Toxicology, 63 (1990) 63--72 Elsevier Scientific Publishers Ireland Ltd.
Subacute toxicity of trichloroacetic acid in male and female rats Mary E. Davis Department of Pharmacology and Toxicology, Health Sciences Center, West Virginia University, Morgantown, WV 26506 (U.S.A.) (Received December 4th, 1989; Accepted March 13th, 1990)
Summary Trichloroacetic acid, TCA, is a water chlorination by-product similar to dichloroacetic acid, DCA. Because DCA has been shown to have effects on intermediary metabolism, TCA was tested to determine if it possesses similar capabilities. The effects were more pronounced in females. High doses of TCA (2.45 tamol/kg three times) decreased plasma glucose and lactate concentrations and liver lactate concentration. DCA had similar, less pronounced effects. In males DCA and TCA each decreased plasma lactate concentrations. Rats were exposed to TCA in drinking water for 14 days. The highest concentration (2.38 g/I) caused decreases of water and food consumption and loss of body weight. At 7 days females had decreased urine volume accompanied by a modest increase of urine osmolality, resulting in a significant decrease of excretion of solute. Concentrations of glucose in plasma and lactate in tissues were not significantly affected by this subchronic TCA exposure. These results indicate that TCA may have effects on intermediary metabolism similar to those of DCA.
Key words: Trichloroacetic acid; Dichloroacetic acid; Lactate metabolism; Water pollutants
Introduction Dichloroacetic acid (DCA) and trichloroacetic acid (TCA) are major by-products of chlorination [1] and they are metabolites of chlorinated ethanes and ethylenes. DCA is produced by demethylation of methoxyflurane [2], oxidation of 1,1,2,2-tetrachloroethane [3], and cysteine conjugate/3-1yase cleavage of the product of glutathione conjugation of tetrachloroethylene, S-(1,2,2-trichlorovinyl)-Lcysteine [4]. TCA is a metabolite of 1,1, l-trichloroethylene and is excreted in the urine of rodents or workers exposed to 1,1,1-trichloroethylene [5] and urine TCA can be used as a dose surrogate for exposure [6]. TCA is formed during the oxidative metabolism of tetrachloroethylene as well [4]. However further metabolism or toxicity of TCA has not been reported. The effects of exposure to DCA in drinking water have been measured [7]. High concentrations (1.875 and 7.5 g/l) were objectionable to rats; water and 0300~3X/90/$03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
63
food consumption both decreased and the animals either lost weight or failed to gain. Concentrations of 0.03--0.5 g/l were well tolerated. In males, urine volume was decreased and osmolality increased on day 7 in the high exposure groups, an appropriate response to decreased fluid intake. Tissue concentrations of lactate were not altered by the DCA treatment, however plasma glucose concentrations tended to be elevated in the high dose groups, suggesting that the high exposures were approaching effective doses [7]. While the effects of DCA on intermediary metabolism have been extensively studied and are well characterized, TCA-induced alterations of intermediary metabolism, or lack of such effects, have not been reported. The main use of TCA has been as an agent to precipitate proteins, both in vitro in laboratory work and in vivo in dermabrasion treatment of acne [8] and other skin disorders [9], including removal of tattoos [10] and warts [11,12]. Since being confirmed as a chlorination by-product, TCA has been found to be a weak promoter and an inducer of peroxisomal proliferation [13--16] and to be genotoxic [17] and a complete hepatocarcinogen in B6C3F1 mice [18]. TCA also induces strand breaks in hepatic DNA [19] and disrupts intracellular communication through gap junctions [20]. The goal of the studies summarized here was to examine specific effects of TCA in rats. Studies were done to determine if TCA has effects on intermediary metabolism similar to those of DCA and to determine if such effects occur after sub-chronic exposure to TCA in drinking water. Materials and methods
Experimental animals Male (275--300 g) and female (225--250 g) rats o f Sprague--Dawley ancestry were used for all studies. They were received from Hilltop Farms (Scottsdale, PA) by truck and were housed in the central animal facility. During holding, the rats were housed in suspended stainless steel group cages with absorbent cardboard beneath the wire mesh cage floor with free access to food (Wayne Rodent Blox) and water.
Gavage experiments Rats were housed in plastic group cages with hardwood chip bedding. Solutions of TCA or DCA (Fisher or Sigma) were administered as 2 ml/kg for three doses (0900 h, 1600 h and 0900 h the next morning) and the rats were killed 3 h after the last dose, or 27 h after the first dose. This time was chosen based upon the studies o f Evans [21] demonstrating peak liver DCA concentrations 3 h after gavage. Because high concentrations of the acids (0.92 and 2.45 ~mol/kg) were used, the solutions were neutralized with sodium hydroxide. The sodium concentration of the solution was measured (using flame photometry) and an equimolar solution of sodium chloride prepared for the controls.
Drinking water experiments Rats were exposed to TCA in their drinking water for 14 days. They were
64
maintained in stainless steel metabolism cages for measurements of food and water consumption and urine output. The pelleted chow was ground up for use in the metabolism cages; the rats were allowed 40 g of food and 200 ml of water or T C A solution. Rats were allowed 2 days to acclimate to the metabolism cages before exposure to T C A for 14 days. Water consumption was estimated as disappearance from the bottle, with no correction for dripping. Food consumption was measured as disappearance of food from the food cup, crumbs caught in the tray beneath the food chute were added back to the food cup before daily weighing. T C A solutions were made in reagent grade water (from a Millipore reverse osmosis and charcoal filter water purification system, yielding water better than 10 MQ) and were replaced twice weekly. The controls received the same water.
