the dogs 24 and 48 hours after administration of the hydrocarbon. The results are shown in Table 3. Here, as in mice (Klaassen and Plaa, 1966), there is a great difference in the ability of these hydrocarbons to produce liver dysfunction. Carbon tetrachloride causes liver dysfunction at much lower doses than the other hydrocarbons. Liver
Histopathology
The hydrocarbons could be divided into two classes according to the changes seen with the light ,microscope at near-lethal doses. In the first class one would group carbon tetrachloride, chloroform, and 1,1,2-trichloroethane from which the most severe damage was seen, consisting of centrolobular necrosis. Carbon tetrachloride caused severe centrolobular necrosis, chloroform caused moderate centrolobular necrosis, and 1,1,2-trichloroethane caused mild centrolobular necrosis. All the hydrocarbons caused slight subcapsular necrosis. The second class would consist of dichloromethane, I,I,l-trichloroethane, trichloroethylene, and tetrachloroethylene which, at near-lethal doses, produced moderate neutrophilic infiltrations in the sinusoids and portal areas, but no necrosis. Near the EDs0 all the hydrocarbons, except l,l,l-trichloroethane, caused vacuolization of centrolobular hepatocytes in about half of the animals. l,l,l-Trichloroethane, at the near-ED,, doses caused subcapsular necrosis but no centrolobular lesion. 1,1,2-Trichloroethane caused centrolobular necrosis in this dose range. With chloroform, 2 of the 7 livers examined had necrosis, but with 1,1,2-trichloroethane all livers exhibited necrosis. Severity
and Length
of Duration
of Liver
Dysfunction.
Figure 1 shows the results obtained when single doses of the hydrocarbons (0.75 X LD6,,) were given to the dogs. Each point is the mean obtained from 3 dogs. Carbon tetrachloride caused the greatest elevation of SGPT activity. Although chloroform did not cause as great an elevation in SGPT, the time needed for the values to return to the normal range was about the same time as that needed for carbon tetrachloride. The rest of the hydrocarbons caused less elevation of SGPT than chloroform and carbon tetrachloride. These SGPT values returned to the normal range within 7 to 10 days compared to 17-18 days with chloroform and carbon tetrachloride. After the SGPT values had returned to the normal range, the livers were removed from the dogs and examined histologically. In most cases the livers exhibited moderate vacuolation of the centrolobular and midzonal hepatocytes as well as traces of brown material in the cytoplasm of centrolobular Kupff er cells. Kidney
Function
In control dogs, 56* following administration - 2 SD) was considered was determined 24 hours
(SD) 8.3% of the PSP was excreted within 30 minutes to dogs. Thus, a PSP excretion of less than 39% (56 to be indicative of significant kidney dysfunction. This after the administration of the hydrocarbon. Only dogs
Dog
(1 Response
Tetrachloroethylene
Trichloroethylene
I,l,%Trichloroethane
is indicated
tetrachloride
I,l,l-Trichloroethane
Carbon
Chloroform
by an “E”
for elevation
N
activity
and
E
N of SGPT
0.84
N
0.45
0.60
E
0.6
0.37
N 0.50 E
E
E 0.75
N 1.05
0.019
0.50 N 0.20 E
2
Dog
CHLORINATED
0.014
E 0.15 N
0.75
Dose (ml/kg) Response” Dose (ml/kg) Response Dose (ml/kg) Response Dose (ml/kg) Response Dose (ml/kg) Response Dose (ml/kg) Response Dose (ml/kg) Response
TABLE
3
an “N”
N
for normal
N
0.60
E
0.6
E
0.37
E
1.05
activity
E
0.84
N
0.45
N
0.24
N
0.75
N
0.019
N
0.014
0.20
N
0.50 N
4
Dog
34 hours
after
N
0.60
N
0.6
E
0.37
E
1.05
E
0.024
E
0.25
E
0.75
Dog 5
the dose.
0.74
0.57
0.35
0.87
0.02
0.20
0.60
(ml/W
EDm
IX DOGSBY A SINGLE INTRAPERITONEAL
0.15
E
0.75
Dog 3
HYDROCARBONS
OFSERUM GLUTAMIC-PYRUVICTRANSAMINASE
Dichloromethane
FOR ELEVATION
1
OFTHE ED,,
Compound
DETERMINATION
DOSE
7.2
6.3
3.8
8.6
0.2
2.5
9.4
(mmole/kg)
EDso
OF VARIOUS
8
2
2 z =I
8 5
5
E !z cn
!4
8
$
U
z
B
a Response
Tetrachlorethylene
l,l,%Trichloroethane
Chloroform
Compound
DETERMINATION
is indicated
OF THE
by a “D”
for diminished
Dose (ml/kg) Responsea Dose (ml/kg) Response Dose (ml/kg) Response
EDSo FOR DIMINUTION
excretion
and an “N”
E
1.05
0.56 D 0.40 D
2
0.4 N 0.28 N 1.45 N
Dog
1
4
for normal
excretion.
