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Absence of potentiation of bromoform hepatotoxicity and lethality by chlordecone
251
Toxicology Letters, 15 (1983) 251-257 Elsevier Biomedical Press
ABSENCE OF POTENTIATION OF BROMOFORM HEPATOTOXICITY AND LETHALITY BY CHLORDECONE (CNS toxicity; phenolphthalein
glucuronide; isocitric dehydrogenase)
A.K. AGARWAL and H.M. MEHENDALE Department
of Pharmacology
State Street, Jackson,
and Toxicology,
The University of Mississippi Medical Center, 2500 N.
MS 39216 (U.S.A.)
(Received June 8th, 1982) (Accepted September 2Oth, 1982)
SUMMARY Our previous studies indicated that the toxicity of chloro- or bromo-methanes is potentiated by chlordecone (CD). The present work was conducted to study the effect of prior dietary exposure to CD on toxicity of bromoform. Male S-D rats (175-200 g) were fed 0 or 10 ppm CD in the powdered ration for 15 days. Bromoform (25 to 300 PI/kg) was given i.p. on day 15.24 h later, hepatotoxicity was assessed by functional, biochemical and histopathological parameters. Excretion of phenolphthalein glucuronide in bile and the rate of bile flow were unaltered by either bromoform or CD-bromoform combination. Serum enzymes (GPT, GOT and isocitric dehydrogenase (ICD) were also not significantly elevated by any treatment. The results suggest that, unlike chloroform, CHBrj does not act as a potent hepatotoxin and that its effects are not potentiated by CD to any significant extent.
INTRODUCTION
Bromoform (CHBr3) shares the properties of its chlorinated analog chloroform (CHCl3) in that it is a mild anesthetic and has been found as a contaminant in drinking water supplies [l]. Very little information is available regarding the hepatotoxicity of this compound. CHBrs is a more potent carcinogen than CHClj [2] suggesting that it may prove to be of greater concern as an environmental health hazard. Previous work has established that hepatotoxicity and lethality of CCL [3, 41 and BrCC13 [S, 61 are potentiated by CD in rats. Hewitt et al. [7] observed the
Abbreviations: CD, chlordecone; phenolphthalein glucuronide. 037%4274/83/OC&-0000/$03.00
CNS, central nervous system; ICD, isocitric dehydrogenase;
0 Elsevier Biomedical Press
PG,
252
potentiation of CHC13 hepatotoxicity by CD in mice. The present studies were conducted to investigate if hepatotoxicity and lethality of the brominated analog of chloroform, CHBr3 are potentiated by prior exposure to CD. Three different doses of CHBr3 were used either alone or in combination with CD pretreatment and hepatotoxicity was assessed using functional, biochemical, and histopathological parameters. 48-h LD5o of CHBr3 given alone or in combination with CD was also determined. MATERIALS
AND
METHODS
Animals and treatment Male Sprague-Dawley rats of CR-l strain (175-200 g; Charles River Breeding Laboratories, Wilmington, MA) were housed in properly ventilated cages under a 12-h photoperiod. Commercial powdered rat chow (Ralston Purina, St. Louis, MO) containing 0 or 10 ppm CD prepared as described previously [3] and water were provided ad lib. Body weights were recorded on days 1 and 15 of treatment. On day 15, CHBr3 (25 to 300 PI/kg) was administered i.p. in corn oil vehicle (1 ml/kg). Controls received corn oil alone. Rats treated with normal diet-CHBr3 combination were compared to normal diet-corn oil controls whereas rats treated with CD-CHBr3 combination were compared to CD-corn oil controls. Hepatotoxicity was assessed 24 h after CHBr3 challenge. Hepatic function Biliary excretion of PG and the rate of bile flow were measured according to previously published procedures [3]. The amount of PG in samples of bile was determined by the method of Gustafson and Benet [lo]. At the termination of the above experiment, blood was collected from dorsal aorta in heparinized tubes and plasma was used for enzyme assays. Serum enzymes GPT and GOT were estimated using the method of Reitman and Frankel [S]. ICD was determined according to King [9]. The enzyme activities expressed as Units/ml plasma were calculated from appropriate standard plots. Liver histopathology Liver samples from control and treated groups of animals were fixed in buffered 10% formaline, embedded in paraffin wax and sectioned. The sections were stained with hematoxylin and eosin (H & E) for light microscopy.