Tissue preparation and assays Rats were killed by exsanguination under ether anesthesia. A large blood sample was taken (from the abdominal aorta) and a piece of liver and one kidney rapidly removed and frozen with Vise-grip clamps at the temperature of liquid nitrogen; the tissues were stored at - - 7 0 ° C until processing for lactate content. If plasma lactate was to be measured, an aliquot of the blood was mixed with an equal volume of perchloric acid (1 M) immediately; the remainder was centrifuged and resulting plasma removed, frozen, and used for various assays. Lactate was measured as production of N A D H during incubation with lactate dehydrogenase and NAD; N A D H was measured spectrophotometrically [22]. Tissues were homogenized in 10% perchloric acid, without being allowed to thaw, as described [221. Glucose was measured colorimetrically with Sigma reagents (kit 115) based upon the hexokinase and glucose-6-phosphatase reactions. Urine osmolality was measured by depression of freezing point using an Osmette S osmometer. TABLE I P L A S M A GL UCOSE C O N C E N T R A T I O N AFTER G A V A G E W I T H C H L O R I N A T E D A C E T I C ACIDS Dose ( v m o l / k g ) 0
0.92
2.45
180 26 152 Il
188 28 165 8
153 8 128 12
168 13 180 6
169 17 162 46
162 17 135* 5
DCA (mg/lO0 mO Males ± S.E. Females _+ S.E.
TCA (mg/lO0 mO Males _+ S.E. Females -+ S.E.
Results are expressed as mean ~: S.E. o f 5 rats. *Denotes different from control, P < 0.05.
65
All results are expressed as mean and standard error of the mean. Data were analyzed by ANOVA using the model appropriate for the experimental design (generally two factor, with repeated measures on one factor for those parameters measured daily in the drinking water exposure studies). SAS Institute software was used for these analyses. The significance o f differences between specific groups was determined using the Student Newman Keul's procedure. The criterion for significance was P < 0.05. Results
Gavage studies: Effects on intermediary metabolism The goal of the acute gavage studies was to determine if TCA has effects on intermediary metabolism similar to those of DCA. The concentration of glucose 1.50
DCA
1.00
X> X>~
>(x ×x
i\/
X"
/xf
XX
)0
x×
E
03 o.
0.50
0.00 X> 0 .g2 2.4.15
×x 0
.92 2.45
0
.g2 2.45
(D 4-'
1.,50
4--,
TCA
0
(D
1.00
0
E 0.50
0.00
0
.g2 2.45
MALES
FEMALES
Fig. 1. Plasma concentration of lactate after garage with DCA or TCA. Rats were gavaged three times with neutralized solution of each acid at the close indicated. They were killed 3 h after the last dose. Results are shown as mean :t: S.E. of 5 rats. *Indicates different from vehicle control, P < 0.05.
66
in plasma (Table I) was significantly decreased only in females treated with TCA. Plasma concentrations o f lactate were decreased by DCA and TCA in male and female rats (Fig. 1). Kidney content o f lactate was not different in any of the groups (not shown). Significant reductions of liver lactate were found in females following TCA (Fig. 2), but the reductions for the DCA group were not significant. These studies have shown that TCA alters plasma concentrations of lactate and glucose similar to DCA and that female rats are more susceptible to this effect.
The effects of TCA exposure in drinking water for 14 days Overall, TCA had significant effects on body weight in both males and females. The high dose groups each had an initial decrease of weight followed by
3
DCA
-
2
¸
L-
> °
~
0
0
.g2 2.~5
MALES
0
3'
0
.g2 2.48
~-E~,4AL~S
TCA
.
0
E
0
0 . g 2 2.4.5 MALES
0
. g 2 2.445
FEMAleS
Fig. 2. Liver concentration of lactate after gavage with DCA or TCA. Rats were gavaged three times with neutralized solution of each acid at the dose indicated. They were killed 3 h after the last dose. Results are shown as mean :l: S.E. of 5 rats, except n = 6 for high dose males. *Indicates different from vehicle control, P < 0.05.