0.4 D 0.28 N 1.45 N
3
Dog
EXCRETION HYDROC.~RBONS
TABLE
Dog
OF PHENOLSULFONEPHTHALEIN CHLORINATED
0.84 N 0.40 N 1.05 E
4
0.4 N 0.52 D 1.45 N
5
Dog
BY A SINGLE
Dog
IN DOGS
IXTIL~PERITONEAL
1.4
0.40
0.43
DOSE
14
4.3
5.4
EDSO (mmole/kg)
OF VARIOUS
CHLORINATED
HYDROCARBONS
AND
TABLE
ORGAN
127
FUNCTION
5
COMPARISON OF VARIOUS CHLORINATED HYDROCARBONS FOR ACUTE LETHALITY AND THEIR EFFECTS ON PHENOLBULFONEPHTHALEIN EXCRETION AND SERUM GLUTAMIC-PYRUVIC TRANSAMINASE ACTIVITY AFTER A SINGLE INTRAPERITONEAL DOSE T O MICE AND DOGS~ PSP excretion
Species
LDSO (ml/kg)
Dichloromethane
Mouse
1.50
Chloroform
Dog Mouse
0.95 1.9 1.0
Compound
Dog
Carbon
tetrachloride
Mouse Dog
l,l,l-Trichloroethane
Mouse Dog
l,l,‘&Trichloroethane Trichloroethylene Tetrachloroethylene
3.1 0.35
Dog
0.45
Mouse Dog Mouse Dog
a.a 1.9 a.9 9.1
0 The data on mice are reproduced * The “potency ratio” is the ratio
given kidney Kidney
chloroform, disfunction
Potency ratio”
0.078 0.45 -
Potency ratio”
0.60
1.6
15
0.18
6.4 5.0
0.17 0.40
a.3
O.&O
-
0.0098 0.019
-
a.5 0.87
2.1
0.10 0.35
1.1 -
1.4
or
-
-
-
from Klaassen and Plaa of the LDso to the EDw.
1,1,2-trichloroethane, (Tables 4 and 5).
ED@ (dkd
elevation
-
a.7 1.5 3.8
Mouse
SGPT
1.5
280 79 1.5 3.5 3.4 1.3
1.6
1.4
0.57
3.8
2.9
0.98
0.74
2.8
(1966).
tetrachloroethylene
demonstrated
Histopathology
At near-lethal doses all the hydrocarbons caused some alteration of the kidneys. The changes do not appear to be as severe in the dog as those seen in mice (Klaassen and Plaa, 1966). Only with l,l,&-trichloroethane was necrosis observed. With carbon tetrachloride, Bowman’s capsules appeared dilated with some glomerular tufts contracted; calcification of a small number of tubules in the medulla was also seen. The rest of the compounds, chloroform, l,l,l-trichloroethane, dichloromethane, trichloroethylene, and tetrachloroethylene, showed slight calcification of the tubules. At the near-PSP excretion-EDbO doses, only slight changes such as mild dilatation of the collecting ducts were seen in some of the kidneys; the rest exhibited no lesions. Efect
of Ethanol on Hydrocarbon-Induced
Liver Dysfunction
Table 6 shows the results when the dogs were pretreated once with ethanol or dextrose 24 hours prior to the administration of an approximate ED50 dose of the hydrocarbon. The SGPT levels were measured 48 hours later. Ethanol potentiated the SGPT activity produced by carbon tetrachloride, chloroform, and trichloroethylene. However, no significant difference could be demonstrated between the SGPT activity of dextrose and alcohol-treated dogs prior to l,l,ltrichloroethane or corn oil. These results were confirmed by histopathologic
oil vehicle
a Difference
Corn
Trichloroethylene
between
tetrachloride
l,l,l-Trichloroethane
Carbon
Chloroform
Compound
TABLE
6
alcohol-pretreated
Yes No Yes No Yes No Yes NO Yes No
Ethanol
Pretreatment
0.20 0.20 0.024 0.024 0.87 0.87 0.57 0.57 -
and dextrose-pretreated
No Yes No Yes No Yes No Yes No Yes
Dextrose
Hydrocarbon dose (ml/kg)
groups,
P < 0.05
400 28 270 as 33 26 63 23 24 20
Dog 1
(one-tail,
Mann-Whitney
5600 57 8200 61 43 28 107 50 32 29
Dog 4
(units)
U test).