253
Determination
of LDSO
32 rats were divided in two groups: one group was fed a diet containing 10 ppm CD for 15 days; the other was maintained on normal but similarly prepared diet. On day 15, the animals of each group were divided in four subgroups comprising 4 animals each. CHBr3 was given i.p. at 4 dose levels in corn oil vehicle and 48-h LDso was determined by the method of Weil [ 111. Statistics Student’s t-test [12] was used to evaluate significance of differences between two groups at a significance level of P 5 0.05. RESULTS
Biological effects Rats treated with 10 ppm dietary CD for 15 days did not exhibit any signs of toxicity. Food and water consumption were unaltered by this treatment. The animals given 200 and 300 ~1 CHBr3 either alone or in combination with CD were in deep sedation and prostration for up to 8 h. Lacrymation was observed in all animals receiving CHBrs. Visceral organs did not show any gross signs of toxicity. Hepatic function
studies
The biliary excretion of PG was unaltered in any of the groups (Fig. 1). Bromoform given alone or in combination with CD did not show any changes in bile flow (Fig. 1). Serum enzymes The elevation in the activity of serum transaminases (GPT and GOT) and ICD seen after CD-CHBr3 combination at the doses of 200 and 300 pi/kg, was not significant (Fig. 2). Histopathology No gross changes in liver histology were noted at any of the three doses used either alone or in combination with CD. Some dilatation of sinusoids was apparent at 300 ~1 CHBrJkg dose and CD treatment did not affect this.
254
17
Control
m
Chlordecone
Control
25
f
9 = 100 5
$ 60 3
20 0
200
300
BROMoFORM [jll/kgJ Fig.
1. Male Sprague-Dawley
feeding
Biliary excretion expressed
rats weighing
0 or 10 ppm CD for 15 days. Excretion of PG as percent
as percent
administered
175-200
g were given bromoform
of PG in bile was measured dose is shown.
i.p. (25-300
24 h after CHBr,
Bile flow was measured
al) after injection.
per unit time and
of control.
600 260
520
220
E440 I ; 360 0 ac 200 E 200
T
e 360 3 ” 200 5 I
P 200 t
120 40 0
120
Catrd
25
200
300
40 0 InJ
BROMOFORM (Cl/kg]
40 0
c&ml
25
1 ~
2( 10 300
Fig. 2. SGPT, SGOT and ICD activities were determined 24 h after i.p. administration oil vehicle. Results were plotted as percent of control. The activity of these enzymes any of the dose levels.
of CHBr, in cornwas not elevated at
255 TABLE I EFFECT OF CHLORDECONE
ON CHBrs LETHALITY IN RATS
Dietary pretreatmenta
48 h LDs,, Cl/kgb
95% confidence limitsb
Control Chlordecone (IO ppm)
414 388
309-554 320-47 1
“Male Sprague-Dawley rats (150-200 g) were maintained on control powdered diet or on diet containing 10 ppm chlordecone. On day 15, the animals received four different doses of CHBrr dissolved in corn oil as a single i.p. injection. bLDsOand confidence limits were calculated according to Weil [ll].