67
weight g a i n p a r a l l e l t o the c o n t r o l s . In the first 2 d a y s the females lost weight ( f r o m 246 ± 5 g d o w n to 231 _+ 6 g) w h e r e a s the c o n t r o l s g a i n e d weight ( f r o m 237 ± 4 g to 239 ± 5 g). C o n s i s t e n t with weight loss, the high e x p o s u r e g r o u p s c o n s u m e d less f o o d (13 ± 1 vs. 7 ± 1 g / d a y ) a n d w a t e r (31 ± 7 vs. 17 _+ 2 m l / d a y ) in the first few d a y s o f e x p o s u r e to T C A . M a l e s o n the high d o s e experienced similar changes. Effects o n urine v o l u m e a n d o s m o l a l i t y , a n d excretion o f solute, were transient. U r i n e o s m o l a l i t y was i n c r e a s e d a n d v o l u m e d e c r e a s e d on d a y 7 in the males o n T C A (Fig. 3). T h e high d o s e g r o u p s excreted s m a l l e r a m o u n t s o f m o r e conc e n t r a t e d urine, an a p p r o p r i a t e r e s p o n s e to d e c r e a s e d water c o n s u m p t i o n . In
0
-o-
.04
DAY
.16
7
-*-
.63
DAY
2..38
14
3000"
1000 0
,
,
.04
•16
DOSiE
TCA
, .63
2.38
(g/L)
Fig. 3. Urine volume and osmolality during TCA exposure of male rats. Urine samples were collected on days 7 and 14 of TCA exposure. Results are shown as mean and S.E. of 5 or 4 (0.04 mg/l group) rats. There were no significant effects on urine volume, for osmolality the TCA treatment and day factors were both significant. *Different from 0 TCA controls, P < 0.05.
68
contrast, o n day 7 o f T C A exposure females had a dose-related decrease o f urine v o l u m e a n d a lesser increase o f o s m o l a l i t y (Fig. 4) such that solute excretion decreased (Table II). In females, T C A appears to i m p a i r the ability to concentrate u r i n e sufficiently to m a t c h u r i n e o u t p u t to decreased fluid intake. There were n o significant effects o f T C A t r e a t m e n t o n either liver or kidney lactate c o n t e n t s or p l a s m a glucose c o n c e n t r a t i o n s .
Discussion T h e goal o f these studies was to d e t e r m i n e if T C A has effects similar to D C A which has been extensively studied. D C A activates the p y r u v a t e d e h y d r o g e n a s e
1
I
0
i
0
-o-
|
DAY
|
i
.04
.63
.16
7
-*-
DAY
|
2.38
14
3500"
-T
150 0
. .04 DOSE
.
. .16 TCA
. .63
9:':18
(g/L)
Fig. 4. Urine volume and osmolality during TCA exposure of female rats. Urine samples were collected on days 7 and 14 of TCA exposure. Results are shown as mean and S.E. of 6 rats. The TCA treatment factor was significant for urine volume and osmolality. *Different from 0 TCA controls, P < 0.05.
69
TABLE II TCA EFFECT ON OSMOTIC EXCRETION TCA concentration in drinking water (g/l) mOsm/day:
0.00
0.04
O. 16
0.63
2.38
Males Day 7 ± S.E. Day 14 ± S.E.
18 1 18 2
19 1 19 2
20 1 19 1
18 I 23 2
19 4 19 I
Females Day 7 S.E. Day 14 ± S.E.
16 1 19 1
17 I 20 2
19 1 18 2
17 1 20 2
12" I 20 1
*Different from highest value, P < 0.05.
complex (PDH), increasing oxidation of glucose and pyruvate through the tricarboxylic acid cycle, which in turn decreases lactate and pyruvate [23]. DCA also increases the clearance and excretion of lactate and pyruvate, further contributing to the decrease of plasma concentrations of both [24]. In liver, DCA is metabolized to glyoxalate and then oxalate [25,26]; oxalate inhibits pyruvate carboxylase [25]. By preventing synthesis of glucose from pyruvate, DCA can decrease glucose availability in states dependent upon gluconeogenesis [26]; however, blood glucose is not decreased in normal humans after DCA administration [27]. The results for tissue lactate are variable, as lactate is metabolized within the body and slight differences in delay of removing the tissue from the animal and the degree of hypoventilation that occurs prior to removing the tissue influence the lactate concentration. Activation of pyruvate dehydrogenase and increased oxidation of glucose and lactate can be shown more readily in isolated tissue preparations. The results for plasma lactate and glucose showed that TCA has effects similar to those of DCA in female rats. The drinking water exposure studies were done to determine if similar effects occur during continuous exposure to lower concentrations. Rats given the highest concentration of T C A consumed less water and food and lost weight. Plasma glucose concentrations were not affected by the 14 day exposure to TCA. Plasma lactate concentrations were not measured, and the effects on tissue lactate were not significant. The highest concentration was objectionable to rats, and likely would be to humans. While TCA has effects similar to DCA, high doses are required to produce these effects, greater than are likely to be present in chlorinated drinking water [1]. Therefore direct toxicity from TCA is unlikely. This agrees with previous results from DCA studies, in that concentrations that were objectionable caused only slight, non-significant decreases of liver lactate or plasma glucose [7]. Healthy adult rats were used for these studies, the young and aged may be at greater risk. Blackshear and co-workers [28] have shown that the
70
effects o f D C A are greater in e x p e r i m e n t a l diabetes, t h e r e f o r e it is likely t h a t diabetics w o u l d be m o r e susceptible to the effects o f T C A as well. W h i l e i n t e r m e d i a r y m e t a b o l i s m was n o t a f f e c t e d b y s u b - c h r o n i c e x p o s u r e to T C A , s o m e s e x - d e p e n d e n t effects o n renal f u n c t i o n were n o t e d . Renal f u n c t i o n was m a i n t a i n e d in the m a l e rats. T h o s e o n the high dose c o n s u m e d less liquid a n d also excreted s m a l l e r a m o u n t s o f a m o r e c o n c e n t r a t e d urine, an a p p r o p r i a t e response to d e c r e a s e d w a t e r c o n s u m p t i o n . In females, however, the urine was not as c o n c e n t r a t e d as it s h o u l d have been for the r e d u c t i o n o f v o l u m e , a n d total excretion o f o s m o l e s d e c r e a s e d , suggesting interference with electrolyte h o m e o s tatic m e c h a n i s m s . T h e results o f these e x p e r i m e n t s suggest t h a t T C A has effects on i n t e r m e d i a r y m e t a b o l i s m that are similar to t h o s e o f D C A . T h a t is, T C A m a y activate p y r u vate d e h y d r o g e n a s e similar to D C A o r be m e t a b o l i z e d to o x a l a t e a n d g l y o x a l a t e similar to D C A . F e m a l e rats e x p e r i e n c e d m o r e effects o f T C A t h a n did the male rats, as was the case with the urine o u t p u t a n d o s m o l a l i t y studies. T h e r e f o r e , there m a y be a sex d i f f e r e n c e in the h a n d l i n g o f T C A o r responsiveness to s o m e o f its effects. This sex d i f f e r e n c e is not a b s o l u t e , however, in that p l a s m a lactate c o n c e n t r a t i o n s were also d e p r e s s e d by T C A in m a l e rats. D i f f e r e n t rates o f m e t a b o l i s m m a y a c c o u n t for this difference.