48 82 500 78 43 34
61 -
Dog 5
120 210 580 310 92 43
-
Dog 6
GLUTAMIC-PYRUVIC TRANSAMINASE CHLORINATED HYDROC LRBONS activity
610 42 7300 48 43 27 82 31 31 2s
Dog 3
SGPT
OF SERUM OF VARIOUS
420 39 400 37 34 26 66 24 31 20
Dog 2
EFFECT OF 24-HOUR PRETRE.
No
Yes
No
Yes
Yes
Significanta
CHLORINATED
HYDROCARBONS
AND
ORGAN
FUNCTION
129
*--a Dichloromethane )r.......~Chloroform Carbon Tetrachloride .+..A I, I, I - Tr ichloroet hone I, I ,2 -Trichloroethone G---O Trichloroethylene o-)-a Tetrochloroethylene
2
4
6
8 IO 12 14 I6 DAYS
I8
FIG. 1. Duration of elevation of serum glutamic-pyruvic transaminase (SGPT) after a single intraperitoneal dose of various chlorinated hydrocarbons to dogs. The dosage of each compound was three-quarters of the single intraperitoneal LD,,. Each point on the curves is the mean of the results from three dogs.
examination, in that alcohol pretreatment caused more severe morphologic changes in the dogs treated with carbon tetrachloride, chloroform, and trichloroethylene, but not in those treated with l,l,l-trichloroethane. DISCUSSION
The method of Brownlee et al. ( 1953) was used in this study in order to conserve animals. This method appears to be quite satisfactory for estimating median effective doses. It has the advantage in that it is possible to determine median doses with as few as 5 animals. The values obtained are also quite reproducible. The liver dysfunction ED 5Ovalues as measured by serum glutamic-pyruvic transaminase (SGPT) demonstrate that all the halogenated hydrocarbons tested are capable of producing some degree of liver dysfunction. Table 5 compares the EDso values obtained in mice and dogs and also contains the SGPT-potency ratio (PR). This ratio compares the dose required to produce death to that required
130
CUFtTIS
D. KLAASSEN
AND
GABRIEL
L. PLAA
to produce organ dysfunction. It is felt (Klaassen and Plaa, 1966) that the PR is more informative about the relative capacities of these agents to produce organ dysfunction than is a comparison of ED 50 values alone. The 24-hour LD50 is largely a measure of the deep narcosis caused by these hydrocarbons. Therefore, a large PR indicates a high capacity for producing organ dysfunction, since the dose required to produce this injury is considerably lower than that required to produce deep narcosis. When one looks at the PR values in Table 5, it can be seen that carbon tetrachloride is vastly different from the other hydrocarbons. This table also shows that when the effects of these halogenated hydrocarbons in dogs are compared to those in mice, there is not much of a species difference in the relative capacity of these agents to produce liver dysfunction. However, it should be noted that dichloromethane produces some liver dysfunction in dogs but not in mice. Chloroform and carbon tetrachloride caused the highest elevation following a dose of three-quarters of the LDsO (Fig. 1). Even though carbon tetrachloride caused a peak SGPT elevation 5 times that of chloroform, both values returned to the normal range at about the same time. The rest of the hydrocarbons produced only moderate SGPT elevation which returned to normal levels in 7-10 days, thus demonstrating a difference in the amount of dysfunction produced. The kidney dysfunction phenolsulfonephthalein (PSP) PR values (Table 5) demonstrate that chloroform, 1,1,2-trichloroethane, and tetrachloroethylene can produce kidney dysfunction in dogs only at doses in the near-lethal range. In the mouse (Plaa and Larson, 1965; Klaassen and Plaa, 1966) only chloroform and 1,1,2-trichloroethane produce kidney damage as measured by PSP excretion. However, chloroform seems to have a greater nephrotoxic capacity in mice. It should be noted that carbon tetrachloride did not produce altered PSP excretion; however, some histologic changes were seen in the kidneys of dogs treated with nearlethal doses of all the hydrocarbons. With chloroform and carbon tetrachloride, which produce liver