Determination
of LDSO
All the animals treated with CHBr3 were in sedation. A 48-h observation period was chosen for these studies on the basis of our earlier experiments which indicated that maximum deaths occurred at 48 h after halomethane administration. The animals which died did not come out of sedation before death. Other animals were lethargic and showed lacrymation. No significant changes in the LD5,, were observed between the animals given bromoform alone or in combination with CD (Table I). DISCUSSION
The propensity of CD in potentiating the hepatotoxicity of several halomethanes (CCld, BrCC13, CHCls) has been demonstrated by a number of investigators [3-71. The increased hepatotoxicity of CCL [3] and BrCC13 [5] culminates in potentiated lethality [4,6]. Hewitt et al. [7] observed that a single dose of CD significantly increased the hepatotoxicity of CHCl3 suggesting that hepatocytes from CDpretreated animals may be more reactive to the noxious effects of CHCls or its toxic metabolite. In the present studies with CHBr3, hepatic damage was not apparent even at the highest dose (300 @kg) when given alone or in combination with CD. This conclusion is supported by the results of the hepatic function tests and the serum GPT, GOT and ICD enzymes. Higher dose of CHBrs (350 pi/kg) resulted in 60% mortality 24 h after administration and hence doses above 300 pi/kg were not used. This mortality appeared to be due to CNS toxicity since the animals were sedated soon after treatment and never recovered consciousness. The results suggest that bromoform does not act as an acute hepatotoxin unlike most halomethanes even at high doses. Observation of animals during the LDso tests indicated that the animals which died of high doses of bromoform remained under sedation until death. These studies were designed to examine if prior treatment with CD resulted in enhanced mortality and the findings were negative in this regard as well. The cause of death in these animals appeared to be secondary to prolonged sedation.
256
The lack of potentiation follows. The bioactivation
of bromoform toxicity by CD could be reconciled of CHBr3 does not lead to a toxic intermediate
as in
contrast to CHClx which is metabolized to a toxic intermediate, phosgene, believed to be responsible for most of its toxic effects [13, 141. The biotransformation of CHBr3
in mammalian
species has not been reported.
this halomethane may occur, it is possible CHCI3. Assuming that CHBr3 is metabolized, by the bioactivation of halomethanes may intermediate. In any case, these findings are Bowman et al. [15] and Chu et al. [16] who thane is more toxic than dibromochloromethane methane.
While
biotransformation
of
that CHBr3 is not bioactivated unlike the chlorinated intermediate formed be more hepatotoxic than brominated supported by the observations made by demonstrated that bromodichloromewhich is more toxic than tribromo-
ACKNOWLEDGEMENTS
This study was supported
by a PHS grant
from NIEHS,
ES-01369.
REFERENCES
1 T.A.
Bellar,
drinking 2 J.C.
J.T.
water,
Theiss,
G.D.
contaminants Cancer
Lichtenberg
and
R.C.
U.S. Environmental Stoner,
M.B.
of United
States
Kroner,
Protection Shimkin
drinking
Curtis,
W.L.
tetrachloride
Williams
following
and
Report,
in chlorinated
EPA-670/4-74-008,
and E.K. Weisburger,
Test of carcinogenicity
waters
tumor
H.M.
preexposure
by pulmonary
Mehendale,
Lett.,
Potentiation
to chlordecone
Pharmacol., 51 (1979) 283-293. 4 J.S. Klingensmith and H.M. Mehendale,
response
1974.
of organic
in strain
A mice,
(Kepone)
Potentiation
of hepatotoxicity
in the male
of CC14 lethality
rat,
of carbon
Toxicol.
by chlordecone,
Appl. Toxicol.
11 (1982) 149-154.
5 J.S. Klingensmith chlordecone 6 A.K. 7 W.R.
and H.M. and
H.M.
by chlordecone Hewitt,
hepato-
Mehendale,
H. Miyajima,
glutamic
9 J. King, Thesis, Institute Methods Gustafson
Cote and G.L.
Toxicol.,
Acute
alteration
Benet,
Biliary
and retrograde
biliary
administration,
calculation
Appl.
hepatotoxicity of chloroform
Biophys.
and
induced
68 (1979) 509-527.
of serum
London,
ed., Academic
oxalacetic
and
kinetics J. Pharm.
of median effective
1967, in H.U. Bergmeyer Press,
of phenolphthalein Pharmacol.,
New York,
1974.
glucuronide
after
26 (1974) 937-944.
dose (LD5a or ED5a) and instructions
8 (1952) 249-263.