Acknowledgements I a m g r a t e f u l to D i a n e L a n e , M a r q u e t a Lewczyk, K a t h l e e n T h a y n e , a n d C r a i g Y o u n g for technical assistance. A l t h o u g h the research d e s c r i b e d in this article has been f u n d e d w h o l l y o r in p a r t b y the U n i t e d States E n v i r o n m e n t a l P r o t e c t i o n A g e n c y t h r o u g h c o o p e r a t i v e a g r e e m e n t CR811906-01, it has n o t been s u b j e c t e d to A g e n c y review a n d t h e r e f o r e d o e s n o t necessarily reflect the views o f the A g e n c y a n d no o f f i c i a l e n d o r s e m e n t s h o u l d be i n f e r r e d .
References I 2 3 4 5 6 7
J.D. Johnson, R.F. Christman, D.L. Norwood and D.S. Millington, Reaction products of aquatic humic substances with chlorine. Environ. Health Perspect., 46 (1982) 63. B.S. Selinsky, M.E. Perlman and R.E. London, In vivo nuclear magnetic resonance studies of hepatic methoxyfluranc metabolism. II. A reevaluation of hepatic metabolic pathways. Mol. Pharmacol., 33 (1988) 567. D.B. Hales, B. Ho and J.A. Thompson, Inter- and intramolecular deuterium isotope effects on the cytochrome P-450 catalyzed oxidative dehalogenation of l,l,2,2-tetrachloroethane. Biochem. Biophys. Res. Commun., 149 (1987) 319. W. Dekant, G. Martens, S. Vamvakas, M. Metzler and D. Henschler, Bioactivation of tetrachloroethylene. Role of glutathione S-transferase-catalyzed conjugation versus cytochrome P-450 dependent phospholipid alkylation. Drug Metab. Dispos., 15 (1987) 702. G. Muller, M. Spassovski and D. Henschler, Metabolism of trichloroethylene in man. Arch. Toxicol., 32 (1974) 283. A.C. Monster, G. Boersma and H. Steenweg, Kinetics of l,l,l-trichloroethane in volunteers; influence of exposure concentration and workload. Int. Arch. Occup. Environ. Health, 42 (1979) 293. M.E. Davis, Effect of chloroacetic acids on the kidneys. Environ. Health Perspect., 69 (1986) 209.
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8
J.J. Stagnone and G.J. Stagnone, A second look at chemabrasion. J. Dermatol. Surg. Oncol., 8 (1982) 701. 9 P.S. Collins, The chemical peel. Clin. Dermatol., 5 (1987) 57. 10 T.A. Piggot and R.W. Norris, The treatment of tattoos with trichloroacetic acid: experience with 670 patients. Br. J. Plast. Surg., 41 (1988) 112. I 1 M.J. Godley, C.S. Bradbeer, M. Gellan and R.N. Thin, Cryotherapy compared with trichloroacetic acid in treating genital warts. Genitourin. Med., 63 (1987) 390. 12 D.B. Schwartz, M.D. Greenberg, Y. Daoud and R. Reid, Genital condylomas in pregnancy: use of trichloroacetic acid and laser therapy. Am. J. Obstet. Gynecol., 158 (1988) 1407. 13 M.J. Parnell, L.D. Koller, J.H. Exon and J.M. Arnzen, Trichloroacetic acid effects on rat liver peroxisomes and enzyme-altered foci. Environ. Health Perspect., 69 (1986) 73. 14 C.R. EIcombe, Species differences in carcinogenicity and peroxisome proliferation due to trichloroethylene: a biochemical human hazard assessment. Arch. Toxicol., 8 (1985) 6. 15 A.M. Mitchell, J.W. Bridges and C.R. Elcombe, Factors influencing peroxisome proliferation in cultured rat hepatocytes. Arch. Toxicol., 55 (1984) 239. 16 J. Odum, T. Green, J.R. Foster and P.M. Hext, The role of trichloroacetic acid and peroxisome proliferation in the differences in carcinogenicity of perchloroethylene in the mouse and rat. Toxicol. Appl. Pharmacol., 92 (1988) 103. 17 S.P. Bhunya and B.C. Behera, Relative genotoxicity of trichloroacetic acid (TCA) as revealed by different cytogenetic assays: bone marrow chromosome aberration, micronucleus and spermhead abnormality in the mouse. Mutat. Res., 188 (1987) 215. 