dysfunction at relatively low doses, a marked potentiation occurs when the dogs are pretreated with ethanol. However, with trichloroethylene and l,l,l-trichloroethane, which produce liver dysfunction at relatively high doses, it is difficult to demonstrate a potentiation of the hydrocarbon-induced liver dysfunction by alcohol. This has also been demonstrated in mice (Klaassen and Plaa, 1966) and in rats (Cornish and Adefuin, 1966). Therefore, from a practical standpoint, it would appear that alcohol intoxication would have its most important effect only on subsequent exposures to carbon tetrachloride or chloroform, agents which cause severe alterations in organ function at relatively low doses. SUMMARY The “up-and-down” statistical method was used to estimate the L&o and liver and kidney dysfunction EDEo values in dogs, and serum glutamic-pyruvic transaminase was used to determine hepatic function following intraperitoneal injection of dichloromethane, chloroform, carbon tetrachloride, l,l,l-trichloroethane, 1,1,2-trichloroethane, trichloroethylene, and tetrachloroethylene. Median effective doses for inducing dysfunction were calculated and compared to the lethal dose. Carbon tetrachloride caused elevated transaminase values at much lower doses than the rest of the hydrocarbons. Renal function was tested by urinary phenol-
CHLORINATJZD
HYDROCARBONS
AND
ORGAN
FUNCTION
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sulfonephthalein excretion, and this test demonstrated that chloroform, 1,1,2-trichloroethane, and tetrachloroethylene produced altered renal function. Enhancement of the hepatic dysfunction produced by the hydrocarbons was assessed using ethanol in intoxicating doses. ACKNOWLEDGMENTS This work was supported by funds from the U.S. Public Health Service (Research Grant AM-05802) and by funds from the Dow Chemical Co., Midland, Michigan. Histologic evaluation of the tissues was performed by Dr. Robert G. Geil, International Research and Development Corporation, Mattawan, Michigan. REFERENCES BROWNLEE, K. A., HODGES, J. L., and ROSENBLAIT, M. ( 1953). The up-and-down method with small samples. Am. Statist. Assoc. J. 48, 262-277. CORNISH, H., and ADEFUIN, J. ( 1966). Ethanol potentiation of halogenated aliphatic solvent toxicity. Am. lnd. Hyg. Assoc. J. 27, 57-61. DEICHMANN, W. B., and LEBLANC, T. J. ( 1943). Determination of the approximate lethal dose with about six animals. J. Ind. Hyg. ToxicoZ. 25, 415417. FINNEY, D. J. ( 1952). Statistical Method in Biological Assay. Charles Griffin, London. GOLDSTEIN, A. ( 1964). Biostatistics: An Introductory Text. ‘Macmillan, New York. KLAASSEN, C. D., and PLAA, G. L. (1966). The relative effects of various chlorinated hydrocarbons on liver and kidney function in mice. Toxicol. Appl. Pharmucot. 9, 139-151. KUTOB, S. D., and PLAA, G. L. ( 1962). A procedure for estimating the hepatotoxic potential of certain industrial solvents. Toxicol. Appl. Phurmucol. 4, 345-361. LITCHFIELD, J. T., JR. and WILCOXON, F. ( 1949). A simplified method for evaluating doseeffect experiments. J. Pharmacol. Exptl. Therup. 96, 99-113. PLAA, G. L., and LARSON, R. E. (1965). Relative nephrotoxic properties of chlorinated methane, ethane, and ethylene derivatives in mice. Toxicol. Appl. Pharmacol. 7, 37-44. PLAA, G. L., EVANS, E. A., and HINE, C. H. ( 1958). Relative hepatotoxicity of seven halogenated hydrocarbons. J. Phurm~.~ol. Exptl. Therap. 123, 224-229. POUTSIAKA, J. W., KEYSSER, C. H., THOMAS, B. G. H., and LINEGAR, C. H. (1962). Simultaneous determination in dogs of liver and kidney functions with bromosulfalein and phenolsulfonephthalein. Toxicol. Appl. Phmmacol. 4, 55-69. REITMAN, S., and FRANK=, S. ( 1957). A calorimetric method for the determination of serum glutamic oxalacetic and glutamic pyruvic transaminases. Am. J. Clin. Puthol. 28, 56-63. VON OETITNGEN, W. F. (1964). The Halogenuted Hydrocarbons of Industrial and Toxicological Importance. Elsevier, Amsterdam.