12 R.R. Sokal and F.J. Rohlf, Biometry, Freeman, San Francisco, 1969, p. 299. 13 L.R. Pohl, B. Bhooshan, N.F. Whittaker and G. Krisha, Phosgene: A metabolite Biochem.
by
28 (1957) 53-56.
and Technology,
excretion
hepatotoxicity
2 (1982) 161-167.
Pharmacol.,
of the determination
Vol. 2, 2nd English
L.Z.
in their use, Biometrics,
Plaa,
Appl.
Toxicol.
method
Laboratory
Analysis,
11 C.S. Weil, Tables for convenient
of bromotrichloromethane
Am. J. Clin. Pathol.,
of Medical
of Enzymatic and
Potentiation
halomethane
61 (1981) 378-384.
in the rat, Fundam.
A calorimetric
transaminases,
of brominated
Pharmacol.,
by mirex and Kepone,
and S. Frankel,
pyruvic
intravenous
M.G.
Potentiation Appl.
preexposure
and nephrotoxicity
8 S. Reitman
Mehendale,
in the male rat, Toxicol.
Agarwal
lethality
10 J.H.
of organohalides
Research
Res., 37 (1977) 2717-2720.
3 L.R.
(Ed.),
The occurrence
Agency
Res. Commun.,
79 (1977) 684-691.
of chloroform,
257
14 L.R. Pohl and G. Krishna, Deuterium isotope effect in hioactivation and hepatotoxicity of chloroform, Life Sci., 23 (1978) 1067-1072. 15 F.J. Bowman, J.F. Borzelleca and A.E. Munson, The toxicity of some halomethanes in mice, Toxicol. Appl. Pharmacol., 44 (1978) 213-215. 16 I. Chu, V. Secours, 1. Mario and D.C. Villeneuve, The acute toxicity of four trihalomethanes in male and female rats, Toxicol. Appl. Pharmacol., 52 (1980) 351-353.
Toxicology Letters, 15 (1983) 251-257 Elsevier Biomedical Press
ABSENCE OF POTENTIATION OF BROMOFORM HEPATOTOXICITY AND LETHALITY BY CHLORDECONE (CNS toxicity; phenolphthalein
glucuronide; isocitric dehydrogenase)
A.K. AGARWAL and H.M. MEHENDALE Department
of Pharmacology
State Street, Jackson,
and Toxicology,
The University of Mississippi Medical Center, 2500 N.
MS 39216 (U.S.A.)
(Received June 8th, 1982) (Accepted September 2Oth, 1982)
SUMMARY Our previous studies indicated that the toxicity of chloro- or bromo-methanes is potentiated by chlordecone (CD). The present work was conducted to study the effect of prior dietary exposure to CD on toxicity of bromoform. Male S-D rats (175-200 g) were fed 0 or 10 ppm CD in the powdered ration for 15 days. Bromoform (25 to 300 PI/kg) was given i.p. on day 15.24 h later, hepatotoxicity was assessed by functional, biochemical and histopathological parameters. Excretion of phenolphthalein glucuronide in bile and the rate of bile flow were unaltered by either bromoform or CD-bromoform combination. Serum enzymes (GPT, GOT and isocitric dehydrogenase (ICD) were also not significantly elevated by any treatment. The results suggest that, unlike chloroform, CHBrj does not act as a potent hepatotoxin and that its effects are not potentiated by CD to any significant extent.