18 S.L. Herren-Freund, M.A. Pereira, M.D. Khoury and G. Olson, The carcinogenicity of trichloroethylene and its metabolites, trichloroacetic acid and dichloroacetic acid, in mouse liver. Toxicol. Appl. Pharmacol., 90 (1987) 183. 19 M.A. Nelson and R.J. Bull, Induction of strand breaks in DNA by trichloroethylene and metabolites in rat and mouse liver in vivo. Toxicol. Appl. Pharmacol., 94 (1988) 45. 20 J.E. Klaunig, R.J. Ruth and E.L.C. Lin, Effects of trichloroethylene and its metabolites on rodent hepatocyte intercellular communication. Toxicol. Appl. Pharmacol., 99 (1989) 454. 21 O.B. Evans, Dichloroacetate tissue concentrations and its relationship to hypolactemia and pyruvate dehydrogenase activation. Biochem. Pharmacol., 31 (1982) 3124. 22 I. Gutman and A.W. Wahlefeld, L-(+)-Lactate determination with lactate dehydrogenase and NAD, in H.U. Bergmeyer (Ed.), Methods of Enzymatic Analysis, Academic Press, New York, 1974, p. 1464. 23 A. McAllister, S.P. Allison and P.J. Randle, Effects of dichloroacetate on the metabolism of glucose, pyruvate, acetate, 3-hydroxybutyrate and palmitate in rat diaphragm and heart muscle in vitro and on extraction of glucose, lactate, pyruvate and free fatty acids by dog heart in vivo. Biochem. J., 134 (1973) 1067. 24 H. Kodama, S. Yamaguchi, 1. Okabe, M. Kodama, T. Orii and S. Kamochita, Renal effects of dichloroacetate in vivo. Clin. Chim. Acta, 169 (1986) 265. 25 F. DeMaugre, C. Cepanec and J.-P. Leroux, Characterization of oxalate as a catabolite of dichloroacetate responsible for the inhibition of gluconeogenesis and pyruvate carboxylation in rat liver cells. Biochem. Biophys. Res. Commun., 85 (1978) l l80. 26 D.W. Crabb and R.A. Harris, Mechanism responsible for the hypoglycemic actions of dichloroacetate and 2-chloropropionate. Arch. Biochem. Biophys., 198 (1979) 145. 27 S.H. Curry, P.I. Chu, T.G. Baumgarmer and P.W. Staepoole, Plasma concentrations and metabolic effects of intravenous sodium dichloroacetate. Clin. Pharmacol. Ther., 37 (1985) 89. 28 P.J. Blackshear, P.A.H. Holloway and K.G.M.M. Alberti, Metabolic interactions of dichloroacetate and insulin in experimental diabetic ketoacidosis. Studies on whole animals and after functional hepatectomy. Biochem. J., 146 (1975) 447.
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Subacute toxicity of trichloroacetic acid in male and female rats Mary E. Davis Department of Pharmacology and Toxicology, Health Sciences Center, West Virginia University, Morgantown, WV 26506 (U.S.A.) (Received December 4th, 1989; Accepted March 13th, 1990)
Summary Trichloroacetic acid, TCA, is a water chlorination by-product similar to dichloroacetic acid, DCA. Because DCA has been shown to have effects on intermediary metabolism, TCA was tested to determine if it possesses similar capabilities. The effects were more pronounced in females. High doses of TCA (2.45 tamol/kg three times) decreased plasma glucose and lactate concentrations and liver lactate concentration. DCA had similar, less pronounced effects. In males DCA and TCA each decreased plasma lactate concentrations. Rats were exposed to TCA in drinking water for 14 days. The highest concentration (2.38 g/I) caused decreases of water and food consumption and loss of body weight. At 7 days females had decreased urine volume accompanied by a modest increase of urine osmolality, resulting in a significant decrease of excretion of solute. Concentrations of glucose in plasma and lactate in tissues were not significantly affected by this subchronic TCA exposure. These results indicate that TCA may have effects on intermediary metabolism similar to those of DCA.
Key words: Trichloroacetic acid; Dichloroacetic acid; Lactate metabolism; Water pollutants
Introduction Dichloroacetic acid (DCA) and trichloroacetic acid (TCA) are major by-products of chlorination [1] and they are metabolites of chlorinated ethanes and ethylenes. DCA is produced by demethylation of methoxyflurane [2], oxidation of 1,1,2,2-tetrachloroethane [3], and cysteine conjugate/3-1yase cleavage of the product of glutathione conjugation of tetrachloroethylene, S-(1,2,2-trichlorovinyl)-Lcysteine [4]. TCA is a metabolite of 1,1, l-trichloroethylene and is excreted in the urine of rodents or workers exposed to 1,1,1-trichloroethylene [5] and urine TCA can be used as a dose surrogate for exposure [6]. TCA is formed during the oxidative metabolism of tetrachloroethylene as well [4]. However further metabolism or toxicity of TCA has not been reported. The effects of exposure to DCA in drinking water have been measured [7]. High concentrations (1.875 and 7.5 g/l) were objectionable to rats; water and 0300~3X/90/$03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
63
food consumption both decreased and the animals either lost weight or failed to gain. Concentrations of 0.03--0.5 g/l were well tolerated. In males, urine volume was decreased and osmolality increased on day 7 in the high exposure groups, an appropriate response to decreased fluid intake. Tissue concentrations of lactate were not altered by the DCA treatment, however plasma glucose concentrations tended to be elevated in the high dose groups, suggesting that the high exposures were approaching effective doses [7]. While the effects of DCA on intermediary metabolism have been extensively studied and are well characterized, TCA-induced alterations of intermediary metabolism, or lack of such effects, have not been reported. The main use of TCA has been as an agent to precipitate proteins, both in vitro in laboratory work and in vivo in dermabrasion treatment of acne [8] and other skin disorders [9], including removal of tattoos [10] and warts [11,12]. Since being confirmed as a chlorination by-product, TCA has been found to be a weak promoter and an inducer of peroxisomal proliferation [13--16] and to be genotoxic [17] and a complete hepatocarcinogen in B6C3F1 mice [18]. TCA also induces strand breaks in hepatic DNA [19] and disrupts intracellular communication through gap junctions [20]. The goal of the studies summarized here was to examine specific effects of TCA in rats. Studies were done to determine if TCA has effects on intermediary metabolism similar to those of DCA and to determine if such effects occur after sub-chronic exposure to TCA in drinking water. Materials and methods
Experimental animals Male (275--300 g) and female (225--250 g) rats o f Sprague--Dawley ancestry were used for all studies. They were received from Hilltop Farms (Scottsdale, PA) by truck and were housed in the central animal facility. During holding, the rats were housed in suspended stainless steel group cages with absorbent cardboard beneath the wire mesh cage floor with free access to food (Wayne Rodent Blox) and water.
Gavage experiments Rats were housed in plastic group cages with hardwood chip bedding. Solutions of TCA or DCA (Fisher or Sigma) were administered as 2 ml/kg for three doses (0900 h, 1600 h and 0900 h the next morning) and the rats were killed 3 h after the last dose, or 27 h after the first dose. This time was chosen based upon the studies o f Evans [21] demonstrating peak liver DCA concentrations 3 h after gavage. Because high concentrations of the acids (0.92 and 2.45 ~mol/kg) were used, the solutions were neutralized with sodium hydroxide. The sodium concentration of the solution was measured (using flame photometry) and an equimolar solution of sodium chloride prepared for the controls.
Drinking water experiments Rats were exposed to TCA in their drinking water for 14 days. They were
64
maintained in stainless steel metabolism cages for measurements of food and water consumption and urine output. The pelleted chow was ground up for use in the metabolism cages; the rats were allowed 40 g of food and 200 ml of water or T C A solution. Rats were allowed 2 days to acclimate to the metabolism cages before exposure to T C A for 14 days. Water consumption was estimated as disappearance from the bottle, with no correction for dripping. Food consumption was measured as disappearance of food from the food cup, crumbs caught in the tray beneath the food chute were added back to the food cup before daily weighing. T C A solutions were made in reagent grade water (from a Millipore reverse osmosis and charcoal filter water purification system, yielding water better than 10 MQ) and were replaced twice weekly. The controls received the same water.
Tissue preparation and assays Rats were killed by exsanguination under ether anesthesia. A large blood sample was taken (from the abdominal aorta) and a piece of liver and one kidney rapidly removed and frozen with Vise-grip clamps at the temperature of liquid nitrogen; the tissues were stored at - - 7 0 ° C until processing for lactate content. If plasma lactate was to be measured, an aliquot of the blood was mixed with an equal volume of perchloric acid (1 M) immediately; the remainder was centrifuged and resulting plasma removed, frozen, and used for various assays. Lactate was measured as production of N A D H during incubation with lactate dehydrogenase and NAD; N A D H was measured spectrophotometrically [22]. Tissues were homogenized in 10% perchloric acid, without being allowed to thaw, as described [221. Glucose was measured colorimetrically with Sigma reagents (kit 115) based upon the hexokinase and glucose-6-phosphatase reactions. Urine osmolality was measured by depression of freezing point using an Osmette S osmometer. TABLE I P L A S M A GL UCOSE C O N C E N T R A T I O N AFTER G A V A G E W I T H C H L O R I N A T E D A C E T I C ACIDS Dose ( v m o l / k g ) 0
0.92
2.45
180 26 152 Il
188 28 165 8
153 8 128 12
168 13 180 6
169 17 162 46
162 17 135* 5
DCA (mg/lO0 mO Males ± S.E. Females _+ S.E.
TCA (mg/lO0 mO Males _+ S.E. Females -+ S.E.
Results are expressed as mean ~: S.E. o f 5 rats. *Denotes different from control, P < 0.05.