INTRODUCTION
Bromoform (CHBr3) shares the properties of its chlorinated analog chloroform (CHCl3) in that it is a mild anesthetic and has been found as a contaminant in drinking water supplies [l]. Very little information is available regarding the hepatotoxicity of this compound. CHBrs is a more potent carcinogen than CHClj [2] suggesting that it may prove to be of greater concern as an environmental health hazard. Previous work has established that hepatotoxicity and lethality of CCL [3, 41 and BrCC13 [S, 61 are potentiated by CD in rats. Hewitt et al. [7] observed the
Abbreviations: CD, chlordecone; phenolphthalein glucuronide. 037%4274/83/OC&-0000/$03.00
CNS, central nervous system; ICD, isocitric dehydrogenase;
0 Elsevier Biomedical Press
PG,
252
potentiation of CHC13 hepatotoxicity by CD in mice. The present studies were conducted to investigate if hepatotoxicity and lethality of the brominated analog of chloroform, CHBr3 are potentiated by prior exposure to CD. Three different doses of CHBr3 were used either alone or in combination with CD pretreatment and hepatotoxicity was assessed using functional, biochemical, and histopathological parameters. 48-h LD5o of CHBr3 given alone or in combination with CD was also determined. MATERIALS
AND
METHODS
Animals and treatment Male Sprague-Dawley rats of CR-l strain (175-200 g; Charles River Breeding Laboratories, Wilmington, MA) were housed in properly ventilated cages under a 12-h photoperiod. Commercial powdered rat chow (Ralston Purina, St. Louis, MO) containing 0 or 10 ppm CD prepared as described previously [3] and water were provided ad lib. Body weights were recorded on days 1 and 15 of treatment. On day 15, CHBr3 (25 to 300 PI/kg) was administered i.p. in corn oil vehicle (1 ml/kg). Controls received corn oil alone. Rats treated with normal diet-CHBr3 combination were compared to normal diet-corn oil controls whereas rats treated with CD-CHBr3 combination were compared to CD-corn oil controls. Hepatotoxicity was assessed 24 h after CHBr3 challenge. Hepatic function Biliary excretion of PG and the rate of bile flow were measured according to previously published procedures [3]. The amount of PG in samples of bile was determined by the method of Gustafson and Benet [lo]. At the termination of the above experiment, blood was collected from dorsal aorta in heparinized tubes and plasma was used for enzyme assays. Serum enzymes GPT and GOT were estimated using the method of Reitman and Frankel [S]. ICD was determined according to King [9]. The enzyme activities expressed as Units/ml plasma were calculated from appropriate standard plots. Liver histopathology Liver samples from control and treated groups of animals were fixed in buffered 10% formaline, embedded in paraffin wax and sectioned. The sections were stained with hematoxylin and eosin (H & E) for light microscopy.
253
Determination
of LDSO
32 rats were divided in two groups: one group was fed a diet containing 10 ppm CD for 15 days; the other was maintained on normal but similarly prepared diet. On day 15, the animals of each group were divided in four subgroups comprising 4 animals each. CHBr3 was given i.p. at 4 dose levels in corn oil vehicle and 48-h LDso was determined by the method of Weil [ 111. Statistics Student’s t-test [12] was used to evaluate significance of differences between two groups at a significance level of P 5 0.05. RESULTS
Biological effects Rats treated with 10 ppm dietary CD for 15 days did not exhibit any signs of toxicity. Food and water consumption were unaltered by this treatment. The animals given 200 and 300 ~1 CHBr3 either alone or in combination with CD were in deep sedation and prostration for up to 8 h. Lacrymation was observed in all animals receiving CHBrs. Visceral organs did not show any gross signs of toxicity. Hepatic function
studies
The biliary excretion of PG was unaltered in any of the groups (Fig. 1). Bromoform given alone or in combination with CD did not show any changes in bile flow (Fig. 1). Serum enzymes The elevation in the activity of serum transaminases (GPT and GOT) and ICD seen after CD-CHBr3 combination at the doses of 200 and 300 pi/kg, was not significant (Fig. 2). Histopathology No gross changes in liver histology were noted at any of the three doses used either alone or in combination with CD. Some dilatation of sinusoids was apparent at 300 ~1 CHBrJkg dose and CD treatment did not affect this.
254
17
Control
m
Chlordecone
Control
25
f
9 = 100 5
$ 60 3
20 0
200
300
BROMoFORM [jll/kgJ Fig.