65
All results are expressed as mean and standard error of the mean. Data were analyzed by ANOVA using the model appropriate for the experimental design (generally two factor, with repeated measures on one factor for those parameters measured daily in the drinking water exposure studies). SAS Institute software was used for these analyses. The significance o f differences between specific groups was determined using the Student Newman Keul's procedure. The criterion for significance was P < 0.05. Results
Gavage studies: Effects on intermediary metabolism The goal of the acute gavage studies was to determine if TCA has effects on intermediary metabolism similar to those of DCA. The concentration of glucose 1.50
DCA
1.00
X> X>~
>(x ×x
i\/
X"
/xf
XX
)0
x×
E
03 o.
0.50
0.00 X> 0 .g2 2.4.15
×x 0
.92 2.45
0
.g2 2.45
(D 4-'
1.,50
4--,
TCA
0
(D
1.00
0
E 0.50
0.00
0
.g2 2.45
MALES
FEMALES
Fig. 1. Plasma concentration of lactate after garage with DCA or TCA. Rats were gavaged three times with neutralized solution of each acid at the close indicated. They were killed 3 h after the last dose. Results are shown as mean :t: S.E. of 5 rats. *Indicates different from vehicle control, P < 0.05.
66
in plasma (Table I) was significantly decreased only in females treated with TCA. Plasma concentrations o f lactate were decreased by DCA and TCA in male and female rats (Fig. 1). Kidney content o f lactate was not different in any of the groups (not shown). Significant reductions of liver lactate were found in females following TCA (Fig. 2), but the reductions for the DCA group were not significant. These studies have shown that TCA alters plasma concentrations of lactate and glucose similar to DCA and that female rats are more susceptible to this effect.
The effects of TCA exposure in drinking water for 14 days Overall, TCA had significant effects on body weight in both males and females. The high dose groups each had an initial decrease of weight followed by
3
DCA
-
2
¸
L-
> °
~
0
0
.g2 2.~5
MALES
0
3'
0
.g2 2.48
~-E~,4AL~S
TCA
.
0
E
0
0 . g 2 2.4.5 MALES
0
. g 2 2.445
FEMAleS
Fig. 2. Liver concentration of lactate after gavage with DCA or TCA. Rats were gavaged three times with neutralized solution of each acid at the dose indicated. They were killed 3 h after the last dose. Results are shown as mean :l: S.E. of 5 rats, except n = 6 for high dose males. *Indicates different from vehicle control, P < 0.05.
67
weight g a i n p a r a l l e l t o the c o n t r o l s . In the first 2 d a y s the females lost weight ( f r o m 246 ± 5 g d o w n to 231 _+ 6 g) w h e r e a s the c o n t r o l s g a i n e d weight ( f r o m 237 ± 4 g to 239 ± 5 g). C o n s i s t e n t with weight loss, the high e x p o s u r e g r o u p s c o n s u m e d less f o o d (13 ± 1 vs. 7 ± 1 g / d a y ) a n d w a t e r (31 ± 7 vs. 17 _+ 2 m l / d a y ) in the first few d a y s o f e x p o s u r e to T C A . M a l e s o n the high d o s e experienced similar changes. Effects o n urine v o l u m e a n d o s m o l a l i t y , a n d excretion o f solute, were transient. U r i n e o s m o l a l i t y was i n c r e a s e d a n d v o l u m e d e c r e a s e d on d a y 7 in the males o n T C A (Fig. 3). T h e high d o s e g r o u p s excreted s m a l l e r a m o u n t s o f m o r e conc e n t r a t e d urine, an a p p r o p r i a t e r e s p o n s e to d e c r e a s e d water c o n s u m p t i o n . In
0
-o-
.04
DAY
.16
7
-*-
.63
DAY
2..38
14
3000"
1000 0
,
,
.04
•16
DOSiE
TCA
, .63
2.38
(g/L)
Fig. 3. Urine volume and osmolality during TCA exposure of male rats. Urine samples were collected on days 7 and 14 of TCA exposure. Results are shown as mean and S.E. of 5 or 4 (0.04 mg/l group) rats. There were no significant effects on urine volume, for osmolality the TCA treatment and day factors were both significant. *Different from 0 TCA controls, P < 0.05.
68
contrast, o n day 7 o f T C A exposure females had a dose-related decrease o f urine v o l u m e a n d a lesser increase o f o s m o l a l i t y (Fig. 4) such that solute excretion decreased (Table II). In females, T C A appears to i m p a i r the ability to concentrate u r i n e sufficiently to m a t c h u r i n e o u t p u t to decreased fluid intake. There were n o significant effects o f T C A t r e a t m e n t o n either liver or kidney lactate c o n t e n t s or p l a s m a glucose c o n c e n t r a t i o n s .
Discussion T h e goal o f these studies was to d e t e r m i n e if T C A has effects similar to D C A which has been extensively studied. D C A activates the p y r u v a t e d e h y d r o g e n a s e
1
I
0
i
0
-o-
|
DAY
|
i
.04
.63
.16
7
-*-
DAY
|
2.38
14
3500"
-T
150 0
. .04 DOSE
.
. .16 TCA
. .63
9:':18
(g/L)
Fig. 4. Urine volume and osmolality during TCA exposure of female rats. Urine samples were collected on days 7 and 14 of TCA exposure. Results are shown as mean and S.E. of 6 rats. The TCA treatment factor was significant for urine volume and osmolality. *Different from 0 TCA controls, P < 0.05.
69
TABLE II TCA EFFECT ON OSMOTIC EXCRETION TCA concentration in drinking water (g/l) mOsm/day:
0.00
0.04
O. 16
0.63
2.38
Males Day 7 ± S.E. Day 14 ± S.E.
18 1 18 2
19 1 19 2
20 1 19 1
18 I 23 2
19 4 19 I
Females Day 7 S.E. Day 14 ± S.E.
16 1 19 1
17 I 20 2
19 1 18 2
17 1 20 2
12" I 20 1
*Different from highest value, P < 0.05.
complex (PDH), increasing oxidation of glucose and pyruvate through the tricarboxylic acid cycle, which in turn decreases lactate and pyruvate [23]. DCA also increases the clearance and excretion of lactate and pyruvate, further contributing to the decrease of plasma concentrations of both [24]. In liver, DCA is metabolized to glyoxalate and then oxalate [25,26]; oxalate inhibits pyruvate carboxylase [25]. By preventing synthesis of glucose from pyruvate, DCA can decrease glucose availability in states dependent upon gluconeogenesis [26]; however, blood glucose is not decreased in normal humans after DCA administration [27]. The results for tissue lactate are variable, as lactate is metabolized within the body and slight differences in delay of removing the tissue from the animal and the degree of hypoventilation that occurs prior to removing the tissue influence the lactate concentration. Activation of pyruvate dehydrogenase and increased oxidation of glucose and lactate can be shown more readily in isolated tissue preparations. The results for plasma lactate and glucose showed that TCA has effects similar to those of DCA in female rats. The drinking water exposure studies were done to determine if similar effects occur during continuous exposure to lower concentrations. Rats given the highest concentration of T C A consumed less water and food and lost weight. Plasma glucose concentrations were not affected by the 14 day exposure to TCA. Plasma lactate concentrations were not measured, and the effects on tissue lactate were not significant. The highest concentration was objectionable to rats, and likely would be to humans. While TCA has effects similar to DCA, high doses are required to produce these effects, greater than are likely to be present in chlorinated drinking water [1]. Therefore direct toxicity from TCA is unlikely. This agrees with previous results from DCA studies, in that concentrations that were objectionable caused only slight, non-significant decreases of liver lactate or plasma glucose [7]. Healthy adult rats were used for these studies, the young and aged may be at greater risk. Blackshear and co-workers [28] have shown that the
70
effects o f D C A are greater in e x p e r i m e n t a l diabetes, t h e r e f o r e it is likely t h a t diabetics w o u l d be m o r e susceptible to the effects o f T C A as well. W h i l e i n t e r m e d i a r y m e t a b o l i s m was n o t a f f e c t e d b y s u b - c h r o n i c e x p o s u r e to T C A , s o m e s e x - d e p e n d e n t effects o n renal f u n c t i o n were n o t e d . Renal f u n c t i o n was m a i n t a i n e d in the m a l e rats. T h o s e o n the high dose c o n s u m e d less liquid a n d also excreted s m a l l e r a m o u n t s o f a m o r e c o n c e n t r a t e d urine, an a p p r o p r i a t e response to d e c r e a s e d w a t e r c o n s u m p t i o n . In females, however, the urine was not as c o n c e n t r a t e d as it s h o u l d have been for the r e d u c t i o n o f v o l u m e , a n d total excretion o f o s m o l e s d e c r e a s e d , suggesting interference with electrolyte h o m e o s tatic m e c h a n i s m s . T h e results o f these e x p e r i m e n t s suggest t h a t T C A has effects on i n t e r m e d i a r y m e t a b o l i s m that are similar to t h o s e o f D C A . T h a t is, T C A m a y activate p y r u vate d e h y d r o g e n a s e similar to D C A o r be m e t a b o l i z e d to o x a l a t e a n d g l y o x a l a t e similar to D C A . F e m a l e rats e x p e r i e n c e d m o r e effects o f T C A t h a n did the male rats, as was the case with the urine o u t p u t a n d o s m o l a l i t y studies. T h e r e f o r e , there m a y be a sex d i f f e r e n c e in the h a n d l i n g o f T C A o r responsiveness to s o m e o f its effects. This sex d i f f e r e n c e is not a b s o l u t e , however, in that p l a s m a lactate c o n c e n t r a t i o n s were also d e p r e s s e d by T C A in m a l e rats. D i f f e r e n t rates o f m e t a b o l i s m m a y a c c o u n t for this difference.
Acknowledgements I a m g r a t e f u l to D i a n e L a n e , M a r q u e t a Lewczyk, K a t h l e e n T h a y n e , a n d C r a i g Y o u n g for technical assistance. A l t h o u g h the research d e s c r i b e d in this article has been f u n d e d w h o l l y o r in p a r t b y the U n i t e d States E n v i r o n m e n t a l P r o t e c t i o n A g e n c y t h r o u g h c o o p e r a t i v e a g r e e m e n t CR811906-01, it has n o t been s u b j e c t e d to A g e n c y review a n d t h e r e f o r e d o e s n o t necessarily reflect the views o f the A g e n c y a n d no o f f i c i a l e n d o r s e m e n t s h o u l d be i n f e r r e d .
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