1. Male Sprague-Dawley
feeding
Biliary excretion expressed
rats weighing
0 or 10 ppm CD for 15 days. Excretion of PG as percent
as percent
administered
175-200
g were given bromoform
of PG in bile was measured dose is shown.
i.p. (25-300
24 h after CHBr,
Bile flow was measured
al) after injection.
per unit time and
of control.
600 260
520
220
E440 I ; 360 0 ac 200 E 200
T
e 360 3 ” 200 5 I
P 200 t
120 40 0
120
Catrd
25
200
300
40 0 InJ
BROMOFORM (Cl/kg]
40 0
c&ml
25
1 ~
2( 10 300
Fig. 2. SGPT, SGOT and ICD activities were determined 24 h after i.p. administration oil vehicle. Results were plotted as percent of control. The activity of these enzymes any of the dose levels.
of CHBr, in cornwas not elevated at
255 TABLE I EFFECT OF CHLORDECONE
ON CHBrs LETHALITY IN RATS
Dietary pretreatmenta
48 h LDs,, Cl/kgb
95% confidence limitsb
Control Chlordecone (IO ppm)
414 388
309-554 320-47 1
“Male Sprague-Dawley rats (150-200 g) were maintained on control powdered diet or on diet containing 10 ppm chlordecone. On day 15, the animals received four different doses of CHBrr dissolved in corn oil as a single i.p. injection. bLDsOand confidence limits were calculated according to Weil [ll].
Determination
of LDSO
All the animals treated with CHBr3 were in sedation. A 48-h observation period was chosen for these studies on the basis of our earlier experiments which indicated that maximum deaths occurred at 48 h after halomethane administration. The animals which died did not come out of sedation before death. Other animals were lethargic and showed lacrymation. No significant changes in the LD5,, were observed between the animals given bromoform alone or in combination with CD (Table I). DISCUSSION
The propensity of CD in potentiating the hepatotoxicity of several halomethanes (CCld, BrCC13, CHCls) has been demonstrated by a number of investigators [3-71. The increased hepatotoxicity of CCL [3] and BrCC13 [5] culminates in potentiated lethality [4,6]. Hewitt et al. [7] observed that a single dose of CD significantly increased the hepatotoxicity of CHCl3 suggesting that hepatocytes from CDpretreated animals may be more reactive to the noxious effects of CHCls or its toxic metabolite. In the present studies with CHBr3, hepatic damage was not apparent even at the highest dose (300 @kg) when given alone or in combination with CD. This conclusion is supported by the results of the hepatic function tests and the serum GPT, GOT and ICD enzymes. Higher dose of CHBrs (350 pi/kg) resulted in 60% mortality 24 h after administration and hence doses above 300 pi/kg were not used. This mortality appeared to be due to CNS toxicity since the animals were sedated soon after treatment and never recovered consciousness. The results suggest that bromoform does not act as an acute hepatotoxin unlike most halomethanes even at high doses. Observation of animals during the LDso tests indicated that the animals which died of high doses of bromoform remained under sedation until death. These studies were designed to examine if prior treatment with CD resulted in enhanced mortality and the findings were negative in this regard as well. The cause of death in these animals appeared to be secondary to prolonged sedation.
256
The lack of potentiation follows. The bioactivation
of bromoform toxicity by CD could be reconciled of CHBr3 does not lead to a toxic intermediate
as in
contrast to CHClx which is metabolized to a toxic intermediate, phosgene, believed to be responsible for most of its toxic effects [13, 141. The biotransformation of CHBr3
in mammalian
species has not been reported.
this halomethane may occur, it is possible CHCI3. Assuming that CHBr3 is metabolized, by the bioactivation of halomethanes may intermediate. In any case, these findings are Bowman et al. [15] and Chu et al. [16] who thane is more toxic than dibromochloromethane methane.
While
biotransformation
of
that CHBr3 is not bioactivated unlike the chlorinated intermediate formed be more hepatotoxic than brominated supported by the observations made by demonstrated that bromodichloromewhich is more toxic than tribromo-
ACKNOWLEDGEMENTS
This study was supported
by a PHS grant
from NIEHS,
ES-01369.
REFERENCES
1 T.A.
Bellar,
drinking 2 J.C.
J.T.
water,
Theiss,
G.D.
contaminants Cancer
Lichtenberg
and
R.C.
U.S. Environmental Stoner,
M.B.
of United
States
Kroner,
Protection Shimkin
drinking
Curtis,
W.L.
tetrachloride
Williams
following
and
Report,
in chlorinated
EPA-670/4-74-008,
and E.K. Weisburger,
Test of carcinogenicity
waters
tumor
H.M.
preexposure
by pulmonary
Mehendale,
Lett.,
Potentiation
to chlordecone
Pharmacol., 51 (1979) 283-293. 4 J.S. Klingensmith and H.M. Mehendale,
response
1974.
of organic
in strain
A mice,
(Kepone)
Potentiation
of hepatotoxicity
in the male
of CC14 lethality
rat,
of carbon
Toxicol.
by chlordecone,
Appl. Toxicol.
11 (1982) 149-154.
5 J.S. Klingensmith chlordecone 6 A.K. 7 W.R.
and H.M. and
H.M.
by chlordecone Hewitt,
hepato-
Mehendale,
H. Miyajima,
glutamic
9 J. King, Thesis, Institute Methods Gustafson
Cote and G.L.
Toxicol.,
Acute
alteration
Benet,
Biliary
and retrograde
biliary
administration,
calculation
Appl.
hepatotoxicity of chloroform
Biophys.
and
induced
68 (1979) 509-527.
of serum
London,
ed., Academic
oxalacetic
and
kinetics J. Pharm.
of median effective
1967, in H.U. Bergmeyer Press,
of phenolphthalein Pharmacol.,
New York,
1974.
glucuronide
after
26 (1974) 937-944.
dose (LD5a or ED5a) and instructions
8 (1952) 249-263.
12 R.R. Sokal and F.J. Rohlf, Biometry, Freeman, San Francisco, 1969, p. 299. 13 L.R. Pohl, B. Bhooshan, N.F. Whittaker and G. Krisha, Phosgene: A metabolite Biochem.
by
28 (1957) 53-56.
and Technology,
excretion
hepatotoxicity
2 (1982) 161-167.
Pharmacol.,
of the determination
Vol. 2, 2nd English
L.Z.
in their use, Biometrics,
Plaa,
Appl.
Toxicol.
method
Laboratory
Analysis,
11 C.S. Weil, Tables for convenient
of bromotrichloromethane
Am. J. Clin. Pathol.,
of Medical
of Enzymatic and
Potentiation
halomethane
61 (1981) 378-384.
in the rat, Fundam.
A calorimetric
transaminases,
of brominated
Pharmacol.,
by mirex and Kepone,
and S. Frankel,
pyruvic
intravenous
M.G.
Potentiation Appl.
preexposure
and nephrotoxicity
8 S. Reitman
Mehendale,
in the male rat, Toxicol.
Agarwal
lethality
10 J.H.
of organohalides
Research
Res., 37 (1977) 2717-2720.
3 L.R.
(Ed.),
The occurrence
Agency
Res. Commun.,
79 (1977) 684-691.
of chloroform,
257
14 L.R. Pohl and G. Krishna, Deuterium isotope effect in hioactivation and hepatotoxicity of chloroform, Life Sci., 23 (1978) 1067-1072. 15 F.J. Bowman, J.F. Borzelleca and A.E. Munson, The toxicity of some halomethanes in mice, Toxicol. Appl. Pharmacol., 44 (1978) 213-215. 16 I. Chu, V. Secours, 1. Mario and D.C. Villeneuve, The acute toxicity of four trihalomethanes in male and female rats, Toxicol. Appl. Pharmacol., 52 (1980) 